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Regulation of secondary wall synthesis and cell death by NAC transcription factors in the monocot Brachypodium distachyon

Regulation of secondary wall synthesis and cell death by NAC transcription factors in the monocot... Abstract In several dicotyledonous species, NAC transcription factors act as master switches capable of turning on programmes of secondary cell-wall synthesis and cell death. This work used an oestradiol-inducible system to overexpress the NAC transcription factor BdSWN5 in the monocot model Brachypodium distachyon. This resulted in ectopic secondary cell-wall formation in both roots and shoots. Some of the genes upregulated in the process were a secondary cell-wall cellulose synthase (BdCESA4), a xylem-specific protease (BdXCP1) and an orthologue of AtMYB46 (BdMYB1). While activation of BdMYB1 may not be direct, this study showed that BdSWN5 is capable of transactivating the BdXCP1 promoter through two conserved binding sites. In the course of Brachypodium development, the BdXCP1 promoter was observed to be active in all types of differentiating tracheary elements. Together, these results suggest that Brachypodium SWNs can act as switches that turn on secondary cell-wall synthesis and programmed cell death. Brachypodium distachyon, cell death, monocot, NAC transcription factor, secondary wall, xylem Introduction The evolution of xylem was a key innovation that allowed land plants to colonize dry habitats. The tracheary elements have thick secondary cell walls that allow them to withstand the negative pressures necessary to elevate groundwater to aboveground organs (Sperry, 2003). The energetic cost of this transport system is minimal since mature tracheary elements undergo programmed cell death (Fukuda, 2000). In addition to tracheary elements with discontinuous secondary cell walls that can facilitate organ elongation and water movement, fibres with more uniform secondary walls are frequent in organs that require additional support (Zhong and Ye, 2009). The main components of the angiosperm secondary cell wall are cellulose, xylan, and lignin. Specific cellulose synthases, together with additional proteins that have yet to be identified, are responsible for secondary cell-wall cellulose biosynthesis (Endler and Persson, 2011). The differentiation of xylem vessels also involves a mechanism of programmed cell death (Bollhöner et al., 2012). Hydrolytic enzymes, which accumulate in the vacuole in parallel with cell-wall synthesis, are released when the membrane is ruptured, resulting in autolysis of the cell contents. Among the Arabidopsis enzymes involved in this process are XCP1 and XCP2, two closely related cysteine proteases specific to tracheary elements (Avci et al., 2008). The network of transcription factors that regulate the process of xylem differentiation has been extensively studied, particularly in Arabidopsis (Caño-Delgado et al., 2010). Seven VND genes from the NAC family of transcription factors are expressed during xylem vessel development (Kubo et al., 2005). Overexpression of either VND6 or VND7 results in ectopic transdifferentiation of many different cell types into vessel elements with their characteristic pattern of secondary cell-wall deposition (Kubo et al., 2005). A large number of putative downstream targets of VND6 and VND7 have been identified (Ohashi-Ito et al., 2010; Zhong et al., 2010b; Yamaguchi et al., 2011). In addition to transcription factors, some of the direct targets are genes involved in the synthesis of cellulose or in programmed cell death, such as XCP1. Both VND7 and VND6 can bind to secondary wall NAC-binding elements (SNBEs), which in some cases encompass the previously identified tracheary element-regulating cis-element (TERE) (Ohashi-Ito et al., 2010; Zhong et al., 2010b; Yamaguchi et al., 2011). The SNBE consensus sequence was identified as WNNYBTNNNNNNNAMGNHW using SND1, a NAC transcription factor expressed in fibres which is closely related to VNDs and shares many of their targets (Zhong et al., 2010b). It has been proposed that secondary wall NACs (SWNs) first appeared to regulate the differentiation of tracheids in early vascular plants and latter diversified to regulate other cell types with secondary walls, such as fibres (Zhong et al., 2010a). The regulation of secondary cell-wall synthesis has an obvious interest for the manipulation of biomass production in second-generation biofuel crops (Demura and Ye, 2010). Some of the more promising species for biomass production are members of the grass family, which includes the model Brachypodium distachyon (Mur et al., 2011). The programme of secondary cell-wall formation is expected to be somewhat similar in grasses and dicots, as the composition of their secondary walls is quite comparable, in contrast with the large divergence found in primary walls. However, some important differences have been found, such as a higher proportion of p-hydroxyphenyl (H) units in grass lignin (Vogel, 2008). Additionally xylan contains more arabinose side chains in grasses than in dicots and it is unclear whether both groups use the same synthesis mechanism (Faik, 2010; Scheller et al., 2010). Another open question is whether grasses use different sets of genes to build xylans in primary and secondary walls. The transcriptional regulation of secondary wall biosynthesis in grasses is very poorly understood (Handakumbura and Hazen, 2012). Coexpression analyses in rice and barley suggested the possible involvement of SWNs in this process (Christiansen et al., 2011; Ruprecht et al., 2011). In addition, it has been recently shown that rice and maize SWNs are expressed in xylem and fibres and that, when overexpressed in Arabidopsis, they can induce the formation of ectopic secondary walls (Zhong et al., 2011). This work reports that inducible overexpression in B. distachyon of the NAC transcription factor BdSWN5 was sufficient to activate secondary cell-wall formation. Among the genes upregulated in the process were a secondary cell-wall cellulose synthase and BdXCP1, an orthologue of AtXCP1. This study also found that the BdXCP1 promoter is normally active in differentiating tracheary elements and that BdSWN5 can directly regulate BdXCP1 transcription by binding two conserved SNBE sites. These findings establish that NAC transcription factors can work as secondary cell-wall master switches in monocots and can be used to study and manipulate this process. Materials and methods Phylogeny Sequences were aligned with clustalw and phylogenetic trees obtained with mega5 (Tamura et al., 2011). Trees were constructed by neighbour joining using Poisson distances and bootstrap values estimated from 500 replicates. Genetic constructions Brachypodium genomic DNA or cDNA was amplified with PrimeSTAR HS (Takara) using primers detailed in Supplementary Table S1 (available at JXB online). Coding sequences and promoter regions were cloned respectively in pENTR/D and pENTR5’ (Invitrogen). BdSWN sequences obtained from cDNA were submitted to Genbank (accession numbers JQ693422–JQ693429). Final constructs were assembled using LR Clonase II (Invitrogen). For overexpression in tobacco, the destination vector was pMDC32 (Curtis and Grossniklaus, 2003). For promoter–reporter constructs, the intron-containing glucuronidase entry clone pEN-L1-SI*-L2 was used (Karimi et al., 2007). This clone was combined with promoter entry clones into pERV2, a double recombination vector obtained by cloning a HindIII–SalI fragment from pH7m24GW,3 (Karimi et al., 2007) into pIPKb001 (Himmelbach et al., 2007). For oestradiol-inducible expression in Brachypodium, pERV1 plasmid was constructed using as a backbone pIJB2, a plasmid kindly provided by Oumaya Bouchabké-Coussa (Institut Jean-Pierre Bourgin, INRA) with the SfiI fragment from pIPKb002 (Himmelbach et al., 2007) inserted into p7U, conferring Basta resistance (DNA Cloning Service). A PCR fragment containing the LEXA promoter was amplified from pMDC7 (Curtis and Grossniklaus, 2003) and cloned between the SpeI and HindIII sites of pIJB2. The actin intron from pIPKb003 was then cloned between the HindIII and AvrII sites in the previous construct. Finally, the XVE coding sequence was amplified from pMDC7 and cloned between the PspXI and SpeI sites. An entry clone of BdSWN5 with its intron was recombined with this vector. The BdXCP1 297-bp promoter fragment in pENTR5’ was used as template to obtain mutated variants by megaprimer PCR (Sarkar and Sommer, 1990). To introduce a mutation at the SNBE1 site, a megaprimer was amplified with XSNBE1R1 and PENTR5L3. In the second amplification, PENTR5R3 was added to obtain a final product that included attL4 and attR1 sites. SNBE2 mutations were introduced by using XSNBE2L1 and PENTR5R3 to produce a megaprimer, adding PENTR5L3 in the second round. Double mutants were obtained by applying the two procedures sequentially. The PCR products were used directly in LR recombination reactions and the final constructs sequenced. Constructs were introduced into Agrobacterium tumefaciens EHA105 (pMDC32) or AGL1 (other plasmids). Tobacco transformation Nicotiana tabacum cv. Petit Havana was grown in soil for 4–6 weeks before infiltration with a suspension of transformed A. tumefaciens cells in infiltration media (10mM MgSO4, 10mM MES at pH 5.5, 0.5% glucose, 100 μM acetosyringone). The OD600 of the suspension was 0.1 for overexpression and 0.05 per construct for transactivation. For overexpression experiments, infiltrated sections were harvested after 5 days and clarified with 70% ethanol and lactic acid. For transactivation experiments, two discs (1cm diameter) were harvested after 2 days and GUS activity against 4-MUG was measured (Kim et al., 2006). All experiments were repeated at least twice with similar results. SYTOX staining Leaf discs (1cm) were incubated in 1 μM SYTOX Orange (Invitrogen) for 15 minutes under vacuum (80 kPa). After several washes, discs were observed on a Leica MZ16 fluorescence stereomicroscope equipped with a GFP Plus filter (460–500nm excitation and 510nm longpass barrier filter). Brachypodium growth and transformation B. distachyon plants (Bd21-3) were grown under a 16/8 light/dark cycle at 24/20 °C and 50–60% humidity. Agrobacterium-mediated transformation was carried out as previously described (Vogel and Hill, 2008) with minor modifications. Briefly, immature seeds were sterilized with 2% bleach and embryos were excised and placed on callus induction media (CIM). Calli were maintained at 26 °C in the dark and subcultured twice before transformation. Agrobacterium cultures scraped from 2-day-old YT plates were resuspended in liquid CIM with 200 μM acetosyringone and the OD600 adjusted to 0.6. About 90 calli per construct were incubated in Agrobacterium suspension for 5 minutes and placed for 5 days on empty plates with sterile filter paper and 750 μl of liquid CIM. Calli were transferred to CIM plates with 150mg l–1 Timentin for 1 week and then for 2 weeks at a time to CIM plates with 150mg l–1 Timentin and either 5mg l–1 ammonium glufosinate (oestradiol-inducible) or 40mg l–1 hygromycin (double recombination). After 6 weeks of selection, calli were moved to regeneration media and incubated at 24 °C in 16h days. Regenerating plantlets were transferred to 50ml glass culture tubes containing media with 0.5 × MS salts with vitamins, 3% sucrose, 0.7% activated charcoal, 0.2% phytagel, and 0.05% MES at pH 5.7. After transfer to soil, plants carrying T-DNA insertions were identified by PCR using Phire Plant Direct PCR Kit (Thermo Scientific) and the primers detailed in Supplementary Table S1. Induction assay Brachypodium seeds were sterilized with 2% bleach and placed in 250ml flasks containing 100ml of 0.25 × MS salts, 0.5g l–1 MES at pH 5.8 and 1% sucrose. Flasks were placed in a rotary shaker at 125rpm under a 16/8 light/dark cycle at 24/20 °C. After 4h of light, flasks were kept in the dark for 44h. Seedlings were induced by adding 100 μl of 10mM β-oestradiol in DMSO or mock induced with 100 μl of DMSO. After 16h in the same conditions, root tips (approx. 8 μm) and aerial parts were collected separately for RNA extraction. Gene expression RNA was extracted using a Plant Total RNA kit (Sigma). cDNA was synthesized using the Transcriptor First Strand cDNA synthesis Kit (Roche). End-point PCR was carried out for 40 cycles with MyTaq (Bioline). Real time PCR was performed for 40 cycles on the Mini Opticon Real-Time PCR system (Bio-rad) using SensiMix SYBR No-Rox Kit (Bioline). Amplification efficiency and Ct values were calculated with LinRegPCR (Ruijter et al., 2009). Statistical analysis was done with REST (Pfaffl et al., 2002) using UBC18 (Bradi4g00660) and SamDC (Bradi5g14640) as references. Primers and annealing temperatures are detailed in Supplementary Table S1. Electrophoretic mobility shift assay BdSWN coding regions were introduced into pET300/NT-DEST (Invitrogen) and used to transform Escherichia coli BL21 DE3 (Novagen). Cell cultures (200ml) were grown to an OD600 of 0.7–1.0 and induced with 1mM IPTG for 6h at 24 °C. Cells were harvested and frozen at –80 °C. Proteins were extracted with B-PER Reagent (Pierce) with 10% glycerol, 300mM NaCl, 5mM imidazole, and Halt Protease Inhibitor Cocktail EDTA-free (Pierce). Purification was carried out with Dynabeads His-Tag Isolation and Pulldown Kit (Novex). Probes and competitors were amplified by PCR or obtained by annealing complementary primers (Supplementary Table S1). Labelling was incorporated with 5’-end biotin-labelled primers. Electrophoretic mobility shift assays were performed using the LightShift Chemiluminescent EMSA Kit (Pierce). Labelled promoter fragments (10fmol) or labelled SNBE motifs (20fmol) were incubated with 150 or 300ng of protein, respectively, for 20min in 10 μl of binding buffer (10 mM Tris at pH 7.5, 100 mM KCl, 1mM DTT, 100ng l–1 poly dI-dC, 0.05% Nonidep P-40). DNA was separated by polyacrylamide gel electrophoresis on 0.5 × Tris-borate-EDTA gels and electroblotted onto Byodine B nylon membranes (Pierce). β-Glucuronidase histochemical staining Tissue samples were incubated in 90% acetone at 4 °C for 20 minutes and transferred to staining buffer (100mM sodium phosphate at pH 7, 10mM EDTA, 0.5mM K3[Fe(CN)6], 0.5mM K4[Fe(CN)6], 0.1% Triton-X, and 1mM X-gluc) and placed under vacuum for 10 minutes. After incubation at 37 °C for approx. 4 hours, samples were cleared in 70% ethanol and lactic acid. Results The Brachypodium genome includes eight SWN genes that are expressed in developing organs A phylogenetic analysis of NAC transcription factors in the Brachypodium genome (Brachypodium Genome Initiative, 2010) revealed the presence of eight SWN genes (Fig. 1A). Six of these genes (SWN1 to SWN6) are orthologues of ArabidopsisVND genes and two (SWN7 and SWN8) are orthologues of SND/NST genes. The phylogeny shows that at least four clades were already differentiated in the last common ancestor or monocots and dicots. The coding regions of all eight genes were amplified from cDNA and sequenced, allowing the correct annotations to be determined (Supplementary Table S2). Fig. 1. Open in new tabDownload slide (A) Phylogenetic tree of SWN proteins from Brachypodium, rice, and Arabidopsis. Annotations for Brachypodium and rice genes can be found in Supplementary Table S1. Bootstrap values are shown at branch nodes. Circles indicate clades differentiated in the last common ancestor of monocots and dicots. (B) Reverse transcription-PCR analysis of BdSWN expression in developing roots (R), leaves (L), and internodes (I). UBC18 was used as a control. Similar results were observed with a second biological replicate. Fig. 1. Open in new tabDownload slide (A) Phylogenetic tree of SWN proteins from Brachypodium, rice, and Arabidopsis. Annotations for Brachypodium and rice genes can be found in Supplementary Table S1. Bootstrap values are shown at branch nodes. Circles indicate clades differentiated in the last common ancestor of monocots and dicots. (B) Reverse transcription-PCR analysis of BdSWN expression in developing roots (R), leaves (L), and internodes (I). UBC18 was used as a control. Similar results were observed with a second biological replicate. The expression level of all eight genes was tested by reverse trancription-PCR in developing roots, leaves, and internodes (Fig. 1B). All the genes were detected in internodes, with SWN3, SWN4, SWN6, and SWN7 reaching their highest level of expression in this organ. On the other hand, expression levels in leaves were generally low or undetectable, with the exception of SWN5 and SWN8. Expression in roots was also generally lower than in other organs with the exceptions of SWN1 and SWN2. Brachypodium SWN genes can induce ectopic cell-wall formation in tobacco The eight Brachypodium SWN genes were transiently expressed in tobacco leaves via infiltration with Agrobacterium cultures carrying overexpression constructs. The SWN genes were under the control of two copies of the CaMV 35S promoter. After five days of co-cultivation, expression of all of the SWN genes had resulted in the ectopic formation of spiral or annular cell-wall thickenings in mesophyll and epidermal cells (Fig. 2). The percentage of cells with this type of response was particularly high in the cases of SWN1, SWN3, SWN4, and SWN7. Fig. 2. Open in new tabDownload slide Ectopic cell-wall formation in tobacco leafs induced by overexpression of different Bd-SWN genes. Bars = 100 μm (SWN7), 50 μm (SWN3, SWN4, and SWN5), and 25 μm (all others) (this figure is available in colour at JXB online). Fig. 2. Open in new tabDownload slide Ectopic cell-wall formation in tobacco leafs induced by overexpression of different Bd-SWN genes. Bars = 100 μm (SWN7), 50 μm (SWN3, SWN4, and SWN5), and 25 μm (all others) (this figure is available in colour at JXB online). Transient expression of some SWN genes also resulted in extensive cell death after 5 days of cocultivation, as evidenced by loss of chlorophyll (Fig. 3A) and membrane permeabilization revealed by SYTOX staining (Fig. 3B). This response was consistently stronger when expressing genes in clades I and II (SWN1, SWN2,SWN3, or SWN4). On the other hand, transient expression of genes in clade IV (SWN7 and SWN8) did not result in additional cell death beyond that caused by Agrobacterium infiltration. In some cases, cells died before any visible wall thickenings had been formed, resulting in the collapse of thin-walled mesophyll cells (Fig. 3C). This was particularly common when SWN2 was transiently expressed, with the number of collapsed cells an order of magnitude above the number of cells with ectopic wall thickenings. Tobacco infiltrations were also performed with overexpression constructs for SWN1–SWN6 that included the native introns within the coding region. Only in the case of SWN5 was the intensity of the response increased, both for cell-wall formation and cell death (data not shown). SWN5 is the only BdSWN gene with a single intron. Fig. 3. Open in new tabDownload slide Cell death in tobacco leafs induced by overexpression of different BdSWN genes. (A) Tobacco leaf infiltrated with Agrobacterium carrying an empty plasmid or different SWN overexpression constructs, as well as with infiltration medium. (B) SYTOX staining of non-viable cells (green) in the abaxial side of leafs transformed with different constructs. (C) Cell collapse caused by SWN2 overexpression in the palisade and spongy mesophylls. Bars = 300 μm (B) and 50 μm (C). Fig. 3. Open in new tabDownload slide Cell death in tobacco leafs induced by overexpression of different BdSWN genes. (A) Tobacco leaf infiltrated with Agrobacterium carrying an empty plasmid or different SWN overexpression constructs, as well as with infiltration medium. (B) SYTOX staining of non-viable cells (green) in the abaxial side of leafs transformed with different constructs. (C) Cell collapse caused by SWN2 overexpression in the palisade and spongy mesophylls. Bars = 300 μm (B) and 50 μm (C). Inducible overexpression of SWN5 is sufficient to cause ectopic cell-wall formation in Brachypodium The oestradiol-inducible transactivation system based on the XVE chimeric transcription factor was used to obtain Brachypodium lines with inducible overexpression of BdSWN5. The original vector is not suitable for grasses due to the G10-90 promoter resulting in low expression levels (Zuo et al., 2006). In order to use this system in Brachypodium, the XVE transcription factor was placed under the control of the maize ubiquitin promoter in pIPKB002 (Fig. 4A). Additionally the actin intron from pPIKB003 was introduced in the 5’-untranscribed region after the OLexA-46 promoter to increase the expression level. BdSWN5 was selected as a phylogenetic orthologue of AtVND7 that showed a strong effect in tobacco when the native intron was included. This intron was also included in the inducible overexpression construct. Fig. 4. Open in new tabDownload slide Inducible overexpression of BdSWN5 in Brachypodium. (A) Plasmid used for oestradiol-inducible overexpression. (B) Arrested development of a 5-day-old transformed seedling from line 6 germinated in media with 10 μM oestradiol (Cp, coleoptile; Cr, coleorhiza; L, leaf; R, root). (C) Root of a transformed seedling from line 9 grown in the same media. (D) Leaf epidermis of a transformed seedling from line 8 grown in the same media. (E) Coleoptile parenchyma of a transformed seedling from line 3 grown in liquid 24h after addition of oestradiol. (F) Coleoptile parenchyma of a similar seedling 24h after mock induction. (G) Root central metaxylem (arrowhead) differentiating less than 2mm from the tip in an induced seedling. (H) Root central metaxylem differentiating approx. 12mm from the tip in a mock-induced seedling. (I) Root cortex in the transition zone of an induced seedling. (J) Mature root cortex in an induced seedling. Bars = 400 μm (B) and 25 μm (all others). (K) Relative expression level of selected genes in induced and mock-induced seedlings grown in liquid. Root tips (approx. 8mm) and aerial parts were collected separately 16h after induction. Error bars are standard errors as calculated by REST software from three biological and two technical replicates. UBC18 and SamDC were used as references. Ratios that are significantly different from 1 are indicated in black (P < 0.005) or grey (P < 0.02) (this figure is available in colour at JXB online). Fig. 4. Open in new tabDownload slide Inducible overexpression of BdSWN5 in Brachypodium. (A) Plasmid used for oestradiol-inducible overexpression. (B) Arrested development of a 5-day-old transformed seedling from line 6 germinated in media with 10 μM oestradiol (Cp, coleoptile; Cr, coleorhiza; L, leaf; R, root). (C) Root of a transformed seedling from line 9 grown in the same media. (D) Leaf epidermis of a transformed seedling from line 8 grown in the same media. (E) Coleoptile parenchyma of a transformed seedling from line 3 grown in liquid 24h after addition of oestradiol. (F) Coleoptile parenchyma of a similar seedling 24h after mock induction. (G) Root central metaxylem (arrowhead) differentiating less than 2mm from the tip in an induced seedling. (H) Root central metaxylem differentiating approx. 12mm from the tip in a mock-induced seedling. (I) Root cortex in the transition zone of an induced seedling. (J) Mature root cortex in an induced seedling. Bars = 400 μm (B) and 25 μm (all others). (K) Relative expression level of selected genes in induced and mock-induced seedlings grown in liquid. Root tips (approx. 8mm) and aerial parts were collected separately 16h after induction. Error bars are standard errors as calculated by REST software from three biological and two technical replicates. UBC18 and SamDC were used as references. Ratios that are significantly different from 1 are indicated in black (P < 0.005) or grey (P < 0.02) (this figure is available in colour at JXB online). Approximately 90 individual calli were transformed and 13 T0 plants were regenerated from 10 independent calli. Plant growth was normal and most plants were fertile. To identify the lines with the strongest response seeds from nine independent lines (T1 generation) were germinated on plates with 10 μM β-oestradiol and grown for 5 days. Five lines showed a very strong response with the development of the majority of the seedlings arrested shortly after germination, before the root had reached 5mm (Fig. 4B). These seedlings showed accelerated vascular development as well as extensive ectopic cell-wall formation in roots, coleoptiles, and leaves, but not the coleorhiza (Fig. 4C and D). In addition, roots tended to curl and the root epidermal cells, which did not show ectopic wall formation, became partially detached from the cortex (Fig. 4C). Three other lines showed a milder response with most seedlings showing normal or partially reduced elongation, but with ectopic wall deposition in many cells, particularly in the root cortex and coleoptile. No phenotype was observed when wild-type seeds were germinated in the same plates. Screening of T1 plants from line 3, one of the lines with a strong response, indicated the presence of a single T-DNA locus, with an insertion in six out of 10 plants. Plant 3–4 was then identified as homozygous when all 26 T2 descendants showed presence of the insertion. Pooled seeds from these T2 plants were grown for 2 days in liquid culture in the dark before addition of β-oestradiol. In comparison with mock-induced seedlings, SWN5 expression severely slowed down seedling development. After 24 hours of induction, ectopic cell-wall depositions in annular or scalariform patterns were visible in the coleoptile parenchyma of induced seedlings (Fig. 4E), but not in mock-induced seedlings (Fig. 4F). No changes were seen in the coleoptile epidermis or in developing leaves, in contrast to what had been observed in seedling germinated on oestradiol plates. In the roots, addition of β-oestradiol resulted in an acceleration of vascular development with all types of xylem vessels differentiating simultaneously very close to the root tip, including the central metaxylem (Fig. 4G). In mock-induced seedlings, cell-wall thickenings in the central metaxylem started to appear more than 1cm away from the tip after the cells had elongated to a length of approx. 350 μm (Fig. 4H). In addition, ectopic cell-wall formation was observed in the root cortex of induced seedlings starting before elongation (Fig. 4I). In mature cortex cells, the pattern of wall deposition was similar to that of pitted xylem vessels (Fig. 4J). Older parts of the roots, more than 1cm away from the tip, did not show any changes, suggesting that only differentiating cells are capable of responding to SWN5 expression. SWN5 overexpression can activate genes involved in secondary cell-wall synthesis and cell death To identify genes activated by SWN5, real-time PCR was used to compare expression levels in induced and mock-induced seedlings 16h after induction (Fig. 4K). Seedlings were grown in liquid as described in the previous section. UBC18 and SamDC were chosen as reference genes as their expression is stable in different organs and growth conditions (Hong et al., 2008). As expected, SWN5 expression was upregulated in response to β-oestradiol in both roots and aerial parts. BdCESA4 (Bradi2g49912) was selected as a putative secondary wall cellulose synthase since it is the only Brachypodium orthologue of OsCESA4 (Supplementary Fig. S1A), a rice gene required for the synthesis of secondary cell walls (Tanaka et al., 2003). BdCESA1 (Bradi2g34240) was selected as a putative primary wall cellulose synthase as an orthologue of both OsCESA1 and AtCESA1 (Supplementary Fig. S1A). The expression pattern of OsCESA1 strongly suggests its involvement in rice primary wall cellulose synthesis (Ruprecht et al., 2011). BdSWN5 strongly upregulated BdCESA4 expression in both roots and aerial parts, 15-fold and 4-fold respectively. In contrast, expression of BdCESA1 was reduced or unchanged. BdXCP1 (Bradi2g39320) was identified as the single orthologue of the xylem-specific cysteine proteases AtXCP1 and AtXCP2 (Supplementary Fig. S1B). Finally BdMYB1 (Bradi4g06317) was chosen as the orthologue of AtMYB46 and AtMYB83 (Supplementary Fig. S1C), both of them direct targets of AtSWNs (McCarthy et al., 2009; Zhong et al., 2010b; Yamaguchi et al., 2011). The expression of both genes was found to be increased in seedlings treated with β-oestradiol, although BdXCP1 upregulation was much higher, reaching 19-fold in roots and 17-fold in aerial parts. Transactivation of BdXCP1 promoter by BdSWN5 is mediated by SNBE sites To identify the binding sites of BdSWN5, this study first tested whether this transcription factor was able to transactivate BdMYB1 and BdXCP1 promoter fragments (2.3kb and 1.5kb respectively). These fragments were placed in front of an intron-containing glucuronidase gene to generate reporter constructs. The effector construct for SWN5 included the native intron (SWN5i), since this intron seemed to enhance expression in tobacco. Tobacco leaves were coinfiltrated with Agrobacterium cultures transformed with effector and reporter plasmids and glucuronidase activity was measured after 2 days. There was no increase in reporter activity from the BdMYB1 promoter–reporter construct when coinfiltrated with SWN5 effector (Fig. 5B). However, SWN5 was able to transactivate a reporter construct that included an AtMYB46 promoter fragment, orthologous to BdMYB1. On the other hand, GUS activity increased to 377 pkat mg–1 when the BdXCP1 promoter–reporter construct was coinfiltrated with the SWN5 effector. A similar result was observed with a truncated reporter construct that contained only the 297bp upstream of the BdXCP1 start codon. This promoter fragment includes two SNBE sites which are highly conserved in sequence and position when compared to the AtXCP1 promoter (Fig. 5A). Fig. 5. Open in new tabDownload slide Transactivation of Brachypodium promoters by BdSWN transcription factors. (A) Alignment of SNBE motifs in the AtXCP1 and BdXCP1 promoters. Conserved nucleotides are boxed in grey. Nucleotide changes introduced in mutated sites are shown below the alignment. TSS = transcription start site. (B–D) Transactivation assays of promoter fragments: (B) reporter constructs were infiltrated on their own (white) or together with an effector construct with a 2×35S promoter driving the expression of BdSWN5 (grey); (C) wild-type and mutated fragments of BdXCP1 promoter; M1 and M2 mutations are indicated in part A; (D) BdMYB1 (white) and BdXCP1 (grey) promoters with eight BdSWN effectors. Error bars indicate SD. Fig. 5. Open in new tabDownload slide Transactivation of Brachypodium promoters by BdSWN transcription factors. (A) Alignment of SNBE motifs in the AtXCP1 and BdXCP1 promoters. Conserved nucleotides are boxed in grey. Nucleotide changes introduced in mutated sites are shown below the alignment. TSS = transcription start site. (B–D) Transactivation assays of promoter fragments: (B) reporter constructs were infiltrated on their own (white) or together with an effector construct with a 2×35S promoter driving the expression of BdSWN5 (grey); (C) wild-type and mutated fragments of BdXCP1 promoter; M1 and M2 mutations are indicated in part A; (D) BdMYB1 (white) and BdXCP1 (grey) promoters with eight BdSWN effectors. Error bars indicate SD. To determine if the SNBE sites were required for the activation of the 297-bp BdXCP1 promoter fragment, the two sites were mutated independently and simultaneously. A total of four point mutations were introduced in each site at highly conserved positions in the SNBE motif (Fig. 5A). Mutation of SNBE1 and SNBE2 resulted in 93% and 98% reductions in activation respectively, suggesting that both sites are required for full activation (Fig. 5C). Mutation of both sites simultaneously abolished transactivation by SWN5. The reporter construct with the 297-bp BdXCP1 promoter fragment was coinfiltrated with effector constructs for the eight BdSWN genes without introns (Fig. 5D). Expression of all the genes resulted in activation of the BdXCP1 promoter, but at very low levels in the case of SWN7 and SWN8. Expression of the remaining genes resulted in much larger increases in reporter activity, with SWN1 consistently giving the highest level. When the same experiment was repeated with the BdMYB1 promoter, the activation level was undetectable for all of the BdSWN genes (Fig. 5D), with the possible exception of SWN1 (P = 0.073). BdSWN5 can bind to SNBE sites in the BdXCP1 promoter To test if SWN5 activates the XCP1 promoter though direct binding to SNBE sites, electrophoretic mobility shift assays were performed. Purified SWN5 protein slowed down the migration of a 142-bp fragment of the XCP1 promoter that included both SNBE sites (Fig. 6A). Two shifted bands were clearly visible, possibly corresponding to the occupation of one or both SNBE sites. An unlabelled wild-type fragment or a fragment with a mutated SNBE1 site (M1) were able to compete with the probe for binding to SWN5. A fragment where SNBE2 was mutated (M2) was a much less effective competitor even at twice the ratio of the wild-type competitor, suggesting a higher binding affinity toward the SNBE2 site than toward the SNBE1 site. The ability to compete was almost abolished when both SNBE sites were mutated (M1M2). Finally, isolated SNBE motifs in 35-bp promoter fragments (S1 and S2) were able to compete for binding to SWN5, with SNBE2 a more effective competitor than SNBE1. Fig. 6. Open in new tabDownload slide Electrophoretic mobility shift assays showing binding of BdSWN proteins to BdXCP1 promoter fragments. (A) A 142-bp XCP1 promoter fragment in the presence of SWN5 protein and a number of different competitors: unlabelled wild-type fragment (WT), mutated fragments (M1, M2, M1M2, see Fig. 4), and isolated SNBE1 and SNBE2 motifs (S1, S2). (B) XCP1 SNBE motifs in the presence of SWN5 protein and a number of different competitors: wild-type and mutated SNBE1 (W1 and M1), and wild-type and mutated SNBE2 (W2 and M2). (C) XCP1 promoter fragment in the presence of SWN7 protein; legend as in A. (D) XCP1 SNBE motifs in the presence of SWN7 protein; legend as in B. Fig. 6. Open in new tabDownload slide Electrophoretic mobility shift assays showing binding of BdSWN proteins to BdXCP1 promoter fragments. (A) A 142-bp XCP1 promoter fragment in the presence of SWN5 protein and a number of different competitors: unlabelled wild-type fragment (WT), mutated fragments (M1, M2, M1M2, see Fig. 4), and isolated SNBE1 and SNBE2 motifs (S1, S2). (B) XCP1 SNBE motifs in the presence of SWN5 protein and a number of different competitors: wild-type and mutated SNBE1 (W1 and M1), and wild-type and mutated SNBE2 (W2 and M2). (C) XCP1 promoter fragment in the presence of SWN7 protein; legend as in A. (D) XCP1 SNBE motifs in the presence of SWN7 protein; legend as in B. When isolated motifs were used as probes, binding of SWN5 protein was also detected (Fig. 6B). Unlabelled wild-type motifs could compete effectively, but not those with four point mutations. Consistent with the results observed with the larger fragment, unlabelled SNBE2 could outcompete labelled SNBE1, while unlabelled SNBE1 was less effective against labelled SNBE2. The higher affinity of SWN5 for SNBE2 fits with the transactivation results obtained with mutated motifs (Fig. 5C), strengthening the case for direct activation. The same experiments were repeated with SWN7 protein since it had shown a reduced capacity to activate the XCP1 promoter in transactivation assays (Fig. 5D). The results obtained with SWN7 were quite similar to those observed for SWN5 both when using as probe the 142bp XCP1 promoter fragment (Fig. 6C) and when using isolated motifs (Fig. 6D). However, the binding affinity of SNW7 toward SNBE1 appeared to be lower than in the case of SWN5, as shown by the poor competition of the M2 fragment or the SNBE1 motif against labelled wild-type fragment (Fig. 6C). This lower affinity was confirmed in the competition of the SNBE1 motif against labelled SNBE2 motif, as well the lower degree of shift observed with labelled SNBE1 (Fig. 6D). BdXCP1 promoter directs expression to developing xylem tracheary elements The same reporter construct containing a 1.5-kb BdXCP1 promoter fragment used in transactivation assays was employed for stable transformation of Brachypodium plants with a promoter–reporter construct. Of a total of 16 independent lines examined, 15 showed a common expression pattern (Fig. 7). In roots, expression was detected in all types of xylem tracheary elements, as they progressively differentiated, starting with protoxylem (Fig. 7A), followed by peripheral metaxylem (Fig. 7B) and later by central metaxylem (Fig. 7C). Staining was also evident in developing xylem of internodes, but not in developing fibres (Fig. 7D and E). In leaves, expression was observed in both longitudinal and commissural vascular bundles (Fig. 7F). Additionally, staining was observed in short leaf epidermal cells with the appearance of cork-silica cell pairs (Fig. 7G). Fig. 7. Open in new tabDownload slide Expression of β-glucuronidase driven by BdXCP1 promoter in transformed Brachypodium plants. Samples were taken from 6-day-old roots (A–C), 15-day-old internodes (D and E), 6-day-old leaves (F), and 15-day-old leaves (G). Bars = 200 μm (A), 100 μm (E and F), 50 μm (C), and 25 μm (all others). Fig. 7. Open in new tabDownload slide Expression of β-glucuronidase driven by BdXCP1 promoter in transformed Brachypodium plants. Samples were taken from 6-day-old roots (A–C), 15-day-old internodes (D and E), 6-day-old leaves (F), and 15-day-old leaves (G). Bars = 200 μm (A), 100 μm (E and F), 50 μm (C), and 25 μm (all others). Discussion The role of NAC transcription factors as key regulators of secondary cell-wall synthesis and programmed cell death has been established in several dicotyledonous species (Zhong et al., 2010a; Wang and Dixon, 2012). The present results now demonstrate that inducible overexpression of a SWN transcription factor in Brachypodium is sufficient to activate the programme of secondary cell-wall synthesis, establishing that SWNs can act as master switches in monocots. This study has shown that overexpression of BdSWN5 leads to accelerated xylem development and deposition of ectopic cell walls in different Brachypodium organs (Fig. 4). The patterns of ectopic cell-wall depositions are reminiscent of tracheary elements. Among the genes that are upregulated by SWN5 are BdCESA4, a secondary cell-wall cellulose synthase, and BdXCP1, a cysteine protease possibly involved in cell death (Fig. 4). Transactivation of the XCP1 promoter by SWN5 depends on the presence of two conserved SNBE motifs, as proven by the effect of point mutations in conserved positions (Fig. 5). Electrophoretic mobility shift assays further show that SWN5 can bind to both motifs whether isolated or paired as in the genome (Fig. 6). Finally, a XCP1 promoter fragment that includes these motifs directs expression of a reporter gene to developing xylem tracheary elements (Fig. 7). It thus appears that a direct target of BdSWNs is activated just as cells undergo the processes of secondary wall deposition and cell death. It is also possible that some epidermal cells share parts of this programme (Fig. 7G). It thus seems likely that SWN genes already functioned as master switches of secondary cell-wall synthesis in the last common ancestor of monocots and dicots and that this role has been preserved in both lineages. Furthermore, the binding specificity of SWN proteins and therefore the motifs present in their target promoters appear to have been conserved. This can explain the activation of both cell-wall deposition and cell death programmes in tobacco leaves through transient expression of BdSWN genes (Figs. 2 and 3). Ectopic cell-wall deposition has also been observed in Arabidopsis when overexpressing rice and maize SWNs (Zhong et al., 2011). Despite the conserved function of SWNs in monocots and dicots, the regulatory network of secondary walls has probably diverged after 140 million years of separate evolution (Chaw et al., 2004). In Arabidopsis, the direct activation of AtMYB46 and AtMYB83 by several AtSWN transcription factors has been demonstrated both in vitro and in vivo (McCarthy et al., 2009; Zhong et al., 2010b; Yamaguchi et al., 2011). In Brachypodium, overexpression of BdSWN5 also upregulates RNA levels of BdMYB1, an orthologue of AtMYB46 and AtMYB83 (Fig. 4). However, this study was unable to detect transactivation of the BdMYB1 promoter by SWN5, even though this Brachypodium transcription factor is capable of transactivating the AtMYB46 promoter (Fig. 5B). Transactivation of the BdMYB1 promoter was also undetectable with the remaining BdSWNs, suggesting the lack of direct activation (Fig. 5D). A recent study showed that SWN proteins from rice and maize could transactivate a promoter with a tandem arrangement of SNBE motifs taken from rice and maize orthologues of BdMYB1 (Zhong et al., 2011). It is unclear, however, if this artificial arrangement reflects the activity of the native promoter. The results presented here can also shed some light on the functional specialization of different SWN genes. Phylogenetic analysis suggests the existence of four clearly differentiated clades in the last common ancestor of monocots and dicots (Fig. 1A). Genes from all four of these clades were capable of inducing ectopic cell-wall thickenings in transiently transformed tobacco leaves (Fig. 2). In Arabidopsis, overexpression of the three genes from clade I does not result in any phenotype (Kubo et al., 2005), while genes from the other three clades are capable of activating the deposition of secondary cell walls (Kubo et al., 2005; Mitsuda et al., 2005; Zhong et al., 2006). In this aspect, Brachypodium seems more similar to poplar, where genes from all four clades induce ectopic wall thickenings in both poplar and Arabidopsis (Ohtani et al., 2011). The pattern of cell-wall deposition caused by transient expression of BdSWNs is similar for all of the genes. This is also the same result obtained with poplar SWNs and again different from Arabidopsis, where overexpression of different SWNs can result in different patterns (Kubo et al., 2005; Ohashi-Ito et al., 2010). In any case, the pattern of ectopic wall deposition is clearly affected by the previous identity of the cell, as shown by the different effects of BdSWN5 expression in coleoptiles and roots (Fig. 4E and J). This had previously been observed for AtSND1 (Zhong et al., 2006; Ohashi-Ito et al., 2010) and poplar SWNs (Ohtani et al., 2011). Overexpression of BdSWN genes in tobacco can also activate programmed cell death (Fig. 3). Interestingly, the cell death and the cell-wall synthesis programmes appear to be partially independent since they are activated with different intensities by different transcription factors. While SWN4 expression results in both extensive cell-wall deposition and cell death, SWN7 expression appears to activate primarily cell-wall synthesis and SWN2 expression mainly results in cell death with no previous cell-wall deposition. It would be reasonable to conclude that SWN7 and SWN2 are activating different sets of genes, possibly due to differences in binding sequences or preferred motif arrangements. The two Brachypodium proteins in clade IV, SWN7 and SWN8, are poor inducers of cell death in tobacco and they also show a very weak capacity to transactivate the BdXCP1 promoter (Fig. 5D). This is unlikely to be caused by a weak activation domain, since SWN7 is very effective inducers of cell-wall synthesis in tobacco (Fig. 2). Alternatively, it could indicate the conservation of a promoter motif or motif arrangement specific to programmed cell death between the two species. Although in electrophoretic mobility shift assays SWN7 can bind the two SNBE motifs in the BdXCP1 promoter, it appears to bind SNBE1 with lower affinity than SWN5 (Fig. 6). Simultaneous binding to both SNBE sites seems to be required for strong activation of the XCP1 promoter, as shown by the effect of point mutations in either of the sites (Fig. 5C). Therefore a low binding affinity for SNBE1 could partly explain the low transactivation observed with SWN7. In poplar, SWN proteins from clade IV are not very efficient at transactivating the PtXCP1 promoter, especially when compared to proteins in clades II and III (Ohtani et al., 2011). Similarly the AtXCP2 promoter can be directly activated by clade II and clade III proteins, but not by the three Arabidopsis clade IV proteins (Zhong et al., 2010b). The situation for AtXCP1 appears to be more complex, with results that depend on the system used (Ohashi-Ito et al., 2010; Zhong et al., 2010b). To explain the lack of AtXCP1 expression in fibres, repression by an additional regulatory mechanism has been suggested (Bollhöner et al., 2012). It is possible that this mechanism acts by blocking SNBE motifs since an artificial promoter with SNBEs from AtXCP1 can be efficiently activated by AtSND1 in transfected protoplasts (Zhong et al., 2010b), but is not active in Arabidopsis fibres (McCarthy et al., 2011). The results presented here suggest that the lower efficiency of clade IV proteins at activating genes involved in cell death could be conserved in monocots. Unlike in Arabidopsis, this apparent functional specialization may not be linked to expression in different cell types as maize and rice genes from clades II, III and IV are expressed indistinctly in fibres and tracheary elements (Zhong et al., 2011). Since the reporter construct suggests that BdXCP1 is not expressed in fibres (Fig. 7E), it is possible that a repression mechanism is also active in monocots. The use of inducible systems capable of activating secondary cell-wall synthesis has generated a wealth of information regarding the transcriptional regulation of this complex developmental process in Arabidopsis (Ohashi-Ito et al., 2010; Zhong et al., 2010b; Yamaguchi et al., 2011). The present study has now extended this approach to the monocots, using the model species Brachypodium dystachion, an important step to understand the regulation of secondary cell-wall synthesis in grasses and to acquire the tools that will be necessary to improve biomass crops. The results indicate that much of what has been learned in Arabidopsis will still be relevant, but point also to some possible differences. Supplementary material Supplementary data are available at JXB online. Supplementary Table S1. Primers used in this work. Supplementary Table S2. Annotation of Brachypodium and rice genes. Supplementary Fig. S1. Phylogenetic trees of genes selected for real-time PCR analysis. Acknowledgements This work was supported by the Ministerio de Ciencia e Innovación (grant no. EUI2008-03693). Results have been achieved within the framework of the Transnational (Germany, France, and Spain) Cooperation within the PLANT-KBBE Initiative, with funding from Agence Nationale pour la Recherche, Bundesministerium für Bildung und Forschung, and Ministerio de Ciencia e Innovación. The authors are grateful to Oumaya Bouchabké-Coussa at the Institut Jean-Pierre Bourgin, INRA for helpful advice in transforming Brachypodium and for plasmid pIJB2. References Avci U Earl Petzold H Ismail IO Beers EP Haigler CH . 2008 . Cysteine proteases XCP1 and XCP2 aid micro-autolysis within the intact central vacuole during xylogenesis in Arabidopsis roots . The Plant Journal 56 , 303 – 315 . Google Scholar Crossref Search ADS PubMed WorldCat Bollhöner B Prestele J Tuominen H . 2012 . Xylem cell death: emerging understanding of regulation and function . Journal of Experimental Botany 63 , 1081 – 1094 . Google Scholar Crossref Search ADS PubMed WorldCat BrachypodiumGenome Initiative 2010 . 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Applications of chemical-inducible expression systems in functional genomics and biotechnology . In: Salinas J Sanchez-Serrano JJ, eds, Arabidopsis protocols , Totowa, NJ : Humana Press pp 329 – 342 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC © The Author(2) [2013]. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] © The Author(2) [2013]. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Experimental Botany Oxford University Press

Regulation of secondary wall synthesis and cell death by NAC transcription factors in the monocot Brachypodium distachyon

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Oxford University Press
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Copyright © 2022 Society for Experimental Biology
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Research Paper
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0022-0957
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10.1093/jxb/ers394
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Abstract

Abstract In several dicotyledonous species, NAC transcription factors act as master switches capable of turning on programmes of secondary cell-wall synthesis and cell death. This work used an oestradiol-inducible system to overexpress the NAC transcription factor BdSWN5 in the monocot model Brachypodium distachyon. This resulted in ectopic secondary cell-wall formation in both roots and shoots. Some of the genes upregulated in the process were a secondary cell-wall cellulose synthase (BdCESA4), a xylem-specific protease (BdXCP1) and an orthologue of AtMYB46 (BdMYB1). While activation of BdMYB1 may not be direct, this study showed that BdSWN5 is capable of transactivating the BdXCP1 promoter through two conserved binding sites. In the course of Brachypodium development, the BdXCP1 promoter was observed to be active in all types of differentiating tracheary elements. Together, these results suggest that Brachypodium SWNs can act as switches that turn on secondary cell-wall synthesis and programmed cell death. Brachypodium distachyon, cell death, monocot, NAC transcription factor, secondary wall, xylem Introduction The evolution of xylem was a key innovation that allowed land plants to colonize dry habitats. The tracheary elements have thick secondary cell walls that allow them to withstand the negative pressures necessary to elevate groundwater to aboveground organs (Sperry, 2003). The energetic cost of this transport system is minimal since mature tracheary elements undergo programmed cell death (Fukuda, 2000). In addition to tracheary elements with discontinuous secondary cell walls that can facilitate organ elongation and water movement, fibres with more uniform secondary walls are frequent in organs that require additional support (Zhong and Ye, 2009). The main components of the angiosperm secondary cell wall are cellulose, xylan, and lignin. Specific cellulose synthases, together with additional proteins that have yet to be identified, are responsible for secondary cell-wall cellulose biosynthesis (Endler and Persson, 2011). The differentiation of xylem vessels also involves a mechanism of programmed cell death (Bollhöner et al., 2012). Hydrolytic enzymes, which accumulate in the vacuole in parallel with cell-wall synthesis, are released when the membrane is ruptured, resulting in autolysis of the cell contents. Among the Arabidopsis enzymes involved in this process are XCP1 and XCP2, two closely related cysteine proteases specific to tracheary elements (Avci et al., 2008). The network of transcription factors that regulate the process of xylem differentiation has been extensively studied, particularly in Arabidopsis (Caño-Delgado et al., 2010). Seven VND genes from the NAC family of transcription factors are expressed during xylem vessel development (Kubo et al., 2005). Overexpression of either VND6 or VND7 results in ectopic transdifferentiation of many different cell types into vessel elements with their characteristic pattern of secondary cell-wall deposition (Kubo et al., 2005). A large number of putative downstream targets of VND6 and VND7 have been identified (Ohashi-Ito et al., 2010; Zhong et al., 2010b; Yamaguchi et al., 2011). In addition to transcription factors, some of the direct targets are genes involved in the synthesis of cellulose or in programmed cell death, such as XCP1. Both VND7 and VND6 can bind to secondary wall NAC-binding elements (SNBEs), which in some cases encompass the previously identified tracheary element-regulating cis-element (TERE) (Ohashi-Ito et al., 2010; Zhong et al., 2010b; Yamaguchi et al., 2011). The SNBE consensus sequence was identified as WNNYBTNNNNNNNAMGNHW using SND1, a NAC transcription factor expressed in fibres which is closely related to VNDs and shares many of their targets (Zhong et al., 2010b). It has been proposed that secondary wall NACs (SWNs) first appeared to regulate the differentiation of tracheids in early vascular plants and latter diversified to regulate other cell types with secondary walls, such as fibres (Zhong et al., 2010a). The regulation of secondary cell-wall synthesis has an obvious interest for the manipulation of biomass production in second-generation biofuel crops (Demura and Ye, 2010). Some of the more promising species for biomass production are members of the grass family, which includes the model Brachypodium distachyon (Mur et al., 2011). The programme of secondary cell-wall formation is expected to be somewhat similar in grasses and dicots, as the composition of their secondary walls is quite comparable, in contrast with the large divergence found in primary walls. However, some important differences have been found, such as a higher proportion of p-hydroxyphenyl (H) units in grass lignin (Vogel, 2008). Additionally xylan contains more arabinose side chains in grasses than in dicots and it is unclear whether both groups use the same synthesis mechanism (Faik, 2010; Scheller et al., 2010). Another open question is whether grasses use different sets of genes to build xylans in primary and secondary walls. The transcriptional regulation of secondary wall biosynthesis in grasses is very poorly understood (Handakumbura and Hazen, 2012). Coexpression analyses in rice and barley suggested the possible involvement of SWNs in this process (Christiansen et al., 2011; Ruprecht et al., 2011). In addition, it has been recently shown that rice and maize SWNs are expressed in xylem and fibres and that, when overexpressed in Arabidopsis, they can induce the formation of ectopic secondary walls (Zhong et al., 2011). This work reports that inducible overexpression in B. distachyon of the NAC transcription factor BdSWN5 was sufficient to activate secondary cell-wall formation. Among the genes upregulated in the process were a secondary cell-wall cellulose synthase and BdXCP1, an orthologue of AtXCP1. This study also found that the BdXCP1 promoter is normally active in differentiating tracheary elements and that BdSWN5 can directly regulate BdXCP1 transcription by binding two conserved SNBE sites. These findings establish that NAC transcription factors can work as secondary cell-wall master switches in monocots and can be used to study and manipulate this process. Materials and methods Phylogeny Sequences were aligned with clustalw and phylogenetic trees obtained with mega5 (Tamura et al., 2011). Trees were constructed by neighbour joining using Poisson distances and bootstrap values estimated from 500 replicates. Genetic constructions Brachypodium genomic DNA or cDNA was amplified with PrimeSTAR HS (Takara) using primers detailed in Supplementary Table S1 (available at JXB online). Coding sequences and promoter regions were cloned respectively in pENTR/D and pENTR5’ (Invitrogen). BdSWN sequences obtained from cDNA were submitted to Genbank (accession numbers JQ693422–JQ693429). Final constructs were assembled using LR Clonase II (Invitrogen). For overexpression in tobacco, the destination vector was pMDC32 (Curtis and Grossniklaus, 2003). For promoter–reporter constructs, the intron-containing glucuronidase entry clone pEN-L1-SI*-L2 was used (Karimi et al., 2007). This clone was combined with promoter entry clones into pERV2, a double recombination vector obtained by cloning a HindIII–SalI fragment from pH7m24GW,3 (Karimi et al., 2007) into pIPKb001 (Himmelbach et al., 2007). For oestradiol-inducible expression in Brachypodium, pERV1 plasmid was constructed using as a backbone pIJB2, a plasmid kindly provided by Oumaya Bouchabké-Coussa (Institut Jean-Pierre Bourgin, INRA) with the SfiI fragment from pIPKb002 (Himmelbach et al., 2007) inserted into p7U, conferring Basta resistance (DNA Cloning Service). A PCR fragment containing the LEXA promoter was amplified from pMDC7 (Curtis and Grossniklaus, 2003) and cloned between the SpeI and HindIII sites of pIJB2. The actin intron from pIPKb003 was then cloned between the HindIII and AvrII sites in the previous construct. Finally, the XVE coding sequence was amplified from pMDC7 and cloned between the PspXI and SpeI sites. An entry clone of BdSWN5 with its intron was recombined with this vector. The BdXCP1 297-bp promoter fragment in pENTR5’ was used as template to obtain mutated variants by megaprimer PCR (Sarkar and Sommer, 1990). To introduce a mutation at the SNBE1 site, a megaprimer was amplified with XSNBE1R1 and PENTR5L3. In the second amplification, PENTR5R3 was added to obtain a final product that included attL4 and attR1 sites. SNBE2 mutations were introduced by using XSNBE2L1 and PENTR5R3 to produce a megaprimer, adding PENTR5L3 in the second round. Double mutants were obtained by applying the two procedures sequentially. The PCR products were used directly in LR recombination reactions and the final constructs sequenced. Constructs were introduced into Agrobacterium tumefaciens EHA105 (pMDC32) or AGL1 (other plasmids). Tobacco transformation Nicotiana tabacum cv. Petit Havana was grown in soil for 4–6 weeks before infiltration with a suspension of transformed A. tumefaciens cells in infiltration media (10mM MgSO4, 10mM MES at pH 5.5, 0.5% glucose, 100 μM acetosyringone). The OD600 of the suspension was 0.1 for overexpression and 0.05 per construct for transactivation. For overexpression experiments, infiltrated sections were harvested after 5 days and clarified with 70% ethanol and lactic acid. For transactivation experiments, two discs (1cm diameter) were harvested after 2 days and GUS activity against 4-MUG was measured (Kim et al., 2006). All experiments were repeated at least twice with similar results. SYTOX staining Leaf discs (1cm) were incubated in 1 μM SYTOX Orange (Invitrogen) for 15 minutes under vacuum (80 kPa). After several washes, discs were observed on a Leica MZ16 fluorescence stereomicroscope equipped with a GFP Plus filter (460–500nm excitation and 510nm longpass barrier filter). Brachypodium growth and transformation B. distachyon plants (Bd21-3) were grown under a 16/8 light/dark cycle at 24/20 °C and 50–60% humidity. Agrobacterium-mediated transformation was carried out as previously described (Vogel and Hill, 2008) with minor modifications. Briefly, immature seeds were sterilized with 2% bleach and embryos were excised and placed on callus induction media (CIM). Calli were maintained at 26 °C in the dark and subcultured twice before transformation. Agrobacterium cultures scraped from 2-day-old YT plates were resuspended in liquid CIM with 200 μM acetosyringone and the OD600 adjusted to 0.6. About 90 calli per construct were incubated in Agrobacterium suspension for 5 minutes and placed for 5 days on empty plates with sterile filter paper and 750 μl of liquid CIM. Calli were transferred to CIM plates with 150mg l–1 Timentin for 1 week and then for 2 weeks at a time to CIM plates with 150mg l–1 Timentin and either 5mg l–1 ammonium glufosinate (oestradiol-inducible) or 40mg l–1 hygromycin (double recombination). After 6 weeks of selection, calli were moved to regeneration media and incubated at 24 °C in 16h days. Regenerating plantlets were transferred to 50ml glass culture tubes containing media with 0.5 × MS salts with vitamins, 3% sucrose, 0.7% activated charcoal, 0.2% phytagel, and 0.05% MES at pH 5.7. After transfer to soil, plants carrying T-DNA insertions were identified by PCR using Phire Plant Direct PCR Kit (Thermo Scientific) and the primers detailed in Supplementary Table S1. Induction assay Brachypodium seeds were sterilized with 2% bleach and placed in 250ml flasks containing 100ml of 0.25 × MS salts, 0.5g l–1 MES at pH 5.8 and 1% sucrose. Flasks were placed in a rotary shaker at 125rpm under a 16/8 light/dark cycle at 24/20 °C. After 4h of light, flasks were kept in the dark for 44h. Seedlings were induced by adding 100 μl of 10mM β-oestradiol in DMSO or mock induced with 100 μl of DMSO. After 16h in the same conditions, root tips (approx. 8 μm) and aerial parts were collected separately for RNA extraction. Gene expression RNA was extracted using a Plant Total RNA kit (Sigma). cDNA was synthesized using the Transcriptor First Strand cDNA synthesis Kit (Roche). End-point PCR was carried out for 40 cycles with MyTaq (Bioline). Real time PCR was performed for 40 cycles on the Mini Opticon Real-Time PCR system (Bio-rad) using SensiMix SYBR No-Rox Kit (Bioline). Amplification efficiency and Ct values were calculated with LinRegPCR (Ruijter et al., 2009). Statistical analysis was done with REST (Pfaffl et al., 2002) using UBC18 (Bradi4g00660) and SamDC (Bradi5g14640) as references. Primers and annealing temperatures are detailed in Supplementary Table S1. Electrophoretic mobility shift assay BdSWN coding regions were introduced into pET300/NT-DEST (Invitrogen) and used to transform Escherichia coli BL21 DE3 (Novagen). Cell cultures (200ml) were grown to an OD600 of 0.7–1.0 and induced with 1mM IPTG for 6h at 24 °C. Cells were harvested and frozen at –80 °C. Proteins were extracted with B-PER Reagent (Pierce) with 10% glycerol, 300mM NaCl, 5mM imidazole, and Halt Protease Inhibitor Cocktail EDTA-free (Pierce). Purification was carried out with Dynabeads His-Tag Isolation and Pulldown Kit (Novex). Probes and competitors were amplified by PCR or obtained by annealing complementary primers (Supplementary Table S1). Labelling was incorporated with 5’-end biotin-labelled primers. Electrophoretic mobility shift assays were performed using the LightShift Chemiluminescent EMSA Kit (Pierce). Labelled promoter fragments (10fmol) or labelled SNBE motifs (20fmol) were incubated with 150 or 300ng of protein, respectively, for 20min in 10 μl of binding buffer (10 mM Tris at pH 7.5, 100 mM KCl, 1mM DTT, 100ng l–1 poly dI-dC, 0.05% Nonidep P-40). DNA was separated by polyacrylamide gel electrophoresis on 0.5 × Tris-borate-EDTA gels and electroblotted onto Byodine B nylon membranes (Pierce). β-Glucuronidase histochemical staining Tissue samples were incubated in 90% acetone at 4 °C for 20 minutes and transferred to staining buffer (100mM sodium phosphate at pH 7, 10mM EDTA, 0.5mM K3[Fe(CN)6], 0.5mM K4[Fe(CN)6], 0.1% Triton-X, and 1mM X-gluc) and placed under vacuum for 10 minutes. After incubation at 37 °C for approx. 4 hours, samples were cleared in 70% ethanol and lactic acid. Results The Brachypodium genome includes eight SWN genes that are expressed in developing organs A phylogenetic analysis of NAC transcription factors in the Brachypodium genome (Brachypodium Genome Initiative, 2010) revealed the presence of eight SWN genes (Fig. 1A). Six of these genes (SWN1 to SWN6) are orthologues of ArabidopsisVND genes and two (SWN7 and SWN8) are orthologues of SND/NST genes. The phylogeny shows that at least four clades were already differentiated in the last common ancestor or monocots and dicots. The coding regions of all eight genes were amplified from cDNA and sequenced, allowing the correct annotations to be determined (Supplementary Table S2). Fig. 1. Open in new tabDownload slide (A) Phylogenetic tree of SWN proteins from Brachypodium, rice, and Arabidopsis. Annotations for Brachypodium and rice genes can be found in Supplementary Table S1. Bootstrap values are shown at branch nodes. Circles indicate clades differentiated in the last common ancestor of monocots and dicots. (B) Reverse transcription-PCR analysis of BdSWN expression in developing roots (R), leaves (L), and internodes (I). UBC18 was used as a control. Similar results were observed with a second biological replicate. Fig. 1. Open in new tabDownload slide (A) Phylogenetic tree of SWN proteins from Brachypodium, rice, and Arabidopsis. Annotations for Brachypodium and rice genes can be found in Supplementary Table S1. Bootstrap values are shown at branch nodes. Circles indicate clades differentiated in the last common ancestor of monocots and dicots. (B) Reverse transcription-PCR analysis of BdSWN expression in developing roots (R), leaves (L), and internodes (I). UBC18 was used as a control. Similar results were observed with a second biological replicate. The expression level of all eight genes was tested by reverse trancription-PCR in developing roots, leaves, and internodes (Fig. 1B). All the genes were detected in internodes, with SWN3, SWN4, SWN6, and SWN7 reaching their highest level of expression in this organ. On the other hand, expression levels in leaves were generally low or undetectable, with the exception of SWN5 and SWN8. Expression in roots was also generally lower than in other organs with the exceptions of SWN1 and SWN2. Brachypodium SWN genes can induce ectopic cell-wall formation in tobacco The eight Brachypodium SWN genes were transiently expressed in tobacco leaves via infiltration with Agrobacterium cultures carrying overexpression constructs. The SWN genes were under the control of two copies of the CaMV 35S promoter. After five days of co-cultivation, expression of all of the SWN genes had resulted in the ectopic formation of spiral or annular cell-wall thickenings in mesophyll and epidermal cells (Fig. 2). The percentage of cells with this type of response was particularly high in the cases of SWN1, SWN3, SWN4, and SWN7. Fig. 2. Open in new tabDownload slide Ectopic cell-wall formation in tobacco leafs induced by overexpression of different Bd-SWN genes. Bars = 100 μm (SWN7), 50 μm (SWN3, SWN4, and SWN5), and 25 μm (all others) (this figure is available in colour at JXB online). Fig. 2. Open in new tabDownload slide Ectopic cell-wall formation in tobacco leafs induced by overexpression of different Bd-SWN genes. Bars = 100 μm (SWN7), 50 μm (SWN3, SWN4, and SWN5), and 25 μm (all others) (this figure is available in colour at JXB online). Transient expression of some SWN genes also resulted in extensive cell death after 5 days of cocultivation, as evidenced by loss of chlorophyll (Fig. 3A) and membrane permeabilization revealed by SYTOX staining (Fig. 3B). This response was consistently stronger when expressing genes in clades I and II (SWN1, SWN2,SWN3, or SWN4). On the other hand, transient expression of genes in clade IV (SWN7 and SWN8) did not result in additional cell death beyond that caused by Agrobacterium infiltration. In some cases, cells died before any visible wall thickenings had been formed, resulting in the collapse of thin-walled mesophyll cells (Fig. 3C). This was particularly common when SWN2 was transiently expressed, with the number of collapsed cells an order of magnitude above the number of cells with ectopic wall thickenings. Tobacco infiltrations were also performed with overexpression constructs for SWN1–SWN6 that included the native introns within the coding region. Only in the case of SWN5 was the intensity of the response increased, both for cell-wall formation and cell death (data not shown). SWN5 is the only BdSWN gene with a single intron. Fig. 3. Open in new tabDownload slide Cell death in tobacco leafs induced by overexpression of different BdSWN genes. (A) Tobacco leaf infiltrated with Agrobacterium carrying an empty plasmid or different SWN overexpression constructs, as well as with infiltration medium. (B) SYTOX staining of non-viable cells (green) in the abaxial side of leafs transformed with different constructs. (C) Cell collapse caused by SWN2 overexpression in the palisade and spongy mesophylls. Bars = 300 μm (B) and 50 μm (C). Fig. 3. Open in new tabDownload slide Cell death in tobacco leafs induced by overexpression of different BdSWN genes. (A) Tobacco leaf infiltrated with Agrobacterium carrying an empty plasmid or different SWN overexpression constructs, as well as with infiltration medium. (B) SYTOX staining of non-viable cells (green) in the abaxial side of leafs transformed with different constructs. (C) Cell collapse caused by SWN2 overexpression in the palisade and spongy mesophylls. Bars = 300 μm (B) and 50 μm (C). Inducible overexpression of SWN5 is sufficient to cause ectopic cell-wall formation in Brachypodium The oestradiol-inducible transactivation system based on the XVE chimeric transcription factor was used to obtain Brachypodium lines with inducible overexpression of BdSWN5. The original vector is not suitable for grasses due to the G10-90 promoter resulting in low expression levels (Zuo et al., 2006). In order to use this system in Brachypodium, the XVE transcription factor was placed under the control of the maize ubiquitin promoter in pIPKB002 (Fig. 4A). Additionally the actin intron from pPIKB003 was introduced in the 5’-untranscribed region after the OLexA-46 promoter to increase the expression level. BdSWN5 was selected as a phylogenetic orthologue of AtVND7 that showed a strong effect in tobacco when the native intron was included. This intron was also included in the inducible overexpression construct. Fig. 4. Open in new tabDownload slide Inducible overexpression of BdSWN5 in Brachypodium. (A) Plasmid used for oestradiol-inducible overexpression. (B) Arrested development of a 5-day-old transformed seedling from line 6 germinated in media with 10 μM oestradiol (Cp, coleoptile; Cr, coleorhiza; L, leaf; R, root). (C) Root of a transformed seedling from line 9 grown in the same media. (D) Leaf epidermis of a transformed seedling from line 8 grown in the same media. (E) Coleoptile parenchyma of a transformed seedling from line 3 grown in liquid 24h after addition of oestradiol. (F) Coleoptile parenchyma of a similar seedling 24h after mock induction. (G) Root central metaxylem (arrowhead) differentiating less than 2mm from the tip in an induced seedling. (H) Root central metaxylem differentiating approx. 12mm from the tip in a mock-induced seedling. (I) Root cortex in the transition zone of an induced seedling. (J) Mature root cortex in an induced seedling. Bars = 400 μm (B) and 25 μm (all others). (K) Relative expression level of selected genes in induced and mock-induced seedlings grown in liquid. Root tips (approx. 8mm) and aerial parts were collected separately 16h after induction. Error bars are standard errors as calculated by REST software from three biological and two technical replicates. UBC18 and SamDC were used as references. Ratios that are significantly different from 1 are indicated in black (P < 0.005) or grey (P < 0.02) (this figure is available in colour at JXB online). Fig. 4. Open in new tabDownload slide Inducible overexpression of BdSWN5 in Brachypodium. (A) Plasmid used for oestradiol-inducible overexpression. (B) Arrested development of a 5-day-old transformed seedling from line 6 germinated in media with 10 μM oestradiol (Cp, coleoptile; Cr, coleorhiza; L, leaf; R, root). (C) Root of a transformed seedling from line 9 grown in the same media. (D) Leaf epidermis of a transformed seedling from line 8 grown in the same media. (E) Coleoptile parenchyma of a transformed seedling from line 3 grown in liquid 24h after addition of oestradiol. (F) Coleoptile parenchyma of a similar seedling 24h after mock induction. (G) Root central metaxylem (arrowhead) differentiating less than 2mm from the tip in an induced seedling. (H) Root central metaxylem differentiating approx. 12mm from the tip in a mock-induced seedling. (I) Root cortex in the transition zone of an induced seedling. (J) Mature root cortex in an induced seedling. Bars = 400 μm (B) and 25 μm (all others). (K) Relative expression level of selected genes in induced and mock-induced seedlings grown in liquid. Root tips (approx. 8mm) and aerial parts were collected separately 16h after induction. Error bars are standard errors as calculated by REST software from three biological and two technical replicates. UBC18 and SamDC were used as references. Ratios that are significantly different from 1 are indicated in black (P < 0.005) or grey (P < 0.02) (this figure is available in colour at JXB online). Approximately 90 individual calli were transformed and 13 T0 plants were regenerated from 10 independent calli. Plant growth was normal and most plants were fertile. To identify the lines with the strongest response seeds from nine independent lines (T1 generation) were germinated on plates with 10 μM β-oestradiol and grown for 5 days. Five lines showed a very strong response with the development of the majority of the seedlings arrested shortly after germination, before the root had reached 5mm (Fig. 4B). These seedlings showed accelerated vascular development as well as extensive ectopic cell-wall formation in roots, coleoptiles, and leaves, but not the coleorhiza (Fig. 4C and D). In addition, roots tended to curl and the root epidermal cells, which did not show ectopic wall formation, became partially detached from the cortex (Fig. 4C). Three other lines showed a milder response with most seedlings showing normal or partially reduced elongation, but with ectopic wall deposition in many cells, particularly in the root cortex and coleoptile. No phenotype was observed when wild-type seeds were germinated in the same plates. Screening of T1 plants from line 3, one of the lines with a strong response, indicated the presence of a single T-DNA locus, with an insertion in six out of 10 plants. Plant 3–4 was then identified as homozygous when all 26 T2 descendants showed presence of the insertion. Pooled seeds from these T2 plants were grown for 2 days in liquid culture in the dark before addition of β-oestradiol. In comparison with mock-induced seedlings, SWN5 expression severely slowed down seedling development. After 24 hours of induction, ectopic cell-wall depositions in annular or scalariform patterns were visible in the coleoptile parenchyma of induced seedlings (Fig. 4E), but not in mock-induced seedlings (Fig. 4F). No changes were seen in the coleoptile epidermis or in developing leaves, in contrast to what had been observed in seedling germinated on oestradiol plates. In the roots, addition of β-oestradiol resulted in an acceleration of vascular development with all types of xylem vessels differentiating simultaneously very close to the root tip, including the central metaxylem (Fig. 4G). In mock-induced seedlings, cell-wall thickenings in the central metaxylem started to appear more than 1cm away from the tip after the cells had elongated to a length of approx. 350 μm (Fig. 4H). In addition, ectopic cell-wall formation was observed in the root cortex of induced seedlings starting before elongation (Fig. 4I). In mature cortex cells, the pattern of wall deposition was similar to that of pitted xylem vessels (Fig. 4J). Older parts of the roots, more than 1cm away from the tip, did not show any changes, suggesting that only differentiating cells are capable of responding to SWN5 expression. SWN5 overexpression can activate genes involved in secondary cell-wall synthesis and cell death To identify genes activated by SWN5, real-time PCR was used to compare expression levels in induced and mock-induced seedlings 16h after induction (Fig. 4K). Seedlings were grown in liquid as described in the previous section. UBC18 and SamDC were chosen as reference genes as their expression is stable in different organs and growth conditions (Hong et al., 2008). As expected, SWN5 expression was upregulated in response to β-oestradiol in both roots and aerial parts. BdCESA4 (Bradi2g49912) was selected as a putative secondary wall cellulose synthase since it is the only Brachypodium orthologue of OsCESA4 (Supplementary Fig. S1A), a rice gene required for the synthesis of secondary cell walls (Tanaka et al., 2003). BdCESA1 (Bradi2g34240) was selected as a putative primary wall cellulose synthase as an orthologue of both OsCESA1 and AtCESA1 (Supplementary Fig. S1A). The expression pattern of OsCESA1 strongly suggests its involvement in rice primary wall cellulose synthesis (Ruprecht et al., 2011). BdSWN5 strongly upregulated BdCESA4 expression in both roots and aerial parts, 15-fold and 4-fold respectively. In contrast, expression of BdCESA1 was reduced or unchanged. BdXCP1 (Bradi2g39320) was identified as the single orthologue of the xylem-specific cysteine proteases AtXCP1 and AtXCP2 (Supplementary Fig. S1B). Finally BdMYB1 (Bradi4g06317) was chosen as the orthologue of AtMYB46 and AtMYB83 (Supplementary Fig. S1C), both of them direct targets of AtSWNs (McCarthy et al., 2009; Zhong et al., 2010b; Yamaguchi et al., 2011). The expression of both genes was found to be increased in seedlings treated with β-oestradiol, although BdXCP1 upregulation was much higher, reaching 19-fold in roots and 17-fold in aerial parts. Transactivation of BdXCP1 promoter by BdSWN5 is mediated by SNBE sites To identify the binding sites of BdSWN5, this study first tested whether this transcription factor was able to transactivate BdMYB1 and BdXCP1 promoter fragments (2.3kb and 1.5kb respectively). These fragments were placed in front of an intron-containing glucuronidase gene to generate reporter constructs. The effector construct for SWN5 included the native intron (SWN5i), since this intron seemed to enhance expression in tobacco. Tobacco leaves were coinfiltrated with Agrobacterium cultures transformed with effector and reporter plasmids and glucuronidase activity was measured after 2 days. There was no increase in reporter activity from the BdMYB1 promoter–reporter construct when coinfiltrated with SWN5 effector (Fig. 5B). However, SWN5 was able to transactivate a reporter construct that included an AtMYB46 promoter fragment, orthologous to BdMYB1. On the other hand, GUS activity increased to 377 pkat mg–1 when the BdXCP1 promoter–reporter construct was coinfiltrated with the SWN5 effector. A similar result was observed with a truncated reporter construct that contained only the 297bp upstream of the BdXCP1 start codon. This promoter fragment includes two SNBE sites which are highly conserved in sequence and position when compared to the AtXCP1 promoter (Fig. 5A). Fig. 5. Open in new tabDownload slide Transactivation of Brachypodium promoters by BdSWN transcription factors. (A) Alignment of SNBE motifs in the AtXCP1 and BdXCP1 promoters. Conserved nucleotides are boxed in grey. Nucleotide changes introduced in mutated sites are shown below the alignment. TSS = transcription start site. (B–D) Transactivation assays of promoter fragments: (B) reporter constructs were infiltrated on their own (white) or together with an effector construct with a 2×35S promoter driving the expression of BdSWN5 (grey); (C) wild-type and mutated fragments of BdXCP1 promoter; M1 and M2 mutations are indicated in part A; (D) BdMYB1 (white) and BdXCP1 (grey) promoters with eight BdSWN effectors. Error bars indicate SD. Fig. 5. Open in new tabDownload slide Transactivation of Brachypodium promoters by BdSWN transcription factors. (A) Alignment of SNBE motifs in the AtXCP1 and BdXCP1 promoters. Conserved nucleotides are boxed in grey. Nucleotide changes introduced in mutated sites are shown below the alignment. TSS = transcription start site. (B–D) Transactivation assays of promoter fragments: (B) reporter constructs were infiltrated on their own (white) or together with an effector construct with a 2×35S promoter driving the expression of BdSWN5 (grey); (C) wild-type and mutated fragments of BdXCP1 promoter; M1 and M2 mutations are indicated in part A; (D) BdMYB1 (white) and BdXCP1 (grey) promoters with eight BdSWN effectors. Error bars indicate SD. To determine if the SNBE sites were required for the activation of the 297-bp BdXCP1 promoter fragment, the two sites were mutated independently and simultaneously. A total of four point mutations were introduced in each site at highly conserved positions in the SNBE motif (Fig. 5A). Mutation of SNBE1 and SNBE2 resulted in 93% and 98% reductions in activation respectively, suggesting that both sites are required for full activation (Fig. 5C). Mutation of both sites simultaneously abolished transactivation by SWN5. The reporter construct with the 297-bp BdXCP1 promoter fragment was coinfiltrated with effector constructs for the eight BdSWN genes without introns (Fig. 5D). Expression of all the genes resulted in activation of the BdXCP1 promoter, but at very low levels in the case of SWN7 and SWN8. Expression of the remaining genes resulted in much larger increases in reporter activity, with SWN1 consistently giving the highest level. When the same experiment was repeated with the BdMYB1 promoter, the activation level was undetectable for all of the BdSWN genes (Fig. 5D), with the possible exception of SWN1 (P = 0.073). BdSWN5 can bind to SNBE sites in the BdXCP1 promoter To test if SWN5 activates the XCP1 promoter though direct binding to SNBE sites, electrophoretic mobility shift assays were performed. Purified SWN5 protein slowed down the migration of a 142-bp fragment of the XCP1 promoter that included both SNBE sites (Fig. 6A). Two shifted bands were clearly visible, possibly corresponding to the occupation of one or both SNBE sites. An unlabelled wild-type fragment or a fragment with a mutated SNBE1 site (M1) were able to compete with the probe for binding to SWN5. A fragment where SNBE2 was mutated (M2) was a much less effective competitor even at twice the ratio of the wild-type competitor, suggesting a higher binding affinity toward the SNBE2 site than toward the SNBE1 site. The ability to compete was almost abolished when both SNBE sites were mutated (M1M2). Finally, isolated SNBE motifs in 35-bp promoter fragments (S1 and S2) were able to compete for binding to SWN5, with SNBE2 a more effective competitor than SNBE1. Fig. 6. Open in new tabDownload slide Electrophoretic mobility shift assays showing binding of BdSWN proteins to BdXCP1 promoter fragments. (A) A 142-bp XCP1 promoter fragment in the presence of SWN5 protein and a number of different competitors: unlabelled wild-type fragment (WT), mutated fragments (M1, M2, M1M2, see Fig. 4), and isolated SNBE1 and SNBE2 motifs (S1, S2). (B) XCP1 SNBE motifs in the presence of SWN5 protein and a number of different competitors: wild-type and mutated SNBE1 (W1 and M1), and wild-type and mutated SNBE2 (W2 and M2). (C) XCP1 promoter fragment in the presence of SWN7 protein; legend as in A. (D) XCP1 SNBE motifs in the presence of SWN7 protein; legend as in B. Fig. 6. Open in new tabDownload slide Electrophoretic mobility shift assays showing binding of BdSWN proteins to BdXCP1 promoter fragments. (A) A 142-bp XCP1 promoter fragment in the presence of SWN5 protein and a number of different competitors: unlabelled wild-type fragment (WT), mutated fragments (M1, M2, M1M2, see Fig. 4), and isolated SNBE1 and SNBE2 motifs (S1, S2). (B) XCP1 SNBE motifs in the presence of SWN5 protein and a number of different competitors: wild-type and mutated SNBE1 (W1 and M1), and wild-type and mutated SNBE2 (W2 and M2). (C) XCP1 promoter fragment in the presence of SWN7 protein; legend as in A. (D) XCP1 SNBE motifs in the presence of SWN7 protein; legend as in B. When isolated motifs were used as probes, binding of SWN5 protein was also detected (Fig. 6B). Unlabelled wild-type motifs could compete effectively, but not those with four point mutations. Consistent with the results observed with the larger fragment, unlabelled SNBE2 could outcompete labelled SNBE1, while unlabelled SNBE1 was less effective against labelled SNBE2. The higher affinity of SWN5 for SNBE2 fits with the transactivation results obtained with mutated motifs (Fig. 5C), strengthening the case for direct activation. The same experiments were repeated with SWN7 protein since it had shown a reduced capacity to activate the XCP1 promoter in transactivation assays (Fig. 5D). The results obtained with SWN7 were quite similar to those observed for SWN5 both when using as probe the 142bp XCP1 promoter fragment (Fig. 6C) and when using isolated motifs (Fig. 6D). However, the binding affinity of SNW7 toward SNBE1 appeared to be lower than in the case of SWN5, as shown by the poor competition of the M2 fragment or the SNBE1 motif against labelled wild-type fragment (Fig. 6C). This lower affinity was confirmed in the competition of the SNBE1 motif against labelled SNBE2 motif, as well the lower degree of shift observed with labelled SNBE1 (Fig. 6D). BdXCP1 promoter directs expression to developing xylem tracheary elements The same reporter construct containing a 1.5-kb BdXCP1 promoter fragment used in transactivation assays was employed for stable transformation of Brachypodium plants with a promoter–reporter construct. Of a total of 16 independent lines examined, 15 showed a common expression pattern (Fig. 7). In roots, expression was detected in all types of xylem tracheary elements, as they progressively differentiated, starting with protoxylem (Fig. 7A), followed by peripheral metaxylem (Fig. 7B) and later by central metaxylem (Fig. 7C). Staining was also evident in developing xylem of internodes, but not in developing fibres (Fig. 7D and E). In leaves, expression was observed in both longitudinal and commissural vascular bundles (Fig. 7F). Additionally, staining was observed in short leaf epidermal cells with the appearance of cork-silica cell pairs (Fig. 7G). Fig. 7. Open in new tabDownload slide Expression of β-glucuronidase driven by BdXCP1 promoter in transformed Brachypodium plants. Samples were taken from 6-day-old roots (A–C), 15-day-old internodes (D and E), 6-day-old leaves (F), and 15-day-old leaves (G). Bars = 200 μm (A), 100 μm (E and F), 50 μm (C), and 25 μm (all others). Fig. 7. Open in new tabDownload slide Expression of β-glucuronidase driven by BdXCP1 promoter in transformed Brachypodium plants. Samples were taken from 6-day-old roots (A–C), 15-day-old internodes (D and E), 6-day-old leaves (F), and 15-day-old leaves (G). Bars = 200 μm (A), 100 μm (E and F), 50 μm (C), and 25 μm (all others). Discussion The role of NAC transcription factors as key regulators of secondary cell-wall synthesis and programmed cell death has been established in several dicotyledonous species (Zhong et al., 2010a; Wang and Dixon, 2012). The present results now demonstrate that inducible overexpression of a SWN transcription factor in Brachypodium is sufficient to activate the programme of secondary cell-wall synthesis, establishing that SWNs can act as master switches in monocots. This study has shown that overexpression of BdSWN5 leads to accelerated xylem development and deposition of ectopic cell walls in different Brachypodium organs (Fig. 4). The patterns of ectopic cell-wall depositions are reminiscent of tracheary elements. Among the genes that are upregulated by SWN5 are BdCESA4, a secondary cell-wall cellulose synthase, and BdXCP1, a cysteine protease possibly involved in cell death (Fig. 4). Transactivation of the XCP1 promoter by SWN5 depends on the presence of two conserved SNBE motifs, as proven by the effect of point mutations in conserved positions (Fig. 5). Electrophoretic mobility shift assays further show that SWN5 can bind to both motifs whether isolated or paired as in the genome (Fig. 6). Finally, a XCP1 promoter fragment that includes these motifs directs expression of a reporter gene to developing xylem tracheary elements (Fig. 7). It thus appears that a direct target of BdSWNs is activated just as cells undergo the processes of secondary wall deposition and cell death. It is also possible that some epidermal cells share parts of this programme (Fig. 7G). It thus seems likely that SWN genes already functioned as master switches of secondary cell-wall synthesis in the last common ancestor of monocots and dicots and that this role has been preserved in both lineages. Furthermore, the binding specificity of SWN proteins and therefore the motifs present in their target promoters appear to have been conserved. This can explain the activation of both cell-wall deposition and cell death programmes in tobacco leaves through transient expression of BdSWN genes (Figs. 2 and 3). Ectopic cell-wall deposition has also been observed in Arabidopsis when overexpressing rice and maize SWNs (Zhong et al., 2011). Despite the conserved function of SWNs in monocots and dicots, the regulatory network of secondary walls has probably diverged after 140 million years of separate evolution (Chaw et al., 2004). In Arabidopsis, the direct activation of AtMYB46 and AtMYB83 by several AtSWN transcription factors has been demonstrated both in vitro and in vivo (McCarthy et al., 2009; Zhong et al., 2010b; Yamaguchi et al., 2011). In Brachypodium, overexpression of BdSWN5 also upregulates RNA levels of BdMYB1, an orthologue of AtMYB46 and AtMYB83 (Fig. 4). However, this study was unable to detect transactivation of the BdMYB1 promoter by SWN5, even though this Brachypodium transcription factor is capable of transactivating the AtMYB46 promoter (Fig. 5B). Transactivation of the BdMYB1 promoter was also undetectable with the remaining BdSWNs, suggesting the lack of direct activation (Fig. 5D). A recent study showed that SWN proteins from rice and maize could transactivate a promoter with a tandem arrangement of SNBE motifs taken from rice and maize orthologues of BdMYB1 (Zhong et al., 2011). It is unclear, however, if this artificial arrangement reflects the activity of the native promoter. The results presented here can also shed some light on the functional specialization of different SWN genes. Phylogenetic analysis suggests the existence of four clearly differentiated clades in the last common ancestor of monocots and dicots (Fig. 1A). Genes from all four of these clades were capable of inducing ectopic cell-wall thickenings in transiently transformed tobacco leaves (Fig. 2). In Arabidopsis, overexpression of the three genes from clade I does not result in any phenotype (Kubo et al., 2005), while genes from the other three clades are capable of activating the deposition of secondary cell walls (Kubo et al., 2005; Mitsuda et al., 2005; Zhong et al., 2006). In this aspect, Brachypodium seems more similar to poplar, where genes from all four clades induce ectopic wall thickenings in both poplar and Arabidopsis (Ohtani et al., 2011). The pattern of cell-wall deposition caused by transient expression of BdSWNs is similar for all of the genes. This is also the same result obtained with poplar SWNs and again different from Arabidopsis, where overexpression of different SWNs can result in different patterns (Kubo et al., 2005; Ohashi-Ito et al., 2010). In any case, the pattern of ectopic wall deposition is clearly affected by the previous identity of the cell, as shown by the different effects of BdSWN5 expression in coleoptiles and roots (Fig. 4E and J). This had previously been observed for AtSND1 (Zhong et al., 2006; Ohashi-Ito et al., 2010) and poplar SWNs (Ohtani et al., 2011). Overexpression of BdSWN genes in tobacco can also activate programmed cell death (Fig. 3). Interestingly, the cell death and the cell-wall synthesis programmes appear to be partially independent since they are activated with different intensities by different transcription factors. While SWN4 expression results in both extensive cell-wall deposition and cell death, SWN7 expression appears to activate primarily cell-wall synthesis and SWN2 expression mainly results in cell death with no previous cell-wall deposition. It would be reasonable to conclude that SWN7 and SWN2 are activating different sets of genes, possibly due to differences in binding sequences or preferred motif arrangements. The two Brachypodium proteins in clade IV, SWN7 and SWN8, are poor inducers of cell death in tobacco and they also show a very weak capacity to transactivate the BdXCP1 promoter (Fig. 5D). This is unlikely to be caused by a weak activation domain, since SWN7 is very effective inducers of cell-wall synthesis in tobacco (Fig. 2). Alternatively, it could indicate the conservation of a promoter motif or motif arrangement specific to programmed cell death between the two species. Although in electrophoretic mobility shift assays SWN7 can bind the two SNBE motifs in the BdXCP1 promoter, it appears to bind SNBE1 with lower affinity than SWN5 (Fig. 6). Simultaneous binding to both SNBE sites seems to be required for strong activation of the XCP1 promoter, as shown by the effect of point mutations in either of the sites (Fig. 5C). Therefore a low binding affinity for SNBE1 could partly explain the low transactivation observed with SWN7. In poplar, SWN proteins from clade IV are not very efficient at transactivating the PtXCP1 promoter, especially when compared to proteins in clades II and III (Ohtani et al., 2011). Similarly the AtXCP2 promoter can be directly activated by clade II and clade III proteins, but not by the three Arabidopsis clade IV proteins (Zhong et al., 2010b). The situation for AtXCP1 appears to be more complex, with results that depend on the system used (Ohashi-Ito et al., 2010; Zhong et al., 2010b). To explain the lack of AtXCP1 expression in fibres, repression by an additional regulatory mechanism has been suggested (Bollhöner et al., 2012). It is possible that this mechanism acts by blocking SNBE motifs since an artificial promoter with SNBEs from AtXCP1 can be efficiently activated by AtSND1 in transfected protoplasts (Zhong et al., 2010b), but is not active in Arabidopsis fibres (McCarthy et al., 2011). The results presented here suggest that the lower efficiency of clade IV proteins at activating genes involved in cell death could be conserved in monocots. Unlike in Arabidopsis, this apparent functional specialization may not be linked to expression in different cell types as maize and rice genes from clades II, III and IV are expressed indistinctly in fibres and tracheary elements (Zhong et al., 2011). Since the reporter construct suggests that BdXCP1 is not expressed in fibres (Fig. 7E), it is possible that a repression mechanism is also active in monocots. The use of inducible systems capable of activating secondary cell-wall synthesis has generated a wealth of information regarding the transcriptional regulation of this complex developmental process in Arabidopsis (Ohashi-Ito et al., 2010; Zhong et al., 2010b; Yamaguchi et al., 2011). The present study has now extended this approach to the monocots, using the model species Brachypodium dystachion, an important step to understand the regulation of secondary cell-wall synthesis in grasses and to acquire the tools that will be necessary to improve biomass crops. The results indicate that much of what has been learned in Arabidopsis will still be relevant, but point also to some possible differences. Supplementary material Supplementary data are available at JXB online. Supplementary Table S1. Primers used in this work. Supplementary Table S2. Annotation of Brachypodium and rice genes. Supplementary Fig. S1. Phylogenetic trees of genes selected for real-time PCR analysis. Acknowledgements This work was supported by the Ministerio de Ciencia e Innovación (grant no. EUI2008-03693). 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Journal

Journal of Experimental Botany – Oxford University Press

Published: Mar 1, 2013

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