Cellulose Synthase Mutants Distinctively Affect Cell Growth and Cell Wall Integrity for Plant Biomass Production in Arabidopsis

Cellulose Synthase Mutants Distinctively Affect Cell Growth and Cell Wall Integrity for Plant... Abstract Cellulose is the most characteristic component of plant cell walls, and plays a central role in plant mechanical strength and morphogenesis. Despite the fact that cellulose synthase (CesA) mutants exhibit a reduction in cellulose level, much remains unknown about their impacts on cell growth (elongation and division) and cell wall integrity that fundamentally determine plant growth. Here, we examined three major types of AtCesA mutants (rsw1, an AtCesA1 mutant; prc1-1 and cesa6, AtCesA6-null mutants; and IRX3, an AtCesA7 mutant) and transgenic mutants that overexpressed AtCesA genes in the background of AtCesA6-null mutants. We found that AtCesA6-null mutants showed a reduced cell elongation of young seedlings with little impact on cell division, which consequently affected cell wall integrity and biomass yield of mature plants. In comparison, rsw1 seedlings exhibited a strong defect in both cell elongation and division at restrictive temperature, whereas the IRX3 mutant showed normal seedling growth. Analyses of transgenic mutants indicated that primary wall AtCesA2, AtCesA3, AtCesA5 and AtCesA9 genes played a partial role in restoration of seedling growth. However, co-overexpression of AtCesA2 and AtCesA5 in AtCesA6-null mutants could greatly enhance cell division and fully restore wall integrity, leading to a significant increase in secondary wall thickness and biomass production in mature plants. Hence, this study has demonstrated distinct functions of AtCesA genes in plant cell growth and cell wall deposition for biomass production, which helps to expalin our recent finding that only three AtCesA6-like genes, rather than other AtCesA genes of the AtCesA family, could greatly enhance biomass production in transgenic Arabidopsis plants. Introduction The plant cell wall basically determines cell size and shape, providing structural support and dynamic protection against biotic and abiotic stresses in plants. It also represents an enormous biomass resource for production of biofuels and chemicals (Somerville et al. 2004, Hématy et al. 2009, Szymanski and Cosgrove 2009, Landrein and Hamant 2013, Malinovsky et al. 2014, Le Gall et al. 2015). In general, plant cell walls are comprised mainly of cellulose, non-cellulosic polysaccharides (hemicellulose and pectin), lignin, proteins and other chemical compounds of two different major types: a thin, pectin-rich primary cell wall (PCW) that surrounds all dividing and expanding cells, and a thicker, lignin-rich secondary cell wall (SCW) that maintains structural support for specialized cells such as vessel elements or fiber cells (Somerville et al. 2004, Harholt et al. 2010, Keegstra 2010, Scheller and Ulvskov 2010, McFarlane et al. 2014). In particular, PCW synthesis is closely associated with cell growth (elongation and division) processes that determine the size of tissues/organs, whereas SCW deposition is initiated during the process of cellular differentiation that contributes to plant mechanical strength and overall biomass production (Keegstra 2010, Schuetz et al. 2013, Hu et al. 2018). Cellulose, the most prominent load-bearing polymer of the plant cell wall, is synthesized at the plasma membrane by cellulose synthase (CesA) complexes (Mueller and Brown 1980, Cosgrove 2005, McFarlane et al. 2014, Liu et al. 2016, Schneider et al. 2016). In Arabidopsis, AtCesA1, AtCesA3 and one of the AtCesA6-like proteins (CesA6, CesA2, CesA5 and CesA9) are required for PCW cellulose synthesis (McFarlane et al. 2014), whereas AtCesA4, CesA7 and CesA8 are essential isoforms for SCW cellulose synthesis (Taylor et al. 1999, Taylor et al. 2000, Taylor et al. 2003). Unlike the null mutants of AtCesA1 and AtCesA3 genes which are gamete lethal (Persson et al. 2007), mutations in any one of the AtCesA6-like genes lead to relatively mild phenotypes (Arioli et al. 1998, Scheible et al. 2001, Cano-Delgado et al. 2003). Specifically, a null mutant of AtCesA6, i.e. the procuste mutants prc1-1, exhibit only slight deficiencies in cell elongation, cellulose synthesis and seedling growth (Fagard et al. 2000a, Fagard et al. 2000b), while cesa2, cesa5 and cesa9 single mutants or cesa2/cesa5 and cesa2/cesa9 double mutants had phenotypes indistinguishable from those of the wild type (WT; Desprez et al. 2007, Persson et al. 2007). However, adult cesa2/prc1-1 double mutants are strongly dwarfed and bushy, cesa5/prc1-1 double or cesa2/cesa5/prc1-1 triple homozygotes were seedling lethal (Desprez et al. 2007) and cesa2/prc1-1/cesa9 triple mutants are gamete lethal due to cesa9 tissue-specific floral expression (Persson et al. 2007). Moreover, AtCesA2 or AtCesA5 expression driven by the AtCesA6 promoter only partially complements prc1-1 mutant phenotypes (Desprez et al. 2007, Persson et al. 2007), suggesting that each AtCesA6-like gene may have its own specialized function. Since the AtCesA1 mutant (rsw1, radially swollen1) was first identified in Arabidopsis (Arioli et al. 1998), several dozen distinct CesA mutants have been characterized in different plant species (Popper et al. 2011). Although most mutants exhibit markedly reduced cellulose levels with strongly defective growth phenotypes, much remains unknown about their function associated with cell growth that fundamentally determines organ/tissue sizes of young seedlings and consequently affects cell wall formation and biomass production of mature plants. Importantly, our recent reports have indicated that only overexpression of three PCW AtCesA6-like genes (AtCesA2, AtCesA5 and AtCesA6), but not AtCesA3, AtCesA9 or SCW AtCesA7, could greatly enhance plant growth and biomass production by increasing cell growth and cell wall thickness in transgenic Arabidopsis plants (Hu et al. 2018). However, it remains to be explained further why three AtCesA6-like genes are unique for enhancing cell growth and biomass production in Arabidopsis. In this study, we performed integrative analyses of three major types of AtCesA mutants (rsw1, an AtCesA1 mutant; prc1-1 and cesa6, AtCesA6-null mutants; and IRX3, an AtCesA7 mutant) and transgenic mutants overexpressing all four AtCesA6-like genes and the PCW AtCesA3 gene in the background of AtCesA6-null mutants using three distinct tissues/organs of Arabidopsis: (i) hypocotyls of dark-grown seedlings, which are optimal for cell length (cell elongation) measurements (Gendreau et al. 1997); (ii) roots of light-grown seedlings that are available for meristem cell number (cell division) assessment (Dolan et al. 1993); and (iii) stems of mature plants, which are major targets for SCW (cell differentiation) observation (Tanaka et al. 2003). We then examined cell proliferation, cell wall formation and plant growth in representative AtCesA mutants and transgenic mutants. This study could distinguish the impacts of three major types of AtCesA mutants on cell elongation, cell division and cell wall integrity, providing insights into cell growth and cell wall formation for plant biomass production in Arabidopsis. Results Primary wall AtCesA1, rather than secondary wall AtCesA7, is essential for cell elongation and division Because AtCesA1, a major isoform of AtCesA complexes for cellulose biosynthesis of PCWs (Persson et al. 2007), displays high gene expression in young seedlings (McFarlane et al. 2014), this study examined its potential function in cell elongation and division. Using the previously identified temperature-sensitive rsw1 (cesa1) mutant (Arioli et al. 1998), we first treated the seedlings under 21°C (normal temperature) and 28°C (restrictive temperature) for several days. The dark- (D) or light-grown (L) seedlings showed much shorter and swollen hypocotyls and roots in the rsw1 mutant under 28°C compared with the WT (Col-0), and the defects of the rsw1 mutant were most remarkable in 7-day-old dark-grown (D7) or 7-day-old light-grown (L7) seedlings under 28°C (Supplementary Fig. S1A, B). We further observed that the rsw1 mutant had extremely retarded D7/L7 seedling growth at 28°C (Fig. 1A, B), and this mutant then became lethal as the growth time was prolonged, compared with the WT (Fig. 1C). Since the number of epidermal cells in a single vertical cell file (parallel to the direction of growth) is genetically fixed to approximately 20 cells in Arabidopsis hypocotyls (Gendreau et al. 1997), we presumed that the changed hypocotyl lengths are mainly due to cell elongation (Hu et al. 2018). Because the root apical meristem (RAM) is the trigger for cell division (Dolan et al. 1993, Beemster et al. 1998, Casamitjana-Martı´nez et al. 2003), we calculated the cortical cell numbers from cell division zones that are distant from the quiescent center (QC) to the transition zone (TZ). As a result, the reduced lengths of D7 hypocotyls and L7 roots at 28°C were due to much shorter cell lengths (by 2-fold) in rsw1 mutant hypocotyls and fewer cells (by 1.9-fold) in the RAM, compared with the WT (Fig. 1D). Taken together, the inability of the dark- or light-grown seedlings to grow at 28 °C suggested that the AtCesA1 gene was essential for both cell elongation and cell division. Fig. 1 View largeDownload slide Observations of cell and seedling growth in AtCesA1 and AtCesA7 mutants. (A, C) Images of Arabidopsis WT (Colombia-0; Col-0) and temperature-sensitive rsw1 mutant seedlings grown at 28°C on 1/2 MS medium for 7 d (A) and 16 d (C) in thge dark or light. Scale bars = 0.5 mm. (B) Hypocotyl and root lengths as shown in (A). (D) Nomarski images of the longest epidermal cells in 7-day-old dark-grown (D7) hypocotyls (the arrowhead indicates one cell length) and 7-day-old light-grown (L7) root meristem boundary (the arrowhead indicate the transition zone of the meristem and elongation zone) grown at 28°C as shown in (A). Scale bars = 100 μm. (E) Nine-day-old dark-grown (D9) or 9-day-old light-grown (L9) Arabidopsis seedlings of the WT (Landsberg erecta, Ler) and IRX3 mutant germinated and grown on 1/2 MS media. Scale bars = 5 mm. (F) Hypocotyl and root lengths as shown in (E). (G, H) Measurement of the longest basal epidermal cells of D9 hypocotyls (G) and cell number in the L9 RAM (H). The data are the mean ± SD of three independent biological replicates; n ≥ 50 seedlings in (B, F) and n ≥ 30 seedlings in (D, G, H) were measured in each replicate, and the asterisks denote statistical significance at P < 0.01 (**) between the WT (Col-0) and rsw1mutant or at P ≥ 0.05 between the WT (Ler) and IRX3 mutant by Student’s t-tests. Fig. 1 View largeDownload slide Observations of cell and seedling growth in AtCesA1 and AtCesA7 mutants. (A, C) Images of Arabidopsis WT (Colombia-0; Col-0) and temperature-sensitive rsw1 mutant seedlings grown at 28°C on 1/2 MS medium for 7 d (A) and 16 d (C) in thge dark or light. Scale bars = 0.5 mm. (B) Hypocotyl and root lengths as shown in (A). (D) Nomarski images of the longest epidermal cells in 7-day-old dark-grown (D7) hypocotyls (the arrowhead indicates one cell length) and 7-day-old light-grown (L7) root meristem boundary (the arrowhead indicate the transition zone of the meristem and elongation zone) grown at 28°C as shown in (A). Scale bars = 100 μm. (E) Nine-day-old dark-grown (D9) or 9-day-old light-grown (L9) Arabidopsis seedlings of the WT (Landsberg erecta, Ler) and IRX3 mutant germinated and grown on 1/2 MS media. Scale bars = 5 mm. (F) Hypocotyl and root lengths as shown in (E). (G, H) Measurement of the longest basal epidermal cells of D9 hypocotyls (G) and cell number in the L9 RAM (H). The data are the mean ± SD of three independent biological replicates; n ≥ 50 seedlings in (B, F) and n ≥ 30 seedlings in (D, G, H) were measured in each replicate, and the asterisks denote statistical significance at P < 0.01 (**) between the WT (Col-0) and rsw1mutant or at P ≥ 0.05 between the WT (Ler) and IRX3 mutant by Student’s t-tests. We also assessed the effect of the SCW major gene AtCesA7 on cell elongation and division using the previously identified IRX3 (cesa7) mutant. Regardless of the defective phenotype of the mature plants reported previously (Taylor et al. 1999), the IRX3 mutant displayed normal seedling growth (Fig. 1E), with D9 hypocotyls and L9 roots having lengths similar to those of the WT (Ler; Fig. 1F). Moreover, the IRX3 mutant showed an insignificant change of cell lengths in the D9 hypocotyls (Fig. 1G) and of cell number in L9 RAM (Fig. 1H) compared with the WT, supporting the finding that AtCesA7 is one essential isoform of cellulose synthase complexes specific for SCW synthesis in Arabidopsis, rather than PCW synthesis related to cell elongation and division. Two AtCesA6-null mutants affect cell elongation rather than cell division To test AtCesA6-like genes, the special clade of AtCesA members, we selected the cesa6 mutant carrying the T-DNA insertion in the N-terminus of AtCesA6 and also examined the previously identified prc1-1 mutant carrying a mutation closer to the C-terminus of AtCesA6 (Fagard et al. 2000a; Fig. 2A). A diagram of the AtCesA6 protein showed that the mutation sites in cesa6 and prc1-1 mutants are the plant-conserved region (P-CR) and class-specific region (CSR), respectively, which are two important regions with potential for CesA isoform interactions (Atanassov et al. 2009; Fig. 2B). As a result, two AtCesA6-null mutants showed similar defective phenotypes, with shorter and swollen D9 hypocotyls and L9 root tissues (Fig. 2C, D). With regards to the defective hypocotyl growth, the AtCesA6-null mutants had remarkably short cell lengths in the D9 hypocotyls compared with the WT (Col-0; Fig. 2E). Unlike the rsw1 mutant (Fig. 1D), both AtCesA6-null mutants showed indistinctive changes in L9 RAM compared with the WT (Fig. 2F). Hence, the results suggested that both the reduced D9 hypocotyl and L9 root lengths in the AtCesA6-null mutants result from a decrease in cell lengths rather than cell numbers. Fig. 2 View largeDownload slide Observations of cell and seedling growth in two AtCesA6-null mutants. (A) Diagrams of AtCesA6 genomic regions showing the insertion site of the T-DNA in the cesa6 mutant and the positions of the nonsense mutation in the prc1-1 mutant, respectively. (B) A diagram of AtCesA6 protein showing mutation sites in cesa6 and prc1-1 mutants. P-CR, plant-conserved region; CSR, class-specific region. (C, D) Images of D9 hypocotyls and L9 roots using a camera (C) and stereoscopic microscope (D) in the WT and AtCesA6-null mutants (prc1-1 and cesa6). Scale bars = 5 mm. (E, F) Nomarski images of the longest epidermal cells in D9 hypocotyls (E) and L9 root meristem boundary (F). Scale bars = 100 μm. The data are the mean ± SD of three independent biological replicates; n ≥ 50 seedlings were measured in each replicate and the asterisks denote statistical significance at P < 0.01 (**) between the WT (Col-0) and mutants by Student’s t-tests. Fig. 2 View largeDownload slide Observations of cell and seedling growth in two AtCesA6-null mutants. (A) Diagrams of AtCesA6 genomic regions showing the insertion site of the T-DNA in the cesa6 mutant and the positions of the nonsense mutation in the prc1-1 mutant, respectively. (B) A diagram of AtCesA6 protein showing mutation sites in cesa6 and prc1-1 mutants. P-CR, plant-conserved region; CSR, class-specific region. (C, D) Images of D9 hypocotyls and L9 roots using a camera (C) and stereoscopic microscope (D) in the WT and AtCesA6-null mutants (prc1-1 and cesa6). Scale bars = 5 mm. (E, F) Nomarski images of the longest epidermal cells in D9 hypocotyls (E) and L9 root meristem boundary (F). Scale bars = 100 μm. The data are the mean ± SD of three independent biological replicates; n ≥ 50 seedlings were measured in each replicate and the asterisks denote statistical significance at P < 0.01 (**) between the WT (Col-0) and mutants by Student’s t-tests. Overexpression of primary wall AtCesA genes partially recovers seedling growth defects of Arabidopsis AtCesA6-null mutants Because AtCesA6-like genes (AtCesA2, AtCesA5 and AtCesA9) driven by the AtCesA6 promoter only partially rescue the phenotype defects of the AtCesA6-null mutant prc1-1 (Desprez et al. 2007, Persson et al. 2007), we further selected homozygous transgenic mutants overexpressing four AtCesA6-like genes (AtCesA6, AtCesA2, AtCesA5 and AtCesA9) and the PCW AtCesA3 gene in AtCesA6-null mutants using the 35S promoter (Fig. 3; Supplementary Fig. S2), with three genetically independent homozygous transgenic lines generated for statistical analysis. To verify the transgenic mutants, we detected remarkably increased transcript levels and protein contents in D9 hypocotyls of all lines by Western blot (Fig. 3E;Supplementary Fig. S3) and Q-PCR (quantitative reverse transcription–PCR; Fig. 4A;Supplementary Fig. S4A) or RT–PCR (reverse transcription–PCR; Supplementary Fig. S2C) analyses. Fig. 3 View largeDownload slide Observations of seedling growth in the transgenic mutants. (A, C) D9 or L9 Arabidopsis seedlings germinated and grown on 1/2 MS media. WT. wild type (Col-0); EV/, transgenic plants transformed with empty vector; A2/, A5/, A6/prc1-1 or A2/, A5/, A6/cesa6 are the homozygous transgenic mutants that overexpress AtCesA2, AtCesA5 and AtCesA6 genes, respectively, in prc1-1 or cesa6 mutants. The A(2 + 5)/prc1-1 or A(2 + 5)/cesa6 is the co-overexpressed mutant obtained by genetically crossing A2/prc1-1 and A5/prc1-1 or A2/cesa6 and A5/cesa6 transgenic mutants. Scale bars = 5 mm. (B, D) Hypocotyls and roots lengths as shown in (A, C). The data are the mean ± SD of three independent biological replicates; n ≥ 50 seedlings were measured in each replicate and the LSD (least significant difference) test was used for multiple comparisons. Different letters above bars indicate that the means differ according to analysis of variance and LSD test (P < 0.01). (E) Western blot analyses of AtCesA2, AtCesA5 and AtCesA6 proteins in D9 seedlings of cesa6 and transgenic mutants. The data are the mean ± SD. Fig. 3 View largeDownload slide Observations of seedling growth in the transgenic mutants. (A, C) D9 or L9 Arabidopsis seedlings germinated and grown on 1/2 MS media. WT. wild type (Col-0); EV/, transgenic plants transformed with empty vector; A2/, A5/, A6/prc1-1 or A2/, A5/, A6/cesa6 are the homozygous transgenic mutants that overexpress AtCesA2, AtCesA5 and AtCesA6 genes, respectively, in prc1-1 or cesa6 mutants. The A(2 + 5)/prc1-1 or A(2 + 5)/cesa6 is the co-overexpressed mutant obtained by genetically crossing A2/prc1-1 and A5/prc1-1 or A2/cesa6 and A5/cesa6 transgenic mutants. Scale bars = 5 mm. (B, D) Hypocotyls and roots lengths as shown in (A, C). The data are the mean ± SD of three independent biological replicates; n ≥ 50 seedlings were measured in each replicate and the LSD (least significant difference) test was used for multiple comparisons. Different letters above bars indicate that the means differ according to analysis of variance and LSD test (P < 0.01). (E) Western blot analyses of AtCesA2, AtCesA5 and AtCesA6 proteins in D9 seedlings of cesa6 and transgenic mutants. The data are the mean ± SD. Fig. 4 View largeDownload slide Q-PCR analyses of AtCesA genes in young seedlings of transgenic mutants. (A, D) AtCesA2, AtCesA5 or AtCesA6 genes in D9 hypocotyls (A) and L9 roots (D). (B, E) AtCesA1 or AtCesA3 genes in D9 hypocotyls (B) and L9 roots (E). (C, F) The AtCesA8 gene in D9 hypocotyls (C) and L9 roots (F). AtGAPDH is used as the internal control, and the expression value of GAPDH is defined as 100. The data are the mean ± SD of three independent biological replicates, and the LSD test was used for multiple comparisons. Different letters above bars indicate that the means differ according to analysis of variance and LSD test (P < 0.01). Fig. 4 View largeDownload slide Q-PCR analyses of AtCesA genes in young seedlings of transgenic mutants. (A, D) AtCesA2, AtCesA5 or AtCesA6 genes in D9 hypocotyls (A) and L9 roots (D). (B, E) AtCesA1 or AtCesA3 genes in D9 hypocotyls (B) and L9 roots (E). (C, F) The AtCesA8 gene in D9 hypocotyls (C) and L9 roots (F). AtGAPDH is used as the internal control, and the expression value of GAPDH is defined as 100. The data are the mean ± SD of three independent biological replicates, and the LSD test was used for multiple comparisons. Different letters above bars indicate that the means differ according to analysis of variance and LSD test (P < 0.01). Compared with two AtCesA6-null mutants, the transgenic mutants (A2/, A5/prc1-1 or A2/, A5/cesa6) overexpressing either the AtCesA2 or AtCesA5 gene could compensate to some extenr for the defective growth phenotypes of the cesa6 or prc1-1 mutants (Fig. 3A–D). The transgenic mutant A9/cesa6 overexpressing AtCesA9, an exceptional AtCesA6-like gene with tissue-specific expression in flowers (Persson et al. 2007), also revealed partial compensatiton of the AtCesA6 gene in seedling growth (Supplementary Fig. S2A, B). The transgenic mutant A3/cesa6 overexpressing AtCesA3, another major isoform (similar to AtCesA1) of the cellulose synthase complex of PCWs (Persson et al. 2007), displayed extensive compensation with increased D9 hypocotyl and L9 root lengths (Supplementary Fig. S2A, B). Furthermore, co-overexpression of AtCesA2 and AtCesA5 in the cesa6 or prc1-1 mutant could lead to much stronger complementation, particularly in terms of L9 root growth in the co-overexpressed transgenic mutant [e.g. A(2 + 5)/cesa6] generated by crossing A2/cesa6 and A5/cesa6 transgenic mutants (Fig. 3A–D). However, full restoration to WT levels was only observed in A6/prc1-1 and A6/cesa6 (overexpressing the AtCesA6 gene) (Fig. 3A–D). Overexpression of three AtCesA6-like genes in AtCesA6-null mutants increases the expression of AtCesA family genes Our recent report has shown that overexpression of any of the three AtCesA6-like genes (AtCesA6, AtCesA2 and AtCesA5) in the Arabidopsis WT could increase the expression of other PCW AtCesA genes to produce more cellulose (Hu et al. 2018). In this study, we further analyzed the expression levels of major AtCesA genes in young seedlings of the transgenic mutants that each overexpressed one of the three AtCesA6-like genes (Fig. 4; Supplementary Fig. S4). We found that overexpression of one of the AtCesA2, AtCesA5 and AtCesA6 genes could enhance expression of the other two in both D9 hypocotyls (Fig. 4A;Supplementary Fig. S4A) and L9 roots in transgenic mutants (Fig. 4D;Supplementary Fig. S4D), compared with the cesa6 or prc1-1 mutant. Notably, the other two major PCW AtCesA genes (AtCesA1 and AtCesA3) also displayed obviously increased expression levels in both D9 hypocotyls (Fig. 4B;Supplementary Fig. S4B) and L9 roots (Fig. 4E;Supplementary Fig. S4E), compared with the cesa6 or prc1-1 mutant. Interestingly, one of the major SCW CesA genes, AtCesA8, also showed markedly increased expression levels in both D9 hypocotyls (Fig. 4C;Supplementary Fig. S4C) and L9 roots of transgenic mutants (Fig. 4F;Supplementary Fig. S4F), compared with the cesa6 or prc1-1 mutant. However, the expression levels of the AtCesA8 gene in mutants and transgenic mutant D9 hypocotyls were still much lower than in the WT (Fig. 4C;Supplementary Fig. S4C). Hence, overexpression of any of the three AtCesA6-like genes in AtCesA6-null mutants could increase the expression of almost all the AtCesA family genes in transgenic mutants, compared with AtCesA6-null mutants. Co-overexpression of AtCesA2 and AtCesA5 largely recovers cellulose levels rather than macrofibril characteristics To assess how AtCesA2 and AtCesA5 genes affect PCW cellulose synthesis in the background of AtCesA6-null mutants, we measured the cellulose levels in D9 and L9 seedlings, and found that the reduced cellulose levels of young seedlings in the AtCesA6-null mutants were partially restored in the transgenic mutants [A2/, A5/prc1-1 or A2/, A5/, A(2 + 5)/cesa6] and completely restored to WT levels in A6/prc1-1 or A6/cesa6 (Fig. 5A, B;Supplementary Fig. S5). Fig. 5 View largeDownload slide Analyses of cellulose characteristics in transgenic mutants. (A, B) Absolute crystalline cellulose contents in D9 seedlings (A) and L9 seedlings (B). The data are the mean ± SD of three independent biological replicates; n = 100 seedlings were measured in each replicate and the asterisks denote statistical significance at P < 0.01 (**) between the WT and mutant or transgenic mutants by Student’s t-tests. The decreased cellulose rate (–%) was calculated by subtraction of values between WT and transgenic lines divided by the WT values. (C) Reassembly of macrofibrils from purified cellulose using AFM. The relative average particle size (width×length) is calculated from randomly selecting 10 particles in each image from three biological replicates. The WT is cited from our recently published article (Hu et al. 2018). Fig. 5 View largeDownload slide Analyses of cellulose characteristics in transgenic mutants. (A, B) Absolute crystalline cellulose contents in D9 seedlings (A) and L9 seedlings (B). The data are the mean ± SD of three independent biological replicates; n = 100 seedlings were measured in each replicate and the asterisks denote statistical significance at P < 0.01 (**) between the WT and mutant or transgenic mutants by Student’s t-tests. The decreased cellulose rate (–%) was calculated by subtraction of values between WT and transgenic lines divided by the WT values. (C) Reassembly of macrofibrils from purified cellulose using AFM. The relative average particle size (width×length) is calculated from randomly selecting 10 particles in each image from three biological replicates. The WT is cited from our recently published article (Hu et al. 2018). Furthermore, we assessed whether AtCesA2 and AtCesA5 co-overexpression affected cellulose macrofibril characteristics by observing the reassembly of macrofibrils of D9 hypocotyls in vitro under atomic force microscopy (AFM). Compared with our recently observed egg-shaped macrofibrils in the WT (Hu et al. 2018), the cesa6 mutant showed rice grain-like macrofibrils, suggesting that cellulose properties may be great altered in the mutant. Notably, the co-overexpressing mutant [A(2 + 5)/cesa6] exhibited very large rice grain-like macrofibrils (Fig. 5C), indicating that AtCesA2 and AtCesA5 co-overexpression in cesa6 mutant may largely restore cellulose levels (Fig. 5A, B), rather than cellulose macrofibril characteristics, compared with the cesa6 mutant. AtCesA2 and AtCesA5 play different compensation roles in cell elongation and division Because AtCesA2 and AtCesA5 co-overexpression largely restored cellulose levels in transgenic AtCesA6-null mutants, as described above, we investigated its impact on cell growth. At the cellular level, the longest cell lengths in the basal D9 hypocotyls were increased in the transgenic mutants [A2/, A5/, A(2 + 5)/prc1-1 or A2/, A5/, A(2 + 5)/cesa6] as a result of the partial complementation of mutants (Fig. 6A, B, E). Interestingly, like AtCesA6-null mutants (Fig. 2F), the transgenic mutants (A2/, A5/, A6/prc1-1 or A2/, A5/, A6/cesa6) maintained a similar cell number (Fig. 6C, D), indicating that the partial complementation in L9 root lengths of transgenic mutants result from an increase in cell lengths rather than cell numbers. Fig. 6 View largeDownload slide Analyses of cell growth in the transgenic mutants. (A–D) The longest epidermal cell lengths of D9 hypocotyls (A, B) and cell number of L9 RAM (C, D). The data are the mean ± SD of three independent biological replicates; n ≥ 30 seedlings were measured in each replicate, and the LSD test is used for multiple comparisons. Different letters above bars indicate that the means differ according to analysis of variance and LSD test (P < 0.01). (E) Confocal laser scanning microscopy images of the longest basal epidermal cells of D4 hypocotyls using PI staining (red fluorescence). Arrowheads indicate a single cell. Scale bars = 100 μm. (F) Typical expression of the G2/M-specific marker proAtCYCB1;1::AtCYCB1;1-GFP (green) of the plant cell cycle in the RAM using PI staining (red fluorescence). Scale bars = 75 μm. Fig. 6 View largeDownload slide Analyses of cell growth in the transgenic mutants. (A–D) The longest epidermal cell lengths of D9 hypocotyls (A, B) and cell number of L9 RAM (C, D). The data are the mean ± SD of three independent biological replicates; n ≥ 30 seedlings were measured in each replicate, and the LSD test is used for multiple comparisons. Different letters above bars indicate that the means differ according to analysis of variance and LSD test (P < 0.01). (E) Confocal laser scanning microscopy images of the longest basal epidermal cells of D4 hypocotyls using PI staining (red fluorescence). Arrowheads indicate a single cell. Scale bars = 100 μm. (F) Typical expression of the G2/M-specific marker proAtCYCB1;1::AtCYCB1;1-GFP (green) of the plant cell cycle in the RAM using PI staining (red fluorescence). Scale bars = 75 μm. Notably, the co-overexpressing mutants [A(2 + 5)/cesa6 or A(2 + 5)/prc1-1] had significantly increased cell numbers compared with AtCesA6-null mutants and the WT (Fig. 6C, D). In addition, genetic crosses with the proCYCB1::CYCB1-GFP transgenic plant, a classic G2/M-specific marker for cell division activity (Ferreira et al. 1994, Colón-Carmona et al. 1999, Ubeda-Tomas et al. 2009), revealed that A(2 + 5)/cesa6 had increased density and intensity of GFP (green fluorescent protein) in the L2 or L4 roots compared with the cesa6 mutant (Fig. 6F), indicating that co-overexpression of AtCesA2 and AtCesA5 could lead to an accumulative enhancement of cell division in the AtCesA6-null mutants. Furthermore, we performed RNA sequencing analyses of 6-day-old dark-grown (D6) WT, cesa6 mutant and transgenic mutants. Compared with the WT, the cesa6 mutant showed transcriptional differences of many genes associated with Gene Ontology-Biological Process (GO-BP) terms related to plant growth (Supplementary Fig. S6B). Further, compared with cesa6, the transgenic mutants also showed different transcriptional levels of some genes related to plant growth (Supplementary Fig. S6A, C–E). AtCesA2 and AtCesA5 have accumulative impacts on secondary wall integrity and plant biomass production To examine how AtCesA2 and AtCesA5 genes affect plant growth and development in the background of AtCesA6-null mutants, we observed plant phenotypes at the flourishing flowering stage. The transgenic mutants (such as A2/, A5/prc1-1) could compensate to some degree for the defective growth phenotypes of the prc1-1 mutants (Supplementary Fig. S7A), whereas the co-overexpressed transgenic mutants [A(2 + 5)/cesa6 or A(2 + 5)/prc1-1] almost fully restored the phenotype of the WT (Fig. 7A; Supplementary Fig. S7A). Fig. 7 View largeDownload slide Observations of secondary cell walls and analyses of mechanical strength and biomass production in mature plants. (A) Plant phenotypes at the flourishing flowering stage. Scale bars = 15 mm. (B) Transverse sections of the first internode of stems of 7-week-old plants are observed under epifluorescence microscopy using Calcofluor staining; co, cortex; ph, phloem; ve, vessel; xf, xylary fiber; if, interfascicular fiber. Scale bars = 50 μm. (C) Observations of the sclerenchyma cell walls in xf tissues in the first the first internode of stems of 7-week-old plants using TEM. Scale bars = 1 μm. (D) Distribution of mechanical strength (Young’s modulus) of reassembled crude cell walls in the first internode of stems of 7-week-old plants using AFM. Bars indicate the means of two biological replicates; 30 cell segments (n = 30) were measured for each replicate, Wilcoxon test is performed. Note, the WT is referenced in our recent paper (Hu et al. 2018). (E, F) Dry weight of 7-week-old mature plants. The data are the mean ± SD of three independent biological replicates; n ≥ 30 seedlings were measured in each replicate, and the asterisks denote statistical significance at P < 0.01 (**) between the WT and others by Student’s t-tests. The rate (%) is calculated by subtraction of values between the WT and other lines divided by the WT value. Fig. 7 View largeDownload slide Observations of secondary cell walls and analyses of mechanical strength and biomass production in mature plants. (A) Plant phenotypes at the flourishing flowering stage. Scale bars = 15 mm. (B) Transverse sections of the first internode of stems of 7-week-old plants are observed under epifluorescence microscopy using Calcofluor staining; co, cortex; ph, phloem; ve, vessel; xf, xylary fiber; if, interfascicular fiber. Scale bars = 50 μm. (C) Observations of the sclerenchyma cell walls in xf tissues in the first the first internode of stems of 7-week-old plants using TEM. Scale bars = 1 μm. (D) Distribution of mechanical strength (Young’s modulus) of reassembled crude cell walls in the first internode of stems of 7-week-old plants using AFM. Bars indicate the means of two biological replicates; 30 cell segments (n = 30) were measured for each replicate, Wilcoxon test is performed. Note, the WT is referenced in our recent paper (Hu et al. 2018). (E, F) Dry weight of 7-week-old mature plants. The data are the mean ± SD of three independent biological replicates; n ≥ 30 seedlings were measured in each replicate, and the asterisks denote statistical significance at P < 0.01 (**) between the WT and others by Student’s t-tests. The rate (%) is calculated by subtraction of values between the WT and other lines divided by the WT value. Because the basal stem tissues are thought to provide mechanical strength and support to entire mature plants (Appenzeller et al. 2004, Hu et al. 2018, Fan et al. 2018), we observed transverse sections of 7-week-old first internode stems using Calcofluor staining. Compared with the WT, the cesa6 and prc1-1 mutants were observed to have irregular expanding pith cells (parenchyma cells); the transgenic mutants could partially rescue the defective phenotype, and co-overexpressed transgenic mutants almost fully restored the phenotype (Fig. 7B; Supplementary Fig. S7B). Using transmission electron microscopy (TEM), we observed the xylary fiber cells that are typical of SCWs in the basal stems of 7-week-old Arabidopsis plants. The cesa6 and prc1-1 mutants, which are defective in cellulose biosynthesis of PCWs, exhibited no boundary lines between the PCW and SCW, and an incomplete cell wall morphology in the xylary fiber cells, and these defects could not be fully rescued by overexpressing single AtCesA2 or AtCesA5 genes in the transgenic mutants (Fig. 7C;Supplementary Fig. S7C). However, co-overexpression of the AtCesA2 and AtCesA5 genes resulted in a complete complementation with respect to cell wall integrity in transgenic mutants such as A(2 + 5)/cesa6 and A(2 + 5)/prc1-1, and the SCWs were even much thicker than those of the WT (Fig. 7C; Supplementary Fig. S7C), suggesting that AtCesA2 and AtCesA5 genes may play accumulative roles in cell wall integrity and thickness in AtCesA6-null mutants. We then extracted crude cell walls from the first internode stem of 7-week-old plants and detected their wall forces (Young’s modulus) using AFM technology (Hu et al. 2018). Compared with the cesa6 mutant, the A(2 + 5)/cesa6 plants exhibited significantly enhanced mechanical strength with a higher proportion of Young’s modulus values in the range from 20–100 GPa (Fig. 7D). Therefore, the A(2 + 5)/cesa6 plants had relatively higher mechanical strength in the basal stems of plants, most probably due to the complete cell wall and enhanced SCW synthesis. Furthermore, we examined total biomass production in mature plants (Fig. 7E, F). Compared with the previously reported WT (Hu et al. 2018), relatively smaller plant sizes and lower biomass yields were detected in both AtCesA6-null mutants (cesa6 and prc1-1) and transgenic mutants (A2/, A5/prc1-1 or A2/, A5/cesa6), probably due to their incomplete cell walls. However, the co-overexpressed mutants [A(2 + 5)/prc1-1 and A(2 + 5)/cesa6] could even have significantly higher biomass yield than that of the WT (Fig. 7E, F), consistent with their thicker SCWs (Fig. 7C; Supplementary Fig. S7C), which represent the major biomass production site (Hu et al. 2018, Li et al. 2017, Fan et al. 2018). In terms of small plant sizes and reduced biomass yields, the AtCesA6-like mutants and transgenic mutants (A2/, A5/prc1-1 or A2/, A5/cesa6) exhibited much lower wall crystalline cellulose levels than those of the WT, but they showed practically no significant changes in non-cellulosic polysaccharides and lignin levels (Fig. 8A–F). Notably, despite the cellulose and lignin contents being similar to those of the WT, the co-overexpressed mutants [A(2 + 5)/prc1-1 and A(2 + 5)/cesa6] had 8% increased non-cellulosic polysaccharides levels (Fig. 8A–F), which should contribute to their higher biomass production (Fig. 7E, F). Using a glycan antibody for immunolabeling wall polymers in situ, we also observed stronger fluorescent signals of antibodies against homogalacturonan in a co-overexpressed mutant [A(2 + 5)/cesa6] and similar patterns and intensities for antibodies against xylan in the WT and A(2 + 5)/cesa6 (Fig. 8G). Fig. 8 View largeDownload slide Determinations of cell wall compositions in mature plants. (A–F) Cell wall compositions of 7-week-old stems including cellulose (A, B), non-cellulosic polysaccharides (C, D) and lignin (E, F). The data are the mean ± SD of three independent biological replicates, and the asterisks denote statistical significance at P < 0.05 (*) and P < 0.01 (**) between the WT and others by Student’s t-tests. The increased/decreased wall polymers rates (%) are calculated by subtraction of values between the WT and others divided by the WT value. (G) Immunofluorescent labeling of stems in 7-week-old plants of the cesa6 mutant and the A(2 + 5)/cesa6 co-overexpressed mutant using plant cell wall glycan-directed monoclonal antibodies. JIM5 antibody labels homogalacturonan and CCRC-M149 labels xylan (green). Calcofluor (white) stains the cell wall (β-glucans). Scale bars = 50 μm. Fig. 8 View largeDownload slide Determinations of cell wall compositions in mature plants. (A–F) Cell wall compositions of 7-week-old stems including cellulose (A, B), non-cellulosic polysaccharides (C, D) and lignin (E, F). The data are the mean ± SD of three independent biological replicates, and the asterisks denote statistical significance at P < 0.05 (*) and P < 0.01 (**) between the WT and others by Student’s t-tests. The increased/decreased wall polymers rates (%) are calculated by subtraction of values between the WT and others divided by the WT value. (G) Immunofluorescent labeling of stems in 7-week-old plants of the cesa6 mutant and the A(2 + 5)/cesa6 co-overexpressed mutant using plant cell wall glycan-directed monoclonal antibodies. JIM5 antibody labels homogalacturonan and CCRC-M149 labels xylan (green). Calcofluor (white) stains the cell wall (β-glucans). Scale bars = 50 μm. In addition, we examined the transgenic mutants (A3/,A9/cesa6) regarding their SCW formation and biomass production in mature plants. As a member of the AtCesA6-like genes, the AtCesA9 gene showed a similarity to the AtCesA2 or AtCesA5 gene in terms of plant size and biomass yield (Supplementary Fig. S2D, E). Although the A3/cesa6 transgenic mutant showed large compensation of seedling growth (Supplementary Fig. S2A, B), the mature plants showed an even smaller plant size and lower biomass yield compared with EV/cesa6 plants (Supplementary Fig. S2D, E), indicating that some difference may exist between AtCesA3 and AtCesA6-like genes for their partial compensations in AtCesA6-null mutants. Discussion Several dozen CesA mutants have shown remarkable defects in cellulose synthesis, but the fundamental functions of CesA genes in cell growth remain largely unexplored (Li et al. 2014). AtCesA1 and AtCesA3 have been characterized as essential isoforms of the PCW CesA complex, as their null mutants are gamete lethal (Arioli et al. 1998, Fagard et al. 2000a, Persson et al. 2007). Nevertheless, mutations in the isoforms that contribute to the third position of the PCW CesA complex, the AtCesA6-like genes, only result in mild growth retardation and cell swelling (Fagard et al. 2000a, Scheible et al. 2001, Cano-Delgado et al. 2003, McFarlane et al. 2014). Despite a reduction in cell elongation in the PCW CesA mutants (Fagard et al. 2000a, Cano-Delgado et al. 2003, Bischoff et al. 2011, Fujita et al. 2013, Chen et al. 2010, Chen et al. 2016) and interference with cell division and cell expansion in embryogenesis of the rsw1 mutant examined (Beeckman et al. 2002), our study showed that rsw1 at the restrictive temperature had strongly negative impacts on cell division, with little impact in the AtCesA6-null mutants in vegetative tissues (Figs. 1, 2), which may provide a fundamental explanation for why the rsw1 seedlings could not grow further and die when incubated for a long time at the restrictive temperature. Moreover, the overexpressed AtCesA3 gene in the background of Arabidopsis WT could not promote seedling growth with unchanged cell division (Hu et al. 2018), and the overexpressed AtCesA3 gene in the background of the cesa6 mutant could not further promote plant growth (Supplementary Fig. S2) with decreased cell division (data not shown), which perhaps reflects the ‘housekeeping’ role of AtCesA1 and AtCesA3 with regards to cell division. Despite the fact that little is known about the impact of CesA on cell division, recent reports have indicated that the cellulose synthase-like D (CSLD) genes, with the highest sequence similarity to CesAs among cellulose synthase-like families, promote cell division in maize leaves, rice and Arabidopsis roots (Hunter et al. 2012, Yoshikawa et al. 2013, Gu et al. 2016). Also, the PCW CesAs are trafficked to and from the growing cell plate, corroborating a function for both the CSLDs and the PCW CesAs in this process (Miart et al. 2014). Furthermore, our recent article has shown that overexpressed single AtCesA2, AtCesA5 and AtCesA6 genes in the background of the Arabidopsis WT could significantly enhance cell division (Hu et al. 2018), while only co-overexpressed AtCesA2 and AtCesA5 genes in the background of AtCesA6-null mutants could dramatically enhance cell division (Fig. 6). It will thus be interesting to unravel the mechanisms of how AtCesA2 and AtCesA5 genes regulate cell division in the absence of the AtCesA6 gene in future studies. In addition, our results showed that the IRX3 mutant had no effect on seedling growth (including cell elongation and division; Fig. 1), which confirms that AtCesA7 is specific for SCW synthesis in Arabidopsis (Taylor et al. 1999). As the first defense barrier against infectious pathogens, maintenance of the cell wall integrity is a prerequisite for plant growth (Hamann 2015). Previous reports have indicated that PCW cellulose-deficient plants have incomplete cell walls, such as in the prc1-1 mutant (Fagard et al. 2000a). In this study, based on observations of the irregular PCWs (Fig. 7B; Supplementary Fig. S7B) and incomplete SCWs (Fig. 7C; Supplementary Fig. S7C) in two AtCesA6-null mutants, the results suggest that the irregular PCWs could not mechanically maintain SCW integrity in the mature plants. Because the PCW is tightly associated with cell elongation and division, and the SCW starts with cell differentiation (Schuetz et al. 2013), it remains to be explored in the future how the PCW impacts on SCW deposition and integrity. In addition, despite this study used a standard method for cell wall observation under TEM, it remains a technique where it is difficult to distinguish whether or not the disrupted cell walls are due to sample preparation. However, we observed small amounts of complete cell walls in the mutants, and found hardly any disrupted walls from observations of the WT, suggesting that most disrupted walls in mutants are not due to sample preparation. Although AtCesA6-like genes (AtCesA2, AtCesA5 and AtCesA9) have been characterized by a partial redundancy of the AtCesA6 gene (Desprez et al. 2007, Persson et al. 2007), it remains unexplored how these genes partially compensate for the AtcesA6 mutant in terms of cell growth and cell wall formation. Here, we first selected transgenic AtCesA6-null mutants that overexpressed three AtCesA6-like genes, and then examined their distinct functions associated with cell growth and cell wall formation. Even though the classic 35S promoter was used for overexpressing target genes in this study, the three transgenic mutants (A2/, A5/ and A9/cesa6) only showed partially restored cell elongation and seedling growth, which confirms previous findings of three AtCesA6-like genes (AtCesA2, AtCesA5 and AtCesA9) showing partial redundancy of the AtCesA6 gene (Desprez et al. 2007, Persson et al. 2007). Interestingly, our results also suggested that each AtCesA6-like gene should have its own independent biological function, which was supported by the following evidence. (i) Overexpression of single AtCesA2 and AtCesA5 genes in the background of the AtCesA6-null mutants exhibited a distinct seedling growth between dark and light incubation conditions (Fig. 3). (ii) A2/cesa6 and A5/cesa6 transgenic mutants showed largely different global gene expression patterns (Fig. 4; Supplementary Fig. S6). (iii) AtCesA9 is specifically expressed in flower tissues. More importantly, this study further confirmed the independent functions of AtCesA6-like genes, especially AtCesA2 and AtCesA5, as a fully restored phenotype of the WT was observed in the co-overexpressed transgenic mutant [A(2 + 5)/prc1-1 or A(2 + 5)/cesa6] that exhibits dramatically increased cell numbers (Fig. 6). Thus we speculated that precise particularities of the three AtCesA6-like genes (AtCesA2, AtCesA5 and AtCesA6) caused the unique effects on cellulose biosynthesis, cell wall deposition and plant cell growth for biomass production (Hu et al. 2018). Furthermore, this study examined whether the A3/cesa6 transgenic mutant exhibited more restored seedlings growth than the A2/cesa6 and A5/cesa6 transgenic mutants, and the findings indicated that the AtCesA3 or AtCesA1 gene should have distinct biological function from that of AtCesA2 and AtCesA5. Hence, the A3/cesa6 or A1/cesa6 transgenic mutants could be used as good samples to explore the distinct roles of AtCesA1, AtCesA3 and AtCesA6 in cellulose synthase complex formation and PCW biosynthesis in the future. In conclusion, this study proposes a model highlighting distinct fucntions of three major types of AtCesA genes in cell elongation, cell division, cell wall integrity and plant growth in mutants and transgenic mutants (Fig. 9): (i) the AtCesA1 mutant (rsw1) has remarkably decreased cell elongation and division for a strongly defective growth phenotype at the restrictive temperature; (ii) the AtCesA7 mutant (IRX3) has little impact on cell and seedling growth, but has a reduced SCW due to the collapsed xylems and forms a small mature plant (Taylor et al. 1999); (iii) two AtCesA6-null mutants (prc1-1 and cesa6) show little impact on cell division, but have reduced cell elongation for affected cell wall integrity and biomass yield of mature plants; (iv) transgenic mutants (e.g. A2/, A5/prc1-1) have a partiallly restored phenotype; and (v) co-overexpressed transgenic mutants [A(2 + 5)/prc1-1 or A(2 + 5)/cesa6] show greatly enhanced cell division and a complete cell wall with a strongly restored phenotype of the WT, leading to markedly increased SCW thickness and biomass production in mature plants. Fig. 9 View largeDownload slide Schematic model outlining distinct functions of AtCesAs in cell elongation, cell division, cell wall integrity and plant biomass production in three major types of AtCesA mutants (rsw1, cesa6/prc1-1 and IRX3) and the transgenic mutants that respectively overexpress AtCesAs or co-overexpress AtCesA2 and AtCesA5 in AtCesA6-null mutants. Fig. 9 View largeDownload slide Schematic model outlining distinct functions of AtCesAs in cell elongation, cell division, cell wall integrity and plant biomass production in three major types of AtCesA mutants (rsw1, cesa6/prc1-1 and IRX3) and the transgenic mutants that respectively overexpress AtCesAs or co-overexpress AtCesA2 and AtCesA5 in AtCesA6-null mutants. Materials and Methods Plant materials and growth conditions Arabidopsis homozygous prc1-1 mutants (AtCesA6; Fagard et al. 2000a), rsw1 (AtCesA1; Arioli et al. 1998) and IRX3 (AtCesA7; Taylor et al. 1999) were used in this study. Seeds of the Atcesa6 (SALK_004589) mutant (T-DNA insertion) were obtained from the Arabidopsis Biological Resource Center at Ohio State University. Identification of cesa6 homozygous lines was carried out using three primers (LBb1.3 + LP + RP) designed in T-DNA Primer Design (http://signal.salk.edu/tdnaprimers.2.html) with the following sequences LBb1.3, 5'-ATTTTGCCGATTTCGGAAC-3'; LP, 5'-ATCTATCCTCTGATTTATGGTCTCTG-3'; and RP, 5'-TACTAACAAATACATCCACAGGGG-3'. For generation of overexpression constructs, the coding region of the complete AtCesA genes (CesA6, CesA2, CesA5, CesA3 and CesA9) driven by the D35S promoter were cloned into the binary vector pD1301s to generate the binary plasmid (Supplementary Table S2). Transgenic plants were generated by introduction of the plant expression constructs into Agrobacterium tumefaciens strain GV3101, and transformation was done by floral dipping (Zhang et al. 2006). All the plant expression constructs were transformed into prc1-1 or cesa6 mutants. T1Arabidopsis transgenic seedlings were selected on 1/2 Murashige and Skoog (MS) medium containing 50 mg l–1 hygromycin and confirmed by RT–PCR or Q-PCR. More than three hygromycin-resistant lines (independent transformation events) for each construct were selected as homozygous. Phenotypic characterization was performed on T5 homozygous transgenic lines. Homozygous transgenic types were crossed with each other and selected as homozygous (F3–F5), and confirmed by Q-PCR. Arabidopsis seeds were surface sterilized using 75% ethanol for 4 min and 10% sodium hypochloride with 0.01% Triton X-100 for 3 min, washed in sterile water several times then imbibed at 4°C in the dark in sterile water containing 0.1% agar for 3 d and germinated on plates containing 1/2 MS media (1% sucrose; pH 5.8) in 1% agar. Plates were incubated in a near vertical position at 22°C under light growth conditions (16 h light/8 h dark) for photomorphogenesis or dark growth conditions (24 h dark) for skotomorphogenesis. The seedlings were transplanted to the soil after the second real leaf was clearly visible. Arabidopsis growth conditions were described previously (Hu et al. 2017, Hu et al. 2018). RNA extraction and Q-PCR measurement Seedlings were germinated and grown on 1/2 MS medium for the indicated number of days under light or dark growth conditions, and seedlings (hypocotyls and roots) were harvested in liquid nitrogen. Total RNA extraction and Q-PCR amplification were carried ou as described previously (Hu et al. 2018). The expression value of GAPDH (glyceraldehyde phosphate dehydrogenase) was defined as 100, and the expression level of CesA genes were thus normalized to the expression level of GAPDH. All of the primers used in these assays are listed in Supplementary Table S1.Three biological replications were performed. Total protein extraction and Western blot analyses Total protein extraction of D9 seedlings was performed as described previously (Hu et al. 2018). The AtCesA2, -5 and -6 protein levels were detected by Western blot analysis as described previously (Li et al. 2017). Purification of primary antibodies was performed using protein A–agarose. Dilutions were 1:250, 1:125 and 1:30 for AtCesA6, AtCesA2 and AtCesA5 antibodies, respectively. The relative protein levels were calculated using Quantity One software and the Rubisco large subunit protein (rbcL) as internal reference for SDS–PAGE. Observation of cellulose macrofibrils by AFM The purified cellulose samples of D9 hypocotyls were prepared as described previously (Hu et al. 2018, Li et al. 2017). The cellulose samples were suspended in ultrahigh purity water, and placed on mica using a pipette. The mica was glued onto a metal disc (15 mm diameter) after removal of extra water under nitrogen, and then placed on the piezo scanner of an atomic force microscope (MultiMode VIII; Bruker). AFM imaging was carried out in ScanAsystAtCesAir mode using BrukerScanAsystAtCesAir probes (tip radius, 2 nm and silicon nitride cantilever; spring constant, 0.4 N m–1) with a slow scan rate of 1 Hz. All AFM images were one-third flattened and analyzed quantitatively by using NanoScope Analysis software (Bruker). Three biological replications were performed for each experiment and 10 dots of each AFM image were randomly selected to measure the width (nm)×length (nm) by NanoScope Analysis software (Bruker). The average particle length/width of each image was calculated from the 10 selected particles. Hypocotyl, root and cell length measurements To observe hypocotyl and root growth, Arabidopsis seedlings were scanned using an HP Scanjet 8300 scanner at 600 d.p.i.; the hypocotyl lengths of vertically grown seedlings were measured from the hypocotyl base to the apical hook and the root length was measured (root tip to hypocotyl base) using the freely available ImageJ 1.32j software (https://imagej.nih.gov/ij/ or http://rsb.info.nih.gov/ij/). Two-tail t-tests were performed with Microsoft Excel software. For images of epidermal cell patterns, D9 hypocotyls were mounted and images of epidermal cells were viewed by using differential interference contrast (80i; Nikon). At least three biological replicates were performed for each experiment and >30 seedlings were measured for each genotype. Cell lengths in recorded images were quantified using Image J, and epidermal cells of the hypocotyl were visualized under confocal laser scanning microscopy (p58; Leica) using D4 hypocotyls incubated in the dark for 10 min in a fresh solution of 15 mM (10 mg ml–1) propidium iodide (PI; Naseer et al. 2012). PI was excited at 488 nm, and fluorescence was detected at 600–700 nm. Observation of cell division To count the number of cortical cells between the QC and the TZ (indicating the position of the first elongating cortical cell; Beemster and Baskin 1998), L9 root tips were mounted, and images were viewed by differential interference contrast (80i, Nikon). At least three biological replications were performed for each experiment and >30 seedlings were measured for each genotype. To visualize cell cycle progression in living cells, the G2/M-specific marker proAtCYCB1;1::AtCYCB1;1-GFP (Ubeda-Tomas et al. 2009) was crossed with different homozygous AtCesA6-like transgenic lines. Measurements of F1 hybrid seedlings were performed using confocal images of light-grown roots stained with PI. GFP was excited at 473 nm, and fluorescence was detected at 485–545 nm. Observation of cell wall structures by TEM TEM was used to observe cell wall structures in the xylary fiber cells of the first inflorescence stems of 7-week-old plants. The samples were post-fixed in 2% (w/v) osmium tetroxide (OsO4) for 1 h after extensively washing in phosphate-buffered saline (PBS) and were embedded with Super Kit (Sigma-Aldrich). Sample sections were cut with an Ultracut E ultramicrotome (Leica) and picked up on formvar-coated copper grids. After post-staining with uranyl acetate and lead citrate, the specimens were viewed under a Hitachi H7500 transmission electron microscope. The width of three relatively fixed points on each cell wall was measured using ImageJ. More than 60 cell walls for each genotype were measured. Significance differences were determined by Student’s t-test. Three biological replications were performed. Crude cell wall extraction and mechanical force measurement by AFM The crude cell wall material of the basal (1 cm) inflorescence stems from 7-week-old plants were prepared as described previously (Hu et al. 2018). The crude cell wall material was suspended in ultrahigh purity water, placed on new mica using a pipette and dried in air overnight. The mica was glued onto a metal disc (15 mm diameter) and placed on the piezo scanner of an atomic force microscope (MultiMode VIII; Bruker). A hard tip (RTESP; Bruker) with radius of 8 nm, and spring constant of 40 Nm–1 was used in measurement of the mechanical properties. The precise spring constant was corrected by the Sader method, and the average deflection sensitivity was determined by measuring a set of force–distance curves on the mica. The scan size was 10 μm×10 μm, 16×16 FD curves were collected for every measurement and 10 different cell segments were randomly selected for mechanical measurements for each sample. The Young’s modulus was calculated by using the Hertz model of the NanoScope analysis software, and the Wilcoxon test was used to test the significance of the average Young’s modulus (He et al. 2015). Two biological replications were performed each experiment. Plant dry weight measurement The homozygous lines were transplanted into soil as individual plants per pot and the plants were grown in a glasshouse at 22°C under light-grown condition for 7 weeks in a fully randomized experimental design. Seven-week-old inflorescence stems were harvested from each plant, dried under a suitable temperature (55°C) for 3–5 d and finally weighed using an analytical balance. Three biological replications were performed for each experiment, and >30 plants were measured for each genotype. Significance analysis was performed by Student’s t-test. Immunolocalization of glycan epitopes Thirty-day-old Arabidopsis petioles were embedded with 4% agar and then cut into 60 μm sections using a microtome (VT1000S, Leica). The first node of 7-week-old Arabidopsis inflorescence internodes were cut into 8 μm sections using a paraffin slicer (RM2265, Leica). For immunolabeling, transverse sections were incubated in 3% (w/v) milk protein in 1× PBS (MP/PBS) for 1 h to block non-specific binding. Sections were then incubated with monoclonal antibodies (JIM5, CCRC-M93 and CCRC-M149 antibodies, which bind to homogalacturonan, xyloglucan and xylan, respectively) diluted in MP-PBS (JIM5, diluted 1:10; CCRC-M93 and CCRC-M149, diluted 1:5; http://glycomics.ccrc.uga.edu/wall2/antibodies/antibodyHome.html) for 1 h. After washing with PBS, sections were incubated with anti-mouse-IgG and observed as described previously (Hu et al. 2017). Composition of plant cell walls Plant cell walls were fractionated and their composition determined as described previously (Jin et al. 2016), with some minor modifications for crystalline cellulose extraction of D9 or L9 seedlings or the dry biomass powder of 7-week-old inflorescence stem (40 mesh) samples, as described previously (Hu et al. 2018). For extraction of wall polysaccharides, dry biomass powder (40 mesh) samples (0.1–1.0 g) were washed twice with 5.0 ml of buffer and twice with 5.0 ml of distilled water. The remaining pellet was stirred with 5.0 ml of chloroform–methanol (1:1, v/v) for 1 h at 40°C and washed twice with 5.0 ml of methanol, followed by 5.0 ml of acetone. The pellet was washed once with 5.0 ml of distilled water. The remaining pellet was added to a 5.0 ml aliquot of dimethylsulfoxide (DMSO)–water (9:1, v/v), vortexed for 3 min and then rocked gently on a shaker overnight. After centrifugation, the pellet was washed twice with 5.0 ml of DMSO–water, and then with 5.0 ml of distilled water three times. The remaining pellet was defined as total wall polysaccharides. Total lignin was determined as described previously (Sun et al. 2017). At least three biological replications were performed. Microscopic observation The first inflorescence stems of 7-week-old Arabidopsis plants were embedded with 4% agar and then cut into sections of 100 μm thick by a microtome (VT1000S, Leica). Stem sections were stained in Calcofluor for 3 min and then rinsed, mounted in water, and observed and photographed under epifluorescence microscopy (Olympus BX-61, Retiga-4000DC digital camera). Living phenotypic observation of seedlings were made under light microscopy using a Leica stereomicroscope (Leica S6 D, Leica DFC295 digital camera). RNA sequencing and analysis Total RNA was isolated from the D6 seedlings (six samples, two biological replications) using Trizol reagent (Invitrogen). RNA sample preparation, cDNA library construction, raw reads, identification of differentially expressed genes (DEGs) and GO-BP terms of DEG analyses were performed as described previously (Hu et al. 2018). Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the National Natural Science Foundation of China [31670296]; the Fundamental Research Funds for the Central Universities of China [2662015PY018, 2013QC042]; the Technical Innovation Special Fund of Hubei Province [2017ACA171]; and the National 111 Project [B08032]. Acknowledgements We would like to thank Dr. Herman Höfte (National Institute for Agricultural Research, INRA, France) for kindly providing the prc1-1 mutant, Dr. Yonghong Zhang (Huazhong Agricultural University, China) for providing proCYCB1;1::CYCB1;1-GFP transgenic plants, and Dr. Staffan Persson for kindly discussing the experiments. We also thank Kexing Xin, Limin He and Qinghua Zhang (Huazhong Agricultural University, China) for technical assistance with the confocal laser scanning microscope, transmission electron microscopy and RNA sequencing, respectively. Disclosures The authors have no conflicts of interest to declare. References Appenzeller L. , Doblin M. , Barreiro R. , Wang H.Y. , Niu X.M. , Kollipara K. ( 2004 ) Cellulose synthesis in maize: isolation and expression analysis of the cellulose synthase (CesA) gene family . Cellulose 11 : 287 – 299 . Google Scholar CrossRef Search ADS Arioli T. , Peng L. , Betzner A.S. , Burn J. , Wittke W. , Herth W. , et al. . 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Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations AFM atomic force microscopy CesA cellulose synthase CSLD cellulose synthase like D D9 9-day-old dark-grown DEG differentially expressed gene GFP green fluorescent protein GO-BP Gene Ontology-Biological Process L9 9-day-old light-grown MS Murashige and Skoog PCW primary cell wall PI propidium iodide prc procuste QC quiescent center Q-PCR quantitative reverse transcription–PCR RAM root apical meristem RT–PCR reverse transcription–PCR rsw1 radially swollen1 SCW secondary cell wall TEM transmission electron microscopy TZ transition zone WT wild type © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. 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Cellulose Synthase Mutants Distinctively Affect Cell Growth and Cell Wall Integrity for Plant Biomass Production in Arabidopsis

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© The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com
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Abstract

Abstract Cellulose is the most characteristic component of plant cell walls, and plays a central role in plant mechanical strength and morphogenesis. Despite the fact that cellulose synthase (CesA) mutants exhibit a reduction in cellulose level, much remains unknown about their impacts on cell growth (elongation and division) and cell wall integrity that fundamentally determine plant growth. Here, we examined three major types of AtCesA mutants (rsw1, an AtCesA1 mutant; prc1-1 and cesa6, AtCesA6-null mutants; and IRX3, an AtCesA7 mutant) and transgenic mutants that overexpressed AtCesA genes in the background of AtCesA6-null mutants. We found that AtCesA6-null mutants showed a reduced cell elongation of young seedlings with little impact on cell division, which consequently affected cell wall integrity and biomass yield of mature plants. In comparison, rsw1 seedlings exhibited a strong defect in both cell elongation and division at restrictive temperature, whereas the IRX3 mutant showed normal seedling growth. Analyses of transgenic mutants indicated that primary wall AtCesA2, AtCesA3, AtCesA5 and AtCesA9 genes played a partial role in restoration of seedling growth. However, co-overexpression of AtCesA2 and AtCesA5 in AtCesA6-null mutants could greatly enhance cell division and fully restore wall integrity, leading to a significant increase in secondary wall thickness and biomass production in mature plants. Hence, this study has demonstrated distinct functions of AtCesA genes in plant cell growth and cell wall deposition for biomass production, which helps to expalin our recent finding that only three AtCesA6-like genes, rather than other AtCesA genes of the AtCesA family, could greatly enhance biomass production in transgenic Arabidopsis plants. Introduction The plant cell wall basically determines cell size and shape, providing structural support and dynamic protection against biotic and abiotic stresses in plants. It also represents an enormous biomass resource for production of biofuels and chemicals (Somerville et al. 2004, Hématy et al. 2009, Szymanski and Cosgrove 2009, Landrein and Hamant 2013, Malinovsky et al. 2014, Le Gall et al. 2015). In general, plant cell walls are comprised mainly of cellulose, non-cellulosic polysaccharides (hemicellulose and pectin), lignin, proteins and other chemical compounds of two different major types: a thin, pectin-rich primary cell wall (PCW) that surrounds all dividing and expanding cells, and a thicker, lignin-rich secondary cell wall (SCW) that maintains structural support for specialized cells such as vessel elements or fiber cells (Somerville et al. 2004, Harholt et al. 2010, Keegstra 2010, Scheller and Ulvskov 2010, McFarlane et al. 2014). In particular, PCW synthesis is closely associated with cell growth (elongation and division) processes that determine the size of tissues/organs, whereas SCW deposition is initiated during the process of cellular differentiation that contributes to plant mechanical strength and overall biomass production (Keegstra 2010, Schuetz et al. 2013, Hu et al. 2018). Cellulose, the most prominent load-bearing polymer of the plant cell wall, is synthesized at the plasma membrane by cellulose synthase (CesA) complexes (Mueller and Brown 1980, Cosgrove 2005, McFarlane et al. 2014, Liu et al. 2016, Schneider et al. 2016). In Arabidopsis, AtCesA1, AtCesA3 and one of the AtCesA6-like proteins (CesA6, CesA2, CesA5 and CesA9) are required for PCW cellulose synthesis (McFarlane et al. 2014), whereas AtCesA4, CesA7 and CesA8 are essential isoforms for SCW cellulose synthesis (Taylor et al. 1999, Taylor et al. 2000, Taylor et al. 2003). Unlike the null mutants of AtCesA1 and AtCesA3 genes which are gamete lethal (Persson et al. 2007), mutations in any one of the AtCesA6-like genes lead to relatively mild phenotypes (Arioli et al. 1998, Scheible et al. 2001, Cano-Delgado et al. 2003). Specifically, a null mutant of AtCesA6, i.e. the procuste mutants prc1-1, exhibit only slight deficiencies in cell elongation, cellulose synthesis and seedling growth (Fagard et al. 2000a, Fagard et al. 2000b), while cesa2, cesa5 and cesa9 single mutants or cesa2/cesa5 and cesa2/cesa9 double mutants had phenotypes indistinguishable from those of the wild type (WT; Desprez et al. 2007, Persson et al. 2007). However, adult cesa2/prc1-1 double mutants are strongly dwarfed and bushy, cesa5/prc1-1 double or cesa2/cesa5/prc1-1 triple homozygotes were seedling lethal (Desprez et al. 2007) and cesa2/prc1-1/cesa9 triple mutants are gamete lethal due to cesa9 tissue-specific floral expression (Persson et al. 2007). Moreover, AtCesA2 or AtCesA5 expression driven by the AtCesA6 promoter only partially complements prc1-1 mutant phenotypes (Desprez et al. 2007, Persson et al. 2007), suggesting that each AtCesA6-like gene may have its own specialized function. Since the AtCesA1 mutant (rsw1, radially swollen1) was first identified in Arabidopsis (Arioli et al. 1998), several dozen distinct CesA mutants have been characterized in different plant species (Popper et al. 2011). Although most mutants exhibit markedly reduced cellulose levels with strongly defective growth phenotypes, much remains unknown about their function associated with cell growth that fundamentally determines organ/tissue sizes of young seedlings and consequently affects cell wall formation and biomass production of mature plants. Importantly, our recent reports have indicated that only overexpression of three PCW AtCesA6-like genes (AtCesA2, AtCesA5 and AtCesA6), but not AtCesA3, AtCesA9 or SCW AtCesA7, could greatly enhance plant growth and biomass production by increasing cell growth and cell wall thickness in transgenic Arabidopsis plants (Hu et al. 2018). However, it remains to be explained further why three AtCesA6-like genes are unique for enhancing cell growth and biomass production in Arabidopsis. In this study, we performed integrative analyses of three major types of AtCesA mutants (rsw1, an AtCesA1 mutant; prc1-1 and cesa6, AtCesA6-null mutants; and IRX3, an AtCesA7 mutant) and transgenic mutants overexpressing all four AtCesA6-like genes and the PCW AtCesA3 gene in the background of AtCesA6-null mutants using three distinct tissues/organs of Arabidopsis: (i) hypocotyls of dark-grown seedlings, which are optimal for cell length (cell elongation) measurements (Gendreau et al. 1997); (ii) roots of light-grown seedlings that are available for meristem cell number (cell division) assessment (Dolan et al. 1993); and (iii) stems of mature plants, which are major targets for SCW (cell differentiation) observation (Tanaka et al. 2003). We then examined cell proliferation, cell wall formation and plant growth in representative AtCesA mutants and transgenic mutants. This study could distinguish the impacts of three major types of AtCesA mutants on cell elongation, cell division and cell wall integrity, providing insights into cell growth and cell wall formation for plant biomass production in Arabidopsis. Results Primary wall AtCesA1, rather than secondary wall AtCesA7, is essential for cell elongation and division Because AtCesA1, a major isoform of AtCesA complexes for cellulose biosynthesis of PCWs (Persson et al. 2007), displays high gene expression in young seedlings (McFarlane et al. 2014), this study examined its potential function in cell elongation and division. Using the previously identified temperature-sensitive rsw1 (cesa1) mutant (Arioli et al. 1998), we first treated the seedlings under 21°C (normal temperature) and 28°C (restrictive temperature) for several days. The dark- (D) or light-grown (L) seedlings showed much shorter and swollen hypocotyls and roots in the rsw1 mutant under 28°C compared with the WT (Col-0), and the defects of the rsw1 mutant were most remarkable in 7-day-old dark-grown (D7) or 7-day-old light-grown (L7) seedlings under 28°C (Supplementary Fig. S1A, B). We further observed that the rsw1 mutant had extremely retarded D7/L7 seedling growth at 28°C (Fig. 1A, B), and this mutant then became lethal as the growth time was prolonged, compared with the WT (Fig. 1C). Since the number of epidermal cells in a single vertical cell file (parallel to the direction of growth) is genetically fixed to approximately 20 cells in Arabidopsis hypocotyls (Gendreau et al. 1997), we presumed that the changed hypocotyl lengths are mainly due to cell elongation (Hu et al. 2018). Because the root apical meristem (RAM) is the trigger for cell division (Dolan et al. 1993, Beemster et al. 1998, Casamitjana-Martı´nez et al. 2003), we calculated the cortical cell numbers from cell division zones that are distant from the quiescent center (QC) to the transition zone (TZ). As a result, the reduced lengths of D7 hypocotyls and L7 roots at 28°C were due to much shorter cell lengths (by 2-fold) in rsw1 mutant hypocotyls and fewer cells (by 1.9-fold) in the RAM, compared with the WT (Fig. 1D). Taken together, the inability of the dark- or light-grown seedlings to grow at 28 °C suggested that the AtCesA1 gene was essential for both cell elongation and cell division. Fig. 1 View largeDownload slide Observations of cell and seedling growth in AtCesA1 and AtCesA7 mutants. (A, C) Images of Arabidopsis WT (Colombia-0; Col-0) and temperature-sensitive rsw1 mutant seedlings grown at 28°C on 1/2 MS medium for 7 d (A) and 16 d (C) in thge dark or light. Scale bars = 0.5 mm. (B) Hypocotyl and root lengths as shown in (A). (D) Nomarski images of the longest epidermal cells in 7-day-old dark-grown (D7) hypocotyls (the arrowhead indicates one cell length) and 7-day-old light-grown (L7) root meristem boundary (the arrowhead indicate the transition zone of the meristem and elongation zone) grown at 28°C as shown in (A). Scale bars = 100 μm. (E) Nine-day-old dark-grown (D9) or 9-day-old light-grown (L9) Arabidopsis seedlings of the WT (Landsberg erecta, Ler) and IRX3 mutant germinated and grown on 1/2 MS media. Scale bars = 5 mm. (F) Hypocotyl and root lengths as shown in (E). (G, H) Measurement of the longest basal epidermal cells of D9 hypocotyls (G) and cell number in the L9 RAM (H). The data are the mean ± SD of three independent biological replicates; n ≥ 50 seedlings in (B, F) and n ≥ 30 seedlings in (D, G, H) were measured in each replicate, and the asterisks denote statistical significance at P < 0.01 (**) between the WT (Col-0) and rsw1mutant or at P ≥ 0.05 between the WT (Ler) and IRX3 mutant by Student’s t-tests. Fig. 1 View largeDownload slide Observations of cell and seedling growth in AtCesA1 and AtCesA7 mutants. (A, C) Images of Arabidopsis WT (Colombia-0; Col-0) and temperature-sensitive rsw1 mutant seedlings grown at 28°C on 1/2 MS medium for 7 d (A) and 16 d (C) in thge dark or light. Scale bars = 0.5 mm. (B) Hypocotyl and root lengths as shown in (A). (D) Nomarski images of the longest epidermal cells in 7-day-old dark-grown (D7) hypocotyls (the arrowhead indicates one cell length) and 7-day-old light-grown (L7) root meristem boundary (the arrowhead indicate the transition zone of the meristem and elongation zone) grown at 28°C as shown in (A). Scale bars = 100 μm. (E) Nine-day-old dark-grown (D9) or 9-day-old light-grown (L9) Arabidopsis seedlings of the WT (Landsberg erecta, Ler) and IRX3 mutant germinated and grown on 1/2 MS media. Scale bars = 5 mm. (F) Hypocotyl and root lengths as shown in (E). (G, H) Measurement of the longest basal epidermal cells of D9 hypocotyls (G) and cell number in the L9 RAM (H). The data are the mean ± SD of three independent biological replicates; n ≥ 50 seedlings in (B, F) and n ≥ 30 seedlings in (D, G, H) were measured in each replicate, and the asterisks denote statistical significance at P < 0.01 (**) between the WT (Col-0) and rsw1mutant or at P ≥ 0.05 between the WT (Ler) and IRX3 mutant by Student’s t-tests. We also assessed the effect of the SCW major gene AtCesA7 on cell elongation and division using the previously identified IRX3 (cesa7) mutant. Regardless of the defective phenotype of the mature plants reported previously (Taylor et al. 1999), the IRX3 mutant displayed normal seedling growth (Fig. 1E), with D9 hypocotyls and L9 roots having lengths similar to those of the WT (Ler; Fig. 1F). Moreover, the IRX3 mutant showed an insignificant change of cell lengths in the D9 hypocotyls (Fig. 1G) and of cell number in L9 RAM (Fig. 1H) compared with the WT, supporting the finding that AtCesA7 is one essential isoform of cellulose synthase complexes specific for SCW synthesis in Arabidopsis, rather than PCW synthesis related to cell elongation and division. Two AtCesA6-null mutants affect cell elongation rather than cell division To test AtCesA6-like genes, the special clade of AtCesA members, we selected the cesa6 mutant carrying the T-DNA insertion in the N-terminus of AtCesA6 and also examined the previously identified prc1-1 mutant carrying a mutation closer to the C-terminus of AtCesA6 (Fagard et al. 2000a; Fig. 2A). A diagram of the AtCesA6 protein showed that the mutation sites in cesa6 and prc1-1 mutants are the plant-conserved region (P-CR) and class-specific region (CSR), respectively, which are two important regions with potential for CesA isoform interactions (Atanassov et al. 2009; Fig. 2B). As a result, two AtCesA6-null mutants showed similar defective phenotypes, with shorter and swollen D9 hypocotyls and L9 root tissues (Fig. 2C, D). With regards to the defective hypocotyl growth, the AtCesA6-null mutants had remarkably short cell lengths in the D9 hypocotyls compared with the WT (Col-0; Fig. 2E). Unlike the rsw1 mutant (Fig. 1D), both AtCesA6-null mutants showed indistinctive changes in L9 RAM compared with the WT (Fig. 2F). Hence, the results suggested that both the reduced D9 hypocotyl and L9 root lengths in the AtCesA6-null mutants result from a decrease in cell lengths rather than cell numbers. Fig. 2 View largeDownload slide Observations of cell and seedling growth in two AtCesA6-null mutants. (A) Diagrams of AtCesA6 genomic regions showing the insertion site of the T-DNA in the cesa6 mutant and the positions of the nonsense mutation in the prc1-1 mutant, respectively. (B) A diagram of AtCesA6 protein showing mutation sites in cesa6 and prc1-1 mutants. P-CR, plant-conserved region; CSR, class-specific region. (C, D) Images of D9 hypocotyls and L9 roots using a camera (C) and stereoscopic microscope (D) in the WT and AtCesA6-null mutants (prc1-1 and cesa6). Scale bars = 5 mm. (E, F) Nomarski images of the longest epidermal cells in D9 hypocotyls (E) and L9 root meristem boundary (F). Scale bars = 100 μm. The data are the mean ± SD of three independent biological replicates; n ≥ 50 seedlings were measured in each replicate and the asterisks denote statistical significance at P < 0.01 (**) between the WT (Col-0) and mutants by Student’s t-tests. Fig. 2 View largeDownload slide Observations of cell and seedling growth in two AtCesA6-null mutants. (A) Diagrams of AtCesA6 genomic regions showing the insertion site of the T-DNA in the cesa6 mutant and the positions of the nonsense mutation in the prc1-1 mutant, respectively. (B) A diagram of AtCesA6 protein showing mutation sites in cesa6 and prc1-1 mutants. P-CR, plant-conserved region; CSR, class-specific region. (C, D) Images of D9 hypocotyls and L9 roots using a camera (C) and stereoscopic microscope (D) in the WT and AtCesA6-null mutants (prc1-1 and cesa6). Scale bars = 5 mm. (E, F) Nomarski images of the longest epidermal cells in D9 hypocotyls (E) and L9 root meristem boundary (F). Scale bars = 100 μm. The data are the mean ± SD of three independent biological replicates; n ≥ 50 seedlings were measured in each replicate and the asterisks denote statistical significance at P < 0.01 (**) between the WT (Col-0) and mutants by Student’s t-tests. Overexpression of primary wall AtCesA genes partially recovers seedling growth defects of Arabidopsis AtCesA6-null mutants Because AtCesA6-like genes (AtCesA2, AtCesA5 and AtCesA9) driven by the AtCesA6 promoter only partially rescue the phenotype defects of the AtCesA6-null mutant prc1-1 (Desprez et al. 2007, Persson et al. 2007), we further selected homozygous transgenic mutants overexpressing four AtCesA6-like genes (AtCesA6, AtCesA2, AtCesA5 and AtCesA9) and the PCW AtCesA3 gene in AtCesA6-null mutants using the 35S promoter (Fig. 3; Supplementary Fig. S2), with three genetically independent homozygous transgenic lines generated for statistical analysis. To verify the transgenic mutants, we detected remarkably increased transcript levels and protein contents in D9 hypocotyls of all lines by Western blot (Fig. 3E;Supplementary Fig. S3) and Q-PCR (quantitative reverse transcription–PCR; Fig. 4A;Supplementary Fig. S4A) or RT–PCR (reverse transcription–PCR; Supplementary Fig. S2C) analyses. Fig. 3 View largeDownload slide Observations of seedling growth in the transgenic mutants. (A, C) D9 or L9 Arabidopsis seedlings germinated and grown on 1/2 MS media. WT. wild type (Col-0); EV/, transgenic plants transformed with empty vector; A2/, A5/, A6/prc1-1 or A2/, A5/, A6/cesa6 are the homozygous transgenic mutants that overexpress AtCesA2, AtCesA5 and AtCesA6 genes, respectively, in prc1-1 or cesa6 mutants. The A(2 + 5)/prc1-1 or A(2 + 5)/cesa6 is the co-overexpressed mutant obtained by genetically crossing A2/prc1-1 and A5/prc1-1 or A2/cesa6 and A5/cesa6 transgenic mutants. Scale bars = 5 mm. (B, D) Hypocotyls and roots lengths as shown in (A, C). The data are the mean ± SD of three independent biological replicates; n ≥ 50 seedlings were measured in each replicate and the LSD (least significant difference) test was used for multiple comparisons. Different letters above bars indicate that the means differ according to analysis of variance and LSD test (P < 0.01). (E) Western blot analyses of AtCesA2, AtCesA5 and AtCesA6 proteins in D9 seedlings of cesa6 and transgenic mutants. The data are the mean ± SD. Fig. 3 View largeDownload slide Observations of seedling growth in the transgenic mutants. (A, C) D9 or L9 Arabidopsis seedlings germinated and grown on 1/2 MS media. WT. wild type (Col-0); EV/, transgenic plants transformed with empty vector; A2/, A5/, A6/prc1-1 or A2/, A5/, A6/cesa6 are the homozygous transgenic mutants that overexpress AtCesA2, AtCesA5 and AtCesA6 genes, respectively, in prc1-1 or cesa6 mutants. The A(2 + 5)/prc1-1 or A(2 + 5)/cesa6 is the co-overexpressed mutant obtained by genetically crossing A2/prc1-1 and A5/prc1-1 or A2/cesa6 and A5/cesa6 transgenic mutants. Scale bars = 5 mm. (B, D) Hypocotyls and roots lengths as shown in (A, C). The data are the mean ± SD of three independent biological replicates; n ≥ 50 seedlings were measured in each replicate and the LSD (least significant difference) test was used for multiple comparisons. Different letters above bars indicate that the means differ according to analysis of variance and LSD test (P < 0.01). (E) Western blot analyses of AtCesA2, AtCesA5 and AtCesA6 proteins in D9 seedlings of cesa6 and transgenic mutants. The data are the mean ± SD. Fig. 4 View largeDownload slide Q-PCR analyses of AtCesA genes in young seedlings of transgenic mutants. (A, D) AtCesA2, AtCesA5 or AtCesA6 genes in D9 hypocotyls (A) and L9 roots (D). (B, E) AtCesA1 or AtCesA3 genes in D9 hypocotyls (B) and L9 roots (E). (C, F) The AtCesA8 gene in D9 hypocotyls (C) and L9 roots (F). AtGAPDH is used as the internal control, and the expression value of GAPDH is defined as 100. The data are the mean ± SD of three independent biological replicates, and the LSD test was used for multiple comparisons. Different letters above bars indicate that the means differ according to analysis of variance and LSD test (P < 0.01). Fig. 4 View largeDownload slide Q-PCR analyses of AtCesA genes in young seedlings of transgenic mutants. (A, D) AtCesA2, AtCesA5 or AtCesA6 genes in D9 hypocotyls (A) and L9 roots (D). (B, E) AtCesA1 or AtCesA3 genes in D9 hypocotyls (B) and L9 roots (E). (C, F) The AtCesA8 gene in D9 hypocotyls (C) and L9 roots (F). AtGAPDH is used as the internal control, and the expression value of GAPDH is defined as 100. The data are the mean ± SD of three independent biological replicates, and the LSD test was used for multiple comparisons. Different letters above bars indicate that the means differ according to analysis of variance and LSD test (P < 0.01). Compared with two AtCesA6-null mutants, the transgenic mutants (A2/, A5/prc1-1 or A2/, A5/cesa6) overexpressing either the AtCesA2 or AtCesA5 gene could compensate to some extenr for the defective growth phenotypes of the cesa6 or prc1-1 mutants (Fig. 3A–D). The transgenic mutant A9/cesa6 overexpressing AtCesA9, an exceptional AtCesA6-like gene with tissue-specific expression in flowers (Persson et al. 2007), also revealed partial compensatiton of the AtCesA6 gene in seedling growth (Supplementary Fig. S2A, B). The transgenic mutant A3/cesa6 overexpressing AtCesA3, another major isoform (similar to AtCesA1) of the cellulose synthase complex of PCWs (Persson et al. 2007), displayed extensive compensation with increased D9 hypocotyl and L9 root lengths (Supplementary Fig. S2A, B). Furthermore, co-overexpression of AtCesA2 and AtCesA5 in the cesa6 or prc1-1 mutant could lead to much stronger complementation, particularly in terms of L9 root growth in the co-overexpressed transgenic mutant [e.g. A(2 + 5)/cesa6] generated by crossing A2/cesa6 and A5/cesa6 transgenic mutants (Fig. 3A–D). However, full restoration to WT levels was only observed in A6/prc1-1 and A6/cesa6 (overexpressing the AtCesA6 gene) (Fig. 3A–D). Overexpression of three AtCesA6-like genes in AtCesA6-null mutants increases the expression of AtCesA family genes Our recent report has shown that overexpression of any of the three AtCesA6-like genes (AtCesA6, AtCesA2 and AtCesA5) in the Arabidopsis WT could increase the expression of other PCW AtCesA genes to produce more cellulose (Hu et al. 2018). In this study, we further analyzed the expression levels of major AtCesA genes in young seedlings of the transgenic mutants that each overexpressed one of the three AtCesA6-like genes (Fig. 4; Supplementary Fig. S4). We found that overexpression of one of the AtCesA2, AtCesA5 and AtCesA6 genes could enhance expression of the other two in both D9 hypocotyls (Fig. 4A;Supplementary Fig. S4A) and L9 roots in transgenic mutants (Fig. 4D;Supplementary Fig. S4D), compared with the cesa6 or prc1-1 mutant. Notably, the other two major PCW AtCesA genes (AtCesA1 and AtCesA3) also displayed obviously increased expression levels in both D9 hypocotyls (Fig. 4B;Supplementary Fig. S4B) and L9 roots (Fig. 4E;Supplementary Fig. S4E), compared with the cesa6 or prc1-1 mutant. Interestingly, one of the major SCW CesA genes, AtCesA8, also showed markedly increased expression levels in both D9 hypocotyls (Fig. 4C;Supplementary Fig. S4C) and L9 roots of transgenic mutants (Fig. 4F;Supplementary Fig. S4F), compared with the cesa6 or prc1-1 mutant. However, the expression levels of the AtCesA8 gene in mutants and transgenic mutant D9 hypocotyls were still much lower than in the WT (Fig. 4C;Supplementary Fig. S4C). Hence, overexpression of any of the three AtCesA6-like genes in AtCesA6-null mutants could increase the expression of almost all the AtCesA family genes in transgenic mutants, compared with AtCesA6-null mutants. Co-overexpression of AtCesA2 and AtCesA5 largely recovers cellulose levels rather than macrofibril characteristics To assess how AtCesA2 and AtCesA5 genes affect PCW cellulose synthesis in the background of AtCesA6-null mutants, we measured the cellulose levels in D9 and L9 seedlings, and found that the reduced cellulose levels of young seedlings in the AtCesA6-null mutants were partially restored in the transgenic mutants [A2/, A5/prc1-1 or A2/, A5/, A(2 + 5)/cesa6] and completely restored to WT levels in A6/prc1-1 or A6/cesa6 (Fig. 5A, B;Supplementary Fig. S5). Fig. 5 View largeDownload slide Analyses of cellulose characteristics in transgenic mutants. (A, B) Absolute crystalline cellulose contents in D9 seedlings (A) and L9 seedlings (B). The data are the mean ± SD of three independent biological replicates; n = 100 seedlings were measured in each replicate and the asterisks denote statistical significance at P < 0.01 (**) between the WT and mutant or transgenic mutants by Student’s t-tests. The decreased cellulose rate (–%) was calculated by subtraction of values between WT and transgenic lines divided by the WT values. (C) Reassembly of macrofibrils from purified cellulose using AFM. The relative average particle size (width×length) is calculated from randomly selecting 10 particles in each image from three biological replicates. The WT is cited from our recently published article (Hu et al. 2018). Fig. 5 View largeDownload slide Analyses of cellulose characteristics in transgenic mutants. (A, B) Absolute crystalline cellulose contents in D9 seedlings (A) and L9 seedlings (B). The data are the mean ± SD of three independent biological replicates; n = 100 seedlings were measured in each replicate and the asterisks denote statistical significance at P < 0.01 (**) between the WT and mutant or transgenic mutants by Student’s t-tests. The decreased cellulose rate (–%) was calculated by subtraction of values between WT and transgenic lines divided by the WT values. (C) Reassembly of macrofibrils from purified cellulose using AFM. The relative average particle size (width×length) is calculated from randomly selecting 10 particles in each image from three biological replicates. The WT is cited from our recently published article (Hu et al. 2018). Furthermore, we assessed whether AtCesA2 and AtCesA5 co-overexpression affected cellulose macrofibril characteristics by observing the reassembly of macrofibrils of D9 hypocotyls in vitro under atomic force microscopy (AFM). Compared with our recently observed egg-shaped macrofibrils in the WT (Hu et al. 2018), the cesa6 mutant showed rice grain-like macrofibrils, suggesting that cellulose properties may be great altered in the mutant. Notably, the co-overexpressing mutant [A(2 + 5)/cesa6] exhibited very large rice grain-like macrofibrils (Fig. 5C), indicating that AtCesA2 and AtCesA5 co-overexpression in cesa6 mutant may largely restore cellulose levels (Fig. 5A, B), rather than cellulose macrofibril characteristics, compared with the cesa6 mutant. AtCesA2 and AtCesA5 play different compensation roles in cell elongation and division Because AtCesA2 and AtCesA5 co-overexpression largely restored cellulose levels in transgenic AtCesA6-null mutants, as described above, we investigated its impact on cell growth. At the cellular level, the longest cell lengths in the basal D9 hypocotyls were increased in the transgenic mutants [A2/, A5/, A(2 + 5)/prc1-1 or A2/, A5/, A(2 + 5)/cesa6] as a result of the partial complementation of mutants (Fig. 6A, B, E). Interestingly, like AtCesA6-null mutants (Fig. 2F), the transgenic mutants (A2/, A5/, A6/prc1-1 or A2/, A5/, A6/cesa6) maintained a similar cell number (Fig. 6C, D), indicating that the partial complementation in L9 root lengths of transgenic mutants result from an increase in cell lengths rather than cell numbers. Fig. 6 View largeDownload slide Analyses of cell growth in the transgenic mutants. (A–D) The longest epidermal cell lengths of D9 hypocotyls (A, B) and cell number of L9 RAM (C, D). The data are the mean ± SD of three independent biological replicates; n ≥ 30 seedlings were measured in each replicate, and the LSD test is used for multiple comparisons. Different letters above bars indicate that the means differ according to analysis of variance and LSD test (P < 0.01). (E) Confocal laser scanning microscopy images of the longest basal epidermal cells of D4 hypocotyls using PI staining (red fluorescence). Arrowheads indicate a single cell. Scale bars = 100 μm. (F) Typical expression of the G2/M-specific marker proAtCYCB1;1::AtCYCB1;1-GFP (green) of the plant cell cycle in the RAM using PI staining (red fluorescence). Scale bars = 75 μm. Fig. 6 View largeDownload slide Analyses of cell growth in the transgenic mutants. (A–D) The longest epidermal cell lengths of D9 hypocotyls (A, B) and cell number of L9 RAM (C, D). The data are the mean ± SD of three independent biological replicates; n ≥ 30 seedlings were measured in each replicate, and the LSD test is used for multiple comparisons. Different letters above bars indicate that the means differ according to analysis of variance and LSD test (P < 0.01). (E) Confocal laser scanning microscopy images of the longest basal epidermal cells of D4 hypocotyls using PI staining (red fluorescence). Arrowheads indicate a single cell. Scale bars = 100 μm. (F) Typical expression of the G2/M-specific marker proAtCYCB1;1::AtCYCB1;1-GFP (green) of the plant cell cycle in the RAM using PI staining (red fluorescence). Scale bars = 75 μm. Notably, the co-overexpressing mutants [A(2 + 5)/cesa6 or A(2 + 5)/prc1-1] had significantly increased cell numbers compared with AtCesA6-null mutants and the WT (Fig. 6C, D). In addition, genetic crosses with the proCYCB1::CYCB1-GFP transgenic plant, a classic G2/M-specific marker for cell division activity (Ferreira et al. 1994, Colón-Carmona et al. 1999, Ubeda-Tomas et al. 2009), revealed that A(2 + 5)/cesa6 had increased density and intensity of GFP (green fluorescent protein) in the L2 or L4 roots compared with the cesa6 mutant (Fig. 6F), indicating that co-overexpression of AtCesA2 and AtCesA5 could lead to an accumulative enhancement of cell division in the AtCesA6-null mutants. Furthermore, we performed RNA sequencing analyses of 6-day-old dark-grown (D6) WT, cesa6 mutant and transgenic mutants. Compared with the WT, the cesa6 mutant showed transcriptional differences of many genes associated with Gene Ontology-Biological Process (GO-BP) terms related to plant growth (Supplementary Fig. S6B). Further, compared with cesa6, the transgenic mutants also showed different transcriptional levels of some genes related to plant growth (Supplementary Fig. S6A, C–E). AtCesA2 and AtCesA5 have accumulative impacts on secondary wall integrity and plant biomass production To examine how AtCesA2 and AtCesA5 genes affect plant growth and development in the background of AtCesA6-null mutants, we observed plant phenotypes at the flourishing flowering stage. The transgenic mutants (such as A2/, A5/prc1-1) could compensate to some degree for the defective growth phenotypes of the prc1-1 mutants (Supplementary Fig. S7A), whereas the co-overexpressed transgenic mutants [A(2 + 5)/cesa6 or A(2 + 5)/prc1-1] almost fully restored the phenotype of the WT (Fig. 7A; Supplementary Fig. S7A). Fig. 7 View largeDownload slide Observations of secondary cell walls and analyses of mechanical strength and biomass production in mature plants. (A) Plant phenotypes at the flourishing flowering stage. Scale bars = 15 mm. (B) Transverse sections of the first internode of stems of 7-week-old plants are observed under epifluorescence microscopy using Calcofluor staining; co, cortex; ph, phloem; ve, vessel; xf, xylary fiber; if, interfascicular fiber. Scale bars = 50 μm. (C) Observations of the sclerenchyma cell walls in xf tissues in the first the first internode of stems of 7-week-old plants using TEM. Scale bars = 1 μm. (D) Distribution of mechanical strength (Young’s modulus) of reassembled crude cell walls in the first internode of stems of 7-week-old plants using AFM. Bars indicate the means of two biological replicates; 30 cell segments (n = 30) were measured for each replicate, Wilcoxon test is performed. Note, the WT is referenced in our recent paper (Hu et al. 2018). (E, F) Dry weight of 7-week-old mature plants. The data are the mean ± SD of three independent biological replicates; n ≥ 30 seedlings were measured in each replicate, and the asterisks denote statistical significance at P < 0.01 (**) between the WT and others by Student’s t-tests. The rate (%) is calculated by subtraction of values between the WT and other lines divided by the WT value. Fig. 7 View largeDownload slide Observations of secondary cell walls and analyses of mechanical strength and biomass production in mature plants. (A) Plant phenotypes at the flourishing flowering stage. Scale bars = 15 mm. (B) Transverse sections of the first internode of stems of 7-week-old plants are observed under epifluorescence microscopy using Calcofluor staining; co, cortex; ph, phloem; ve, vessel; xf, xylary fiber; if, interfascicular fiber. Scale bars = 50 μm. (C) Observations of the sclerenchyma cell walls in xf tissues in the first the first internode of stems of 7-week-old plants using TEM. Scale bars = 1 μm. (D) Distribution of mechanical strength (Young’s modulus) of reassembled crude cell walls in the first internode of stems of 7-week-old plants using AFM. Bars indicate the means of two biological replicates; 30 cell segments (n = 30) were measured for each replicate, Wilcoxon test is performed. Note, the WT is referenced in our recent paper (Hu et al. 2018). (E, F) Dry weight of 7-week-old mature plants. The data are the mean ± SD of three independent biological replicates; n ≥ 30 seedlings were measured in each replicate, and the asterisks denote statistical significance at P < 0.01 (**) between the WT and others by Student’s t-tests. The rate (%) is calculated by subtraction of values between the WT and other lines divided by the WT value. Because the basal stem tissues are thought to provide mechanical strength and support to entire mature plants (Appenzeller et al. 2004, Hu et al. 2018, Fan et al. 2018), we observed transverse sections of 7-week-old first internode stems using Calcofluor staining. Compared with the WT, the cesa6 and prc1-1 mutants were observed to have irregular expanding pith cells (parenchyma cells); the transgenic mutants could partially rescue the defective phenotype, and co-overexpressed transgenic mutants almost fully restored the phenotype (Fig. 7B; Supplementary Fig. S7B). Using transmission electron microscopy (TEM), we observed the xylary fiber cells that are typical of SCWs in the basal stems of 7-week-old Arabidopsis plants. The cesa6 and prc1-1 mutants, which are defective in cellulose biosynthesis of PCWs, exhibited no boundary lines between the PCW and SCW, and an incomplete cell wall morphology in the xylary fiber cells, and these defects could not be fully rescued by overexpressing single AtCesA2 or AtCesA5 genes in the transgenic mutants (Fig. 7C;Supplementary Fig. S7C). However, co-overexpression of the AtCesA2 and AtCesA5 genes resulted in a complete complementation with respect to cell wall integrity in transgenic mutants such as A(2 + 5)/cesa6 and A(2 + 5)/prc1-1, and the SCWs were even much thicker than those of the WT (Fig. 7C; Supplementary Fig. S7C), suggesting that AtCesA2 and AtCesA5 genes may play accumulative roles in cell wall integrity and thickness in AtCesA6-null mutants. We then extracted crude cell walls from the first internode stem of 7-week-old plants and detected their wall forces (Young’s modulus) using AFM technology (Hu et al. 2018). Compared with the cesa6 mutant, the A(2 + 5)/cesa6 plants exhibited significantly enhanced mechanical strength with a higher proportion of Young’s modulus values in the range from 20–100 GPa (Fig. 7D). Therefore, the A(2 + 5)/cesa6 plants had relatively higher mechanical strength in the basal stems of plants, most probably due to the complete cell wall and enhanced SCW synthesis. Furthermore, we examined total biomass production in mature plants (Fig. 7E, F). Compared with the previously reported WT (Hu et al. 2018), relatively smaller plant sizes and lower biomass yields were detected in both AtCesA6-null mutants (cesa6 and prc1-1) and transgenic mutants (A2/, A5/prc1-1 or A2/, A5/cesa6), probably due to their incomplete cell walls. However, the co-overexpressed mutants [A(2 + 5)/prc1-1 and A(2 + 5)/cesa6] could even have significantly higher biomass yield than that of the WT (Fig. 7E, F), consistent with their thicker SCWs (Fig. 7C; Supplementary Fig. S7C), which represent the major biomass production site (Hu et al. 2018, Li et al. 2017, Fan et al. 2018). In terms of small plant sizes and reduced biomass yields, the AtCesA6-like mutants and transgenic mutants (A2/, A5/prc1-1 or A2/, A5/cesa6) exhibited much lower wall crystalline cellulose levels than those of the WT, but they showed practically no significant changes in non-cellulosic polysaccharides and lignin levels (Fig. 8A–F). Notably, despite the cellulose and lignin contents being similar to those of the WT, the co-overexpressed mutants [A(2 + 5)/prc1-1 and A(2 + 5)/cesa6] had 8% increased non-cellulosic polysaccharides levels (Fig. 8A–F), which should contribute to their higher biomass production (Fig. 7E, F). Using a glycan antibody for immunolabeling wall polymers in situ, we also observed stronger fluorescent signals of antibodies against homogalacturonan in a co-overexpressed mutant [A(2 + 5)/cesa6] and similar patterns and intensities for antibodies against xylan in the WT and A(2 + 5)/cesa6 (Fig. 8G). Fig. 8 View largeDownload slide Determinations of cell wall compositions in mature plants. (A–F) Cell wall compositions of 7-week-old stems including cellulose (A, B), non-cellulosic polysaccharides (C, D) and lignin (E, F). The data are the mean ± SD of three independent biological replicates, and the asterisks denote statistical significance at P < 0.05 (*) and P < 0.01 (**) between the WT and others by Student’s t-tests. The increased/decreased wall polymers rates (%) are calculated by subtraction of values between the WT and others divided by the WT value. (G) Immunofluorescent labeling of stems in 7-week-old plants of the cesa6 mutant and the A(2 + 5)/cesa6 co-overexpressed mutant using plant cell wall glycan-directed monoclonal antibodies. JIM5 antibody labels homogalacturonan and CCRC-M149 labels xylan (green). Calcofluor (white) stains the cell wall (β-glucans). Scale bars = 50 μm. Fig. 8 View largeDownload slide Determinations of cell wall compositions in mature plants. (A–F) Cell wall compositions of 7-week-old stems including cellulose (A, B), non-cellulosic polysaccharides (C, D) and lignin (E, F). The data are the mean ± SD of three independent biological replicates, and the asterisks denote statistical significance at P < 0.05 (*) and P < 0.01 (**) between the WT and others by Student’s t-tests. The increased/decreased wall polymers rates (%) are calculated by subtraction of values between the WT and others divided by the WT value. (G) Immunofluorescent labeling of stems in 7-week-old plants of the cesa6 mutant and the A(2 + 5)/cesa6 co-overexpressed mutant using plant cell wall glycan-directed monoclonal antibodies. JIM5 antibody labels homogalacturonan and CCRC-M149 labels xylan (green). Calcofluor (white) stains the cell wall (β-glucans). Scale bars = 50 μm. In addition, we examined the transgenic mutants (A3/,A9/cesa6) regarding their SCW formation and biomass production in mature plants. As a member of the AtCesA6-like genes, the AtCesA9 gene showed a similarity to the AtCesA2 or AtCesA5 gene in terms of plant size and biomass yield (Supplementary Fig. S2D, E). Although the A3/cesa6 transgenic mutant showed large compensation of seedling growth (Supplementary Fig. S2A, B), the mature plants showed an even smaller plant size and lower biomass yield compared with EV/cesa6 plants (Supplementary Fig. S2D, E), indicating that some difference may exist between AtCesA3 and AtCesA6-like genes for their partial compensations in AtCesA6-null mutants. Discussion Several dozen CesA mutants have shown remarkable defects in cellulose synthesis, but the fundamental functions of CesA genes in cell growth remain largely unexplored (Li et al. 2014). AtCesA1 and AtCesA3 have been characterized as essential isoforms of the PCW CesA complex, as their null mutants are gamete lethal (Arioli et al. 1998, Fagard et al. 2000a, Persson et al. 2007). Nevertheless, mutations in the isoforms that contribute to the third position of the PCW CesA complex, the AtCesA6-like genes, only result in mild growth retardation and cell swelling (Fagard et al. 2000a, Scheible et al. 2001, Cano-Delgado et al. 2003, McFarlane et al. 2014). Despite a reduction in cell elongation in the PCW CesA mutants (Fagard et al. 2000a, Cano-Delgado et al. 2003, Bischoff et al. 2011, Fujita et al. 2013, Chen et al. 2010, Chen et al. 2016) and interference with cell division and cell expansion in embryogenesis of the rsw1 mutant examined (Beeckman et al. 2002), our study showed that rsw1 at the restrictive temperature had strongly negative impacts on cell division, with little impact in the AtCesA6-null mutants in vegetative tissues (Figs. 1, 2), which may provide a fundamental explanation for why the rsw1 seedlings could not grow further and die when incubated for a long time at the restrictive temperature. Moreover, the overexpressed AtCesA3 gene in the background of Arabidopsis WT could not promote seedling growth with unchanged cell division (Hu et al. 2018), and the overexpressed AtCesA3 gene in the background of the cesa6 mutant could not further promote plant growth (Supplementary Fig. S2) with decreased cell division (data not shown), which perhaps reflects the ‘housekeeping’ role of AtCesA1 and AtCesA3 with regards to cell division. Despite the fact that little is known about the impact of CesA on cell division, recent reports have indicated that the cellulose synthase-like D (CSLD) genes, with the highest sequence similarity to CesAs among cellulose synthase-like families, promote cell division in maize leaves, rice and Arabidopsis roots (Hunter et al. 2012, Yoshikawa et al. 2013, Gu et al. 2016). Also, the PCW CesAs are trafficked to and from the growing cell plate, corroborating a function for both the CSLDs and the PCW CesAs in this process (Miart et al. 2014). Furthermore, our recent article has shown that overexpressed single AtCesA2, AtCesA5 and AtCesA6 genes in the background of the Arabidopsis WT could significantly enhance cell division (Hu et al. 2018), while only co-overexpressed AtCesA2 and AtCesA5 genes in the background of AtCesA6-null mutants could dramatically enhance cell division (Fig. 6). It will thus be interesting to unravel the mechanisms of how AtCesA2 and AtCesA5 genes regulate cell division in the absence of the AtCesA6 gene in future studies. In addition, our results showed that the IRX3 mutant had no effect on seedling growth (including cell elongation and division; Fig. 1), which confirms that AtCesA7 is specific for SCW synthesis in Arabidopsis (Taylor et al. 1999). As the first defense barrier against infectious pathogens, maintenance of the cell wall integrity is a prerequisite for plant growth (Hamann 2015). Previous reports have indicated that PCW cellulose-deficient plants have incomplete cell walls, such as in the prc1-1 mutant (Fagard et al. 2000a). In this study, based on observations of the irregular PCWs (Fig. 7B; Supplementary Fig. S7B) and incomplete SCWs (Fig. 7C; Supplementary Fig. S7C) in two AtCesA6-null mutants, the results suggest that the irregular PCWs could not mechanically maintain SCW integrity in the mature plants. Because the PCW is tightly associated with cell elongation and division, and the SCW starts with cell differentiation (Schuetz et al. 2013), it remains to be explored in the future how the PCW impacts on SCW deposition and integrity. In addition, despite this study used a standard method for cell wall observation under TEM, it remains a technique where it is difficult to distinguish whether or not the disrupted cell walls are due to sample preparation. However, we observed small amounts of complete cell walls in the mutants, and found hardly any disrupted walls from observations of the WT, suggesting that most disrupted walls in mutants are not due to sample preparation. Although AtCesA6-like genes (AtCesA2, AtCesA5 and AtCesA9) have been characterized by a partial redundancy of the AtCesA6 gene (Desprez et al. 2007, Persson et al. 2007), it remains unexplored how these genes partially compensate for the AtcesA6 mutant in terms of cell growth and cell wall formation. Here, we first selected transgenic AtCesA6-null mutants that overexpressed three AtCesA6-like genes, and then examined their distinct functions associated with cell growth and cell wall formation. Even though the classic 35S promoter was used for overexpressing target genes in this study, the three transgenic mutants (A2/, A5/ and A9/cesa6) only showed partially restored cell elongation and seedling growth, which confirms previous findings of three AtCesA6-like genes (AtCesA2, AtCesA5 and AtCesA9) showing partial redundancy of the AtCesA6 gene (Desprez et al. 2007, Persson et al. 2007). Interestingly, our results also suggested that each AtCesA6-like gene should have its own independent biological function, which was supported by the following evidence. (i) Overexpression of single AtCesA2 and AtCesA5 genes in the background of the AtCesA6-null mutants exhibited a distinct seedling growth between dark and light incubation conditions (Fig. 3). (ii) A2/cesa6 and A5/cesa6 transgenic mutants showed largely different global gene expression patterns (Fig. 4; Supplementary Fig. S6). (iii) AtCesA9 is specifically expressed in flower tissues. More importantly, this study further confirmed the independent functions of AtCesA6-like genes, especially AtCesA2 and AtCesA5, as a fully restored phenotype of the WT was observed in the co-overexpressed transgenic mutant [A(2 + 5)/prc1-1 or A(2 + 5)/cesa6] that exhibits dramatically increased cell numbers (Fig. 6). Thus we speculated that precise particularities of the three AtCesA6-like genes (AtCesA2, AtCesA5 and AtCesA6) caused the unique effects on cellulose biosynthesis, cell wall deposition and plant cell growth for biomass production (Hu et al. 2018). Furthermore, this study examined whether the A3/cesa6 transgenic mutant exhibited more restored seedlings growth than the A2/cesa6 and A5/cesa6 transgenic mutants, and the findings indicated that the AtCesA3 or AtCesA1 gene should have distinct biological function from that of AtCesA2 and AtCesA5. Hence, the A3/cesa6 or A1/cesa6 transgenic mutants could be used as good samples to explore the distinct roles of AtCesA1, AtCesA3 and AtCesA6 in cellulose synthase complex formation and PCW biosynthesis in the future. In conclusion, this study proposes a model highlighting distinct fucntions of three major types of AtCesA genes in cell elongation, cell division, cell wall integrity and plant growth in mutants and transgenic mutants (Fig. 9): (i) the AtCesA1 mutant (rsw1) has remarkably decreased cell elongation and division for a strongly defective growth phenotype at the restrictive temperature; (ii) the AtCesA7 mutant (IRX3) has little impact on cell and seedling growth, but has a reduced SCW due to the collapsed xylems and forms a small mature plant (Taylor et al. 1999); (iii) two AtCesA6-null mutants (prc1-1 and cesa6) show little impact on cell division, but have reduced cell elongation for affected cell wall integrity and biomass yield of mature plants; (iv) transgenic mutants (e.g. A2/, A5/prc1-1) have a partiallly restored phenotype; and (v) co-overexpressed transgenic mutants [A(2 + 5)/prc1-1 or A(2 + 5)/cesa6] show greatly enhanced cell division and a complete cell wall with a strongly restored phenotype of the WT, leading to markedly increased SCW thickness and biomass production in mature plants. Fig. 9 View largeDownload slide Schematic model outlining distinct functions of AtCesAs in cell elongation, cell division, cell wall integrity and plant biomass production in three major types of AtCesA mutants (rsw1, cesa6/prc1-1 and IRX3) and the transgenic mutants that respectively overexpress AtCesAs or co-overexpress AtCesA2 and AtCesA5 in AtCesA6-null mutants. Fig. 9 View largeDownload slide Schematic model outlining distinct functions of AtCesAs in cell elongation, cell division, cell wall integrity and plant biomass production in three major types of AtCesA mutants (rsw1, cesa6/prc1-1 and IRX3) and the transgenic mutants that respectively overexpress AtCesAs or co-overexpress AtCesA2 and AtCesA5 in AtCesA6-null mutants. Materials and Methods Plant materials and growth conditions Arabidopsis homozygous prc1-1 mutants (AtCesA6; Fagard et al. 2000a), rsw1 (AtCesA1; Arioli et al. 1998) and IRX3 (AtCesA7; Taylor et al. 1999) were used in this study. Seeds of the Atcesa6 (SALK_004589) mutant (T-DNA insertion) were obtained from the Arabidopsis Biological Resource Center at Ohio State University. Identification of cesa6 homozygous lines was carried out using three primers (LBb1.3 + LP + RP) designed in T-DNA Primer Design (http://signal.salk.edu/tdnaprimers.2.html) with the following sequences LBb1.3, 5'-ATTTTGCCGATTTCGGAAC-3'; LP, 5'-ATCTATCCTCTGATTTATGGTCTCTG-3'; and RP, 5'-TACTAACAAATACATCCACAGGGG-3'. For generation of overexpression constructs, the coding region of the complete AtCesA genes (CesA6, CesA2, CesA5, CesA3 and CesA9) driven by the D35S promoter were cloned into the binary vector pD1301s to generate the binary plasmid (Supplementary Table S2). Transgenic plants were generated by introduction of the plant expression constructs into Agrobacterium tumefaciens strain GV3101, and transformation was done by floral dipping (Zhang et al. 2006). All the plant expression constructs were transformed into prc1-1 or cesa6 mutants. T1Arabidopsis transgenic seedlings were selected on 1/2 Murashige and Skoog (MS) medium containing 50 mg l–1 hygromycin and confirmed by RT–PCR or Q-PCR. More than three hygromycin-resistant lines (independent transformation events) for each construct were selected as homozygous. Phenotypic characterization was performed on T5 homozygous transgenic lines. Homozygous transgenic types were crossed with each other and selected as homozygous (F3–F5), and confirmed by Q-PCR. Arabidopsis seeds were surface sterilized using 75% ethanol for 4 min and 10% sodium hypochloride with 0.01% Triton X-100 for 3 min, washed in sterile water several times then imbibed at 4°C in the dark in sterile water containing 0.1% agar for 3 d and germinated on plates containing 1/2 MS media (1% sucrose; pH 5.8) in 1% agar. Plates were incubated in a near vertical position at 22°C under light growth conditions (16 h light/8 h dark) for photomorphogenesis or dark growth conditions (24 h dark) for skotomorphogenesis. The seedlings were transplanted to the soil after the second real leaf was clearly visible. Arabidopsis growth conditions were described previously (Hu et al. 2017, Hu et al. 2018). RNA extraction and Q-PCR measurement Seedlings were germinated and grown on 1/2 MS medium for the indicated number of days under light or dark growth conditions, and seedlings (hypocotyls and roots) were harvested in liquid nitrogen. Total RNA extraction and Q-PCR amplification were carried ou as described previously (Hu et al. 2018). The expression value of GAPDH (glyceraldehyde phosphate dehydrogenase) was defined as 100, and the expression level of CesA genes were thus normalized to the expression level of GAPDH. All of the primers used in these assays are listed in Supplementary Table S1.Three biological replications were performed. Total protein extraction and Western blot analyses Total protein extraction of D9 seedlings was performed as described previously (Hu et al. 2018). The AtCesA2, -5 and -6 protein levels were detected by Western blot analysis as described previously (Li et al. 2017). Purification of primary antibodies was performed using protein A–agarose. Dilutions were 1:250, 1:125 and 1:30 for AtCesA6, AtCesA2 and AtCesA5 antibodies, respectively. The relative protein levels were calculated using Quantity One software and the Rubisco large subunit protein (rbcL) as internal reference for SDS–PAGE. Observation of cellulose macrofibrils by AFM The purified cellulose samples of D9 hypocotyls were prepared as described previously (Hu et al. 2018, Li et al. 2017). The cellulose samples were suspended in ultrahigh purity water, and placed on mica using a pipette. The mica was glued onto a metal disc (15 mm diameter) after removal of extra water under nitrogen, and then placed on the piezo scanner of an atomic force microscope (MultiMode VIII; Bruker). AFM imaging was carried out in ScanAsystAtCesAir mode using BrukerScanAsystAtCesAir probes (tip radius, 2 nm and silicon nitride cantilever; spring constant, 0.4 N m–1) with a slow scan rate of 1 Hz. All AFM images were one-third flattened and analyzed quantitatively by using NanoScope Analysis software (Bruker). Three biological replications were performed for each experiment and 10 dots of each AFM image were randomly selected to measure the width (nm)×length (nm) by NanoScope Analysis software (Bruker). The average particle length/width of each image was calculated from the 10 selected particles. Hypocotyl, root and cell length measurements To observe hypocotyl and root growth, Arabidopsis seedlings were scanned using an HP Scanjet 8300 scanner at 600 d.p.i.; the hypocotyl lengths of vertically grown seedlings were measured from the hypocotyl base to the apical hook and the root length was measured (root tip to hypocotyl base) using the freely available ImageJ 1.32j software (https://imagej.nih.gov/ij/ or http://rsb.info.nih.gov/ij/). Two-tail t-tests were performed with Microsoft Excel software. For images of epidermal cell patterns, D9 hypocotyls were mounted and images of epidermal cells were viewed by using differential interference contrast (80i; Nikon). At least three biological replicates were performed for each experiment and >30 seedlings were measured for each genotype. Cell lengths in recorded images were quantified using Image J, and epidermal cells of the hypocotyl were visualized under confocal laser scanning microscopy (p58; Leica) using D4 hypocotyls incubated in the dark for 10 min in a fresh solution of 15 mM (10 mg ml–1) propidium iodide (PI; Naseer et al. 2012). PI was excited at 488 nm, and fluorescence was detected at 600–700 nm. Observation of cell division To count the number of cortical cells between the QC and the TZ (indicating the position of the first elongating cortical cell; Beemster and Baskin 1998), L9 root tips were mounted, and images were viewed by differential interference contrast (80i, Nikon). At least three biological replications were performed for each experiment and >30 seedlings were measured for each genotype. To visualize cell cycle progression in living cells, the G2/M-specific marker proAtCYCB1;1::AtCYCB1;1-GFP (Ubeda-Tomas et al. 2009) was crossed with different homozygous AtCesA6-like transgenic lines. Measurements of F1 hybrid seedlings were performed using confocal images of light-grown roots stained with PI. GFP was excited at 473 nm, and fluorescence was detected at 485–545 nm. Observation of cell wall structures by TEM TEM was used to observe cell wall structures in the xylary fiber cells of the first inflorescence stems of 7-week-old plants. The samples were post-fixed in 2% (w/v) osmium tetroxide (OsO4) for 1 h after extensively washing in phosphate-buffered saline (PBS) and were embedded with Super Kit (Sigma-Aldrich). Sample sections were cut with an Ultracut E ultramicrotome (Leica) and picked up on formvar-coated copper grids. After post-staining with uranyl acetate and lead citrate, the specimens were viewed under a Hitachi H7500 transmission electron microscope. The width of three relatively fixed points on each cell wall was measured using ImageJ. More than 60 cell walls for each genotype were measured. Significance differences were determined by Student’s t-test. Three biological replications were performed. Crude cell wall extraction and mechanical force measurement by AFM The crude cell wall material of the basal (1 cm) inflorescence stems from 7-week-old plants were prepared as described previously (Hu et al. 2018). The crude cell wall material was suspended in ultrahigh purity water, placed on new mica using a pipette and dried in air overnight. The mica was glued onto a metal disc (15 mm diameter) and placed on the piezo scanner of an atomic force microscope (MultiMode VIII; Bruker). A hard tip (RTESP; Bruker) with radius of 8 nm, and spring constant of 40 Nm–1 was used in measurement of the mechanical properties. The precise spring constant was corrected by the Sader method, and the average deflection sensitivity was determined by measuring a set of force–distance curves on the mica. The scan size was 10 μm×10 μm, 16×16 FD curves were collected for every measurement and 10 different cell segments were randomly selected for mechanical measurements for each sample. The Young’s modulus was calculated by using the Hertz model of the NanoScope analysis software, and the Wilcoxon test was used to test the significance of the average Young’s modulus (He et al. 2015). Two biological replications were performed each experiment. Plant dry weight measurement The homozygous lines were transplanted into soil as individual plants per pot and the plants were grown in a glasshouse at 22°C under light-grown condition for 7 weeks in a fully randomized experimental design. Seven-week-old inflorescence stems were harvested from each plant, dried under a suitable temperature (55°C) for 3–5 d and finally weighed using an analytical balance. Three biological replications were performed for each experiment, and >30 plants were measured for each genotype. Significance analysis was performed by Student’s t-test. Immunolocalization of glycan epitopes Thirty-day-old Arabidopsis petioles were embedded with 4% agar and then cut into 60 μm sections using a microtome (VT1000S, Leica). The first node of 7-week-old Arabidopsis inflorescence internodes were cut into 8 μm sections using a paraffin slicer (RM2265, Leica). For immunolabeling, transverse sections were incubated in 3% (w/v) milk protein in 1× PBS (MP/PBS) for 1 h to block non-specific binding. Sections were then incubated with monoclonal antibodies (JIM5, CCRC-M93 and CCRC-M149 antibodies, which bind to homogalacturonan, xyloglucan and xylan, respectively) diluted in MP-PBS (JIM5, diluted 1:10; CCRC-M93 and CCRC-M149, diluted 1:5; http://glycomics.ccrc.uga.edu/wall2/antibodies/antibodyHome.html) for 1 h. After washing with PBS, sections were incubated with anti-mouse-IgG and observed as described previously (Hu et al. 2017). Composition of plant cell walls Plant cell walls were fractionated and their composition determined as described previously (Jin et al. 2016), with some minor modifications for crystalline cellulose extraction of D9 or L9 seedlings or the dry biomass powder of 7-week-old inflorescence stem (40 mesh) samples, as described previously (Hu et al. 2018). For extraction of wall polysaccharides, dry biomass powder (40 mesh) samples (0.1–1.0 g) were washed twice with 5.0 ml of buffer and twice with 5.0 ml of distilled water. The remaining pellet was stirred with 5.0 ml of chloroform–methanol (1:1, v/v) for 1 h at 40°C and washed twice with 5.0 ml of methanol, followed by 5.0 ml of acetone. The pellet was washed once with 5.0 ml of distilled water. The remaining pellet was added to a 5.0 ml aliquot of dimethylsulfoxide (DMSO)–water (9:1, v/v), vortexed for 3 min and then rocked gently on a shaker overnight. After centrifugation, the pellet was washed twice with 5.0 ml of DMSO–water, and then with 5.0 ml of distilled water three times. The remaining pellet was defined as total wall polysaccharides. Total lignin was determined as described previously (Sun et al. 2017). At least three biological replications were performed. Microscopic observation The first inflorescence stems of 7-week-old Arabidopsis plants were embedded with 4% agar and then cut into sections of 100 μm thick by a microtome (VT1000S, Leica). Stem sections were stained in Calcofluor for 3 min and then rinsed, mounted in water, and observed and photographed under epifluorescence microscopy (Olympus BX-61, Retiga-4000DC digital camera). Living phenotypic observation of seedlings were made under light microscopy using a Leica stereomicroscope (Leica S6 D, Leica DFC295 digital camera). RNA sequencing and analysis Total RNA was isolated from the D6 seedlings (six samples, two biological replications) using Trizol reagent (Invitrogen). RNA sample preparation, cDNA library construction, raw reads, identification of differentially expressed genes (DEGs) and GO-BP terms of DEG analyses were performed as described previously (Hu et al. 2018). Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the National Natural Science Foundation of China [31670296]; the Fundamental Research Funds for the Central Universities of China [2662015PY018, 2013QC042]; the Technical Innovation Special Fund of Hubei Province [2017ACA171]; and the National 111 Project [B08032]. Acknowledgements We would like to thank Dr. Herman Höfte (National Institute for Agricultural Research, INRA, France) for kindly providing the prc1-1 mutant, Dr. Yonghong Zhang (Huazhong Agricultural University, China) for providing proCYCB1;1::CYCB1;1-GFP transgenic plants, and Dr. Staffan Persson for kindly discussing the experiments. We also thank Kexing Xin, Limin He and Qinghua Zhang (Huazhong Agricultural University, China) for technical assistance with the confocal laser scanning microscope, transmission electron microscopy and RNA sequencing, respectively. Disclosures The authors have no conflicts of interest to declare. References Appenzeller L. , Doblin M. , Barreiro R. , Wang H.Y. , Niu X.M. , Kollipara K. ( 2004 ) Cellulose synthesis in maize: isolation and expression analysis of the cellulose synthase (CesA) gene family . Cellulose 11 : 287 – 299 . Google Scholar CrossRef Search ADS Arioli T. , Peng L. , Betzner A.S. , Burn J. , Wittke W. , Herth W. , et al. . 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Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations AFM atomic force microscopy CesA cellulose synthase CSLD cellulose synthase like D D9 9-day-old dark-grown DEG differentially expressed gene GFP green fluorescent protein GO-BP Gene Ontology-Biological Process L9 9-day-old light-grown MS Murashige and Skoog PCW primary cell wall PI propidium iodide prc procuste QC quiescent center Q-PCR quantitative reverse transcription–PCR RAM root apical meristem RT–PCR reverse transcription–PCR rsw1 radially swollen1 SCW secondary cell wall TEM transmission electron microscopy TZ transition zone WT wild type © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. 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Plant and Cell PhysiologyOxford University Press

Published: Mar 5, 2018

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