TY - JOUR
AU - Suh, Mi Chung
AB - Abstract Cuticular waxes are synthesized by the extensive export of intracellular lipids from epidermal cells. However, it is still not known how hydrophobic cuticular lipids are exported to the plant surface through the hydrophilic cell wall. The LTPG2 gene was isolated based on Arabidopsis microarray analysis; this gene is predominantly expressed in stem epidermal peels as compared with in stems. The expression of LTPG2 transcripts was observed in various organs, including stem epidermis and silique walls. The composition of the cuticular wax was significantly altered in the stems and siliques of the ltpg2 mutant and ltpg1 ltpg2 double mutant. In particular, the reduced level of the C29 alkane, which is the major component of cuticular waxes in ltpg1 ltpg2 stems and siliques, was similar to the sum of reduced values of either parent. The total cuticular wax load was reduced by approximately 13% and 20% in both ltpg2 and ltpg1 ltpg2 siliques, respectively, and by approximately 14% in ltpg1 ltpg2 stems when compared with the wild-type. Similarly, severe alterations in the cuticular layer structure of epidermal cells of ltpg2 and ltpg1 ltpg2 stems and silique walls were observed. In tobacco epidermal cells, intracellular trafficking of the fluorescent LTPG/LTPG1 and LTPG2 to the plasma membrane was prevented by a dominant-negative mutant form of ADP-ribosylation factor 1, ARF1(T31N). Taken together, these results indicate that LTPG2 is functionally overlapped with LTPG/LTPG1 during cuticular wax export or accumulation and LTPG/LTPG1 and LTPG2 are targeted to the plasma membrane via the vesicular trafficking system. Introduction Plant surfaces are covered by a lipophilic cuticle layer that protects them from environmental stresses, including drought, cold, exposure to UV light and pathogen attack (Pollard et al. 2008). It has been suggested that the biosynthesis of cuticular lipids occurs exclusively within epidermal cells. Approximately 60% (in mass) of total lipids synthesized in stem epidermal cells are exported to the plant surface and accumulate as extracellular lipids, such as cuticular waxes and cutin polyesters in Arabidopsis (Suh et al. 2005). However, it is still not known how hydrophobic cuticular lipids are exported to the plant surface through the hydrophilic cell wall layer. Cuticular waxes consist of very-long-chain fatty acids (VLCFAs, C20–C34) and their derivatives, alkanes, aldehydes, primary and secondary alcohols, ketones, and wax esters. C16 and C18 fatty acids are synthesized in the plastids, followed by elongation to VLCFAs in the range of 20–34 carbons via the fatty acid elongase complex in the endoplasmic reticulum (ER) (Dietrich et al. 2005, Fiebig et al. 2000, Hooker et al. 2002, Lee et al. 2009b, Millar and Kunst 1997, Rowland et al. 2006, Todd et al. 1999, Yephremov et al. 1999, Zheng et al. 2005). The synthesized VLCFAs are further modified by a series of enzymes that are involved in the decarbonylation pathway and acyl reduction pathway in the ER (Aarts et al. 1995, Kunst and Samuels 2003, Rowland et al. 2006). The movement of wax precursors from the ER to the plasma membrane is known to be processed by vesicular or non-vesicular trafficking in Arabidopsis (McFarlane et al. 2010). The transport of the synthesized wax molecules onto the epidermal surface is mediated via adenosine triphosphate binding cassette (ABC) transporters in the plasma membrane of the epidermis (Bird et al. 2007). Recently, ABCG11/WBC11 and ABCG12/CER5 heterodimers were shown to be likely involved in wax export, which provided valuable information on the mechanisms of cuticular wax transport in Arabidopsis (McFarlane et al. 2010, Panikashvili et al. 2010). One hypothesis for the cuticular lipid transport from the plasma membrane to the plant surface is the involvement of lipid transfer proteins (LTPs), which are located in the extracellular space (Pyee and Kolattududy 1995, Somerville et al. 2000). Because plant LTPs contain a hydrophobic pocket that is capable of accommodating fatty acids or lysophospholipid molecules, they are able to transfer phospholipids between membranes in vitro (Kader 1996). Recently, disruption of Arabidopsis glycosylphosphatidylinositol (GPI)-anchored LTP (LTPG/LTPG1) was shown to alter the ultrastructure of the cuticle layer of the stem epidermis and reduction of C29 alkane, which is a major component of cuticular waxes in the stems and siliques, but no significant alteration of cutin monomer composition and amount was observed (Lee et al. 2009a). In addition, Debono et al. (2009) found that the total wax load was decreased in knock-down mutants with decreased LTPG/LTPG1 expression. LTPG/LTPG1, which displays lipid-binding activity, is localized to the plasma membrane of the epidermis in the growing regions of inflorescence stems, where cuticular lipids are actively secreted. These combined results indicate that LTPG/LTPG1 directly or indirectly contributes to cuticular wax export or accumulation. GPI-anchored proteins (GAPs) are known to be attached to the external surface of the plasma membrane via a GPI anchor. GPI anchoring is catalyzed in the ER lumen by GPI transamidase through the removal of the N-terminal ER targeting sequence and the C-terminal GPI signal sequence, and the subsequent addition of a core structure comprising ethanolamine phosphate, trimannoside, glucosamine and inositol phospholipid. The glycan and lipid moieties of protein-bound GPIs are further modified in the secretory pathway. In yeast, the remodeling of a GPI’s lipid moiety, which involves the removal of the shorter acyl chain at the sn-2 position and the introduction of either a C26:0 fatty acid or ceramide consisting of C18:0 phytosphingosine and a hydroxyl-C26:0 fatty acid (Orlean and Menon 2007), is required for the transport of GAPs from the ER to the Golgi and their interaction with membrane lipids in lipid rafts or microdomains, where cholesterols and sphingolipids are abundant. Finally, the remodeled GAPs are sorted into coat protein complex II-coated vesicles that move from the ER to the Golgi (Fujita et al. 2011). Liu et al. (2006) reported that the ADP-ribosylation factor (ARF)-like protein is involved in the transport of GPI-anchored plasma membrane-resident proteins from the Golgi to the plasma membrane. Plasma membrane-localized GAPs are known to be internalized by dynamin-dependent pathways and return to the plasma membrane via recycling endosomes (Kumari and Mayor 2008). Arabidopsis harbors >20 LTPG proteins (Borner et al. 2003). Based on the transcriptome analysis of Arabidopsis stems and stem epidermal cells (Suh et al. 2005), seven LTP candidates, including LTPG1 (At1g27950), that might play a role in wax and/or cutin monomer transport were isolated. Although disruption of LTPG1 altered the cuticular wax composition and reduced the total stem wax loads (Debono et al. 2009), >70% of total stem wax loads were still secreted onto the plant surface, suggesting that other LTPGs might be involved in cuticular wax export. In this study, LTPG2 was characterized in regard to cuticular lipid accumulation or export. In addition, the functional relationship between LTPG/LTPG1 and LTPG2 was investigated using ltpg1 ltpg2 double-knockout mutants. LTPG/LTPG1 and LTPG2 were identified to be localized to the plasma membrane via vesicular trafficking, similar to other GAPs. Results Isolation of the LTPG2 gene encoding GPI-anchored LTP LTPG2, a homolog of LTPG/LTPG1, was isolated based on the mRNA expression pattern in microarray analysis of Arabidopsis stem and stem epidermal peels (Suh et al. 2005) and its protein structure similarity with LTPG/LTPG1 (Debono et al. 2009, Lee et al. 2009a). LTPG2 (At3g43720) contains three different domains, which correspond to a signal peptide domain, a lipid transfer domain and a transmembrane domain (Fig. 1A). The signal peptide domain, which was predicted to be required for protein secretion, is composed of 18 amino acid residues (5–22 amino acid residues) at the N-terminal end. The lipid transfer domain contains eight highly conserved cysteine residues at positions 38, 48, 67, 68, 81, 83, 110 and 120. Finally, the C-terminal end (170–192 amino acid residues) was predicted to encode a transmembrane domain. According to the prediction of the potential C-terminal GPI-modification site (http://mendel.imp.ac.at/gpi/gpi_server.html, Eisenhaber et al. 2000), the sequence position of the omega site is a serine at position 170. The transmembrane domain of LTPG2 was predicted to be cleaved and anchored with GPI in the ER and then the modified LTPG2 is targeted to the plasma membrane (Fig. 1B). Fig. 1 View largeDownload slide Description of LTPG protein. (A) Schematic representation of the LTPG2 domain. SP, signal peptide. GPID, GPI-anchored domain. The numbers indicate amino acid sequences of the LTPG2 protein. (B) Amino acid sequences of the LTPG2 protein. The thin underline and thick underline indicate the signal peptide domain and the transmembrane domain, respectively. Asterisks are the conserved Cys residues. (C) RT–PCR analysis of LTPG2 transcripts in stems and stem epidermal peels. The ACTIN2 gene was used to determine the quantity and quality of the cDNAs. (D) Quantitative real-time RT–PCR of LTPG2 transcripts in stems and stem epidermal peels. The relative transcript abundance of eIF4a-1 and PP2A in each sample was determined and used to normalize for differences in cDNAs. Fig. 1 View largeDownload slide Description of LTPG protein. (A) Schematic representation of the LTPG2 domain. SP, signal peptide. GPID, GPI-anchored domain. The numbers indicate amino acid sequences of the LTPG2 protein. (B) Amino acid sequences of the LTPG2 protein. The thin underline and thick underline indicate the signal peptide domain and the transmembrane domain, respectively. Asterisks are the conserved Cys residues. (C) RT–PCR analysis of LTPG2 transcripts in stems and stem epidermal peels. The ACTIN2 gene was used to determine the quantity and quality of the cDNAs. (D) Quantitative real-time RT–PCR of LTPG2 transcripts in stems and stem epidermal peels. The relative transcript abundance of eIF4a-1 and PP2A in each sample was determined and used to normalize for differences in cDNAs. To investigate whether the LTPG2 gene also exhibits a higher expression level in stem epidermal peels than in stems, RT–PCR and quantitative real-time RT–PCR analyses were performed. As has been observed for LTPG/LTPG1, the expression levels of the LTPG2 transcripts were approximately 5-fold higher in the stem epidermal peels than in the stems (Fig. 1C, D). This result was consistent with the microarray results of Arabidopsis stem and stem epidermal peels (Suh et al. 2005). Isolation of ltpg2 and ltpg1 ltpg2 knockout mutants To investigate the in planta role of the LTPG2 gene, T-DNA-inserted ltpg2-1 (SALK_016947), ltpg2-2 (SALK_100010) and ltpg2-3 (SALK_023228) Arabidopsis mutants were obtained from the Arabidopsis Biological Resource Center (http://www.arabidopsis.org). The ltpg2-1, ltpg2-2 and ltpg2-3 alleles contain T-DNA insertions in the first exon, the third exon and the second intron of At3g43720, at positions +320, +1522 and +1388 bp relative to the transcriptional initiation site, respectively (Fig. 2A). No phenotypic alterations were visible during growth and development of all single mutants under normal growing conditions. To further investigate the functional relationships between LTPG/LTPG1 and LTPG2 in planta, double mutants were created by crossing ltpg2-1, ltpg2-2 and ltpg2-3 with null ltpg1 (Fig. 2A) (Lee et al. 2009a). Fig. 2 View largeDownload slide Isolation of T-DNA-inserted ltpg1, ltpg2 and ltpg1 ltpg2 mutants. (A) Genomic organization of T-DNA-inserted LTPG1 and LTPG2 genes. Gray boxes, black boxes and white boxes indicate untranslated region (UTR), exon and intron, respectively. The positions of T-DNA insertion are represented by triangles and the numbers are indicated as bp downstream of the transcriptional initiation site. (B) RT–PCR analysis of the LTPG1 and LTPG2 transcripts levels in the wild-type and the ltpg1, ltpg2 and ltpg1 ltpg2 mutants. The ACTIN7 gene was used to determine the quantity and quality of the cDNAs. (C) Quantitative RT–PCR analysis of LTPG1 and LTPG2 expression in 6-week-old stem and silique wall of the wild-type and the ltpg1 and ltpg2-1 mutants. The relative transcript abundance of PP2A in each sample was determined and used to normalize the differences in the total RNA amount. Each value represents means ± SE of three replicates. Fig. 2 View largeDownload slide Isolation of T-DNA-inserted ltpg1, ltpg2 and ltpg1 ltpg2 mutants. (A) Genomic organization of T-DNA-inserted LTPG1 and LTPG2 genes. Gray boxes, black boxes and white boxes indicate untranslated region (UTR), exon and intron, respectively. The positions of T-DNA insertion are represented by triangles and the numbers are indicated as bp downstream of the transcriptional initiation site. (B) RT–PCR analysis of the LTPG1 and LTPG2 transcripts levels in the wild-type and the ltpg1, ltpg2 and ltpg1 ltpg2 mutants. The ACTIN7 gene was used to determine the quantity and quality of the cDNAs. (C) Quantitative RT–PCR analysis of LTPG1 and LTPG2 expression in 6-week-old stem and silique wall of the wild-type and the ltpg1 and ltpg2-1 mutants. The relative transcript abundance of PP2A in each sample was determined and used to normalize the differences in the total RNA amount. Each value represents means ± SE of three replicates. We examined the transcript levels of the LTPG/LTPG1 and LTPG2 genes in the single and double mutants. Total RNA was isolated from 3-week-old mature leaves of wild-type, single and double mutants, and then subjected to RT–PCR analysis. LTPG2 transcripts were expressed in the wild-type and ltpg1, while the ltpg2-1, ltpg2-2 and ltpg2-3 mutants did not contain any detectable LTPG2 transcripts. Both LTPG/LTPG1 and LTPG2 transcripts were not detected in all ltpg1 ltpg2 double-mutant alleles (Fig. 2B). When the levels of LTPG1 and LTPG2 transcripts in wild-type, ltpg1 and ltpg2-1 stems and silique walls were analyzed by quantitative RT–PCR, the level of the LTPG1 transcripts was increased by approximately 1.8-fold in the ltpg2-1 stem, but decreased by approximately 2-fold in ltpg2-1 silique walls compared with the wild-type (Fig. 2C). However, the levels of LTPG2 transcripts in ltpg1 stem and silique walls were not significantly changed relative to the wild-type level (Fig. 2C). LTPG1 and LTPG2 are associated with cuticular wax accumulation To investigate the role of LTPG2 in cuticular wax export or accumulation and its functional relationship with LTPG/LTPG1, the cuticular waxes extracted from the stems, siliques and leaves of the wild-type and the ltpg1, ltpg2 and ltpg1 ltpg2 mutants were analyzed by using gas chromatography (GC) and GC–mass spectrometry (MS). The amount of C29 alkane (nonacosane), the major component of cuticular waxes in the stems and siliques, was reduced in all three ltpg2 knockout mutants (Supplementary Fig. S1). The amount of C29 alkane was decreased by 4.4% in ltpg2-1 stems and 19.6% in ltpg2-1 siliques when compared with the wild-type (Fig. 3A, B). The decreased proportion in ltpg1 ltpg2-1 stems and siliques reflects the sum of the reduced C29 alkane content of ltpg1 and ltpg2-1 single mutants, indicating that LTPG/LTPG1 and LTPG2 are functionally overlapped (Fig. 3A, B). No alteration in the C29 alkane content was observed in ltpg2-1 and ltpg1 ltpg2-1 leaves (Fig. 3C). Fig. 3 View largeDownload slide Cuticular wax composition (A–C) and amount (D–F) in the wild-type and the ltpg1, ltpg2-1 and ltpg1 ltpg2-1 mutants. Cuticular waxes were extracted from stems (A, D), siliques (B, E) and leaves (C, F), and analyzed by GC and GC–MS. The right and left panels show the major and minor components in the cuticular waxes in each organ, respectively. Each value is the mean of four independent measurements. Error bars indicate SE. Asterisks (*P < 0.05, **P < 0.01) denote statistical differences with respect to the wild-type using Student’s t-test. Fig. 3 View largeDownload slide Cuticular wax composition (A–C) and amount (D–F) in the wild-type and the ltpg1, ltpg2-1 and ltpg1 ltpg2-1 mutants. Cuticular waxes were extracted from stems (A, D), siliques (B, E) and leaves (C, F), and analyzed by GC and GC–MS. The right and left panels show the major and minor components in the cuticular waxes in each organ, respectively. Each value is the mean of four independent measurements. Error bars indicate SE. Asterisks (*P < 0.05, **P < 0.01) denote statistical differences with respect to the wild-type using Student’s t-test. Interestingly, the amount of C29 secondary alcohol and C29 ketone was increased in ltpg1 stems and siliques, but not significantly altered in ltpg2-1 when compared with the wild-type. The amount of C29 secondary alcohol and C29 ketone in ltpg1 ltpg2-1 stems and siliques was similar to the sum of the changed value of the two single mutants (Fig. 3A, B). In addition, the amount of C28 and C30 aldehydes was not altered in ltpg2-1 stems, but was significantly reduced in ltpg2-1 siliques by approximately 38% and 35.1%, respectively, when compared with the wild-type. The reduced amount of C28 and C30 aldehydes and C27 alkane in ltpg1 ltpg2-1 stems and siliques was similar to the sum of the reduced values of the two single mutants (Fig. 3A, B). A similar reduction pattern in the amount of C28 aldehyde and C30 primary alcohol was observed in both ltpg2-1 and ltpg1 ltpg2-1 leaves (Fig. 3C). The total wax loads were reduced by approximately 14% in ltpg1 ltpg2-1 stems when compared with the wild-type (Fig. 3D). The total wax loads were also decreased in ltpg2-1 (∼13%) and ltpg1 ltpg2-1 (∼20%) siliques compared with in the wild-type (Fig. 3E). No change in the total wax amount was observed in both the ltpg single and double mutant leaves (Fig. 3F). These results indicate that LTPG/LTPG1 and LTPG2 might play a predominant role in different organs. In the analysis of cutin monomers, which are major extracellular lipid components, no significant differences in the total amounts were observed between the ltpg2 mutant and wild-type, and between the ltpg1 ltpg2 mutant and each single mutant. However, the composition of cutin monomers was altered in ltpg2-1 and ltpg1 ltpg2-1 mutants compared with the wild-type (Supplementary Fig. S2). Disruption of LTPG1 and LTPG2 caused alteration of the cuticular layer structure Because the cuticle layer consisted of wax and cutin, changes in the cuticular wax composition and content in the mutants led us to predict that changes in the structure of the cuticular layers occurred. The ultrastructure of the cuticle layer of the stem and silique epidermal cells of the wild-type and the ltpg1, ltpg2-1 and ltpg1 ltpg2-1 mutants was observed using transmission electron microscopy (TEM). In accordance with the previous observation in ltpg1 (Lee et al. 2009a), the cuticular layers of ltpg2 and ltpg1 ltpg2-1 stem and silique epidermal cells were disorganized and more diffused, whereas the wild-type cuticle was thin and continuously electron dense. The thickness of the cuticle layer was increased by 11% and 34.5% in ltpg2-1 and ltpg1 ltpg2-1 stem epidermal cells when compared with the wild-type, respectively (Fig. 4A–H). In silique epidermal cells, the diffusion of the cuticle layers was more severe and the thickness of the cuticle layer was 2-fold greater in ltpg2-1 and 2.5-fold greater in ltpg1 ltpg2-1 than in the wild-type (Fig. 4I–P). Additionally, unusual phenotypes, such as accumulated starch and increased number of plastoglobules (Fig. 4Q–X), were observed in the chloroplasts of the ltpg2-1 and ltpg1 ltpg2-1 silique mesocarp. Fig. 4 View largeDownload slide Ultrastructure of cuticular layers (A–P) and silique mesocarp (Q–X) of the ltpg1, ltpg2-1 and ltpg1 ltpg2-1 mutants. (A–H) Transmission electron micrographs of stem epidermal cells from wild-type (A, E), ltpg1 (B, F), ltpg2-1 (C, G) and ltpg1 ltpg2-1 (D, H). (I–P) Transmission electron micrographs of silique epidermal cells from wild-type (I, M), ltpg1 (J, N), ltpg2-1 (K, O) and ltpg1 ltpg2-1 (L, P). (Q–X) Transmission electron micrographs of chloroplasts in mesocarp of wild-type (Q, U), ltpg1 (R, V), ltpg2-1 (S, W) and ltpg1 ltpg2-1 (T, X) siliques. (E–H), (M–P) and (U–X) are enlarged images of the white boxes in (A–D), (I–L) and (Q–T), respectively. Bars = 2 µm in (A–D, I–L and Q–T) and 200 nm in (E–H, M–P and U–X). Fig. 4 View largeDownload slide Ultrastructure of cuticular layers (A–P) and silique mesocarp (Q–X) of the ltpg1, ltpg2-1 and ltpg1 ltpg2-1 mutants. (A–H) Transmission electron micrographs of stem epidermal cells from wild-type (A, E), ltpg1 (B, F), ltpg2-1 (C, G) and ltpg1 ltpg2-1 (D, H). (I–P) Transmission electron micrographs of silique epidermal cells from wild-type (I, M), ltpg1 (J, N), ltpg2-1 (K, O) and ltpg1 ltpg2-1 (L, P). (Q–X) Transmission electron micrographs of chloroplasts in mesocarp of wild-type (Q, U), ltpg1 (R, V), ltpg2-1 (S, W) and ltpg1 ltpg2-1 (T, X) siliques. (E–H), (M–P) and (U–X) are enlarged images of the white boxes in (A–D), (I–L) and (Q–T), respectively. Bars = 2 µm in (A–D, I–L and Q–T) and 200 nm in (E–H, M–P and U–X). LTPG2 and LTPG1 are targeted to the plasma membrane via intracellular vesicular trafficking According to previous reports (Sherrier et al. 1999, Udenfriend and Kodukula 1995), GAPs are mainly found in the plasma membrane. Also, LTPG/LTPG1 was reported to be localized to the plasma membrane (Debono et al. 2009, Lee et al. 2009a). Because LTPG2 harbors the GPI-anchored domain at its C-terminal end, we hypothesized that LTPG2 is localized to the plasma membrane. To verify the subcellular localization of LTPG2, the enhanced yellow fluorescent protein (EYFP) was inserted between the signal peptide and the LTP domain of the LTPG2 protein (between 37 and 38 amino acid residues, Fig. 5A), and then the resulting constructs were transiently introduced into tobacco epidermal cells via an Agrobacterium infiltration system. LTPG1:monomeric red fluorescent protein (mRFP), which was used as a positive control of plasma membrane localization, was co-infiltrated with LTPG2:EYFP and the fluorescent signals were visualized under a confocal fluorescence microscope. As expected, the LTPG1:mRFP and LTPG2:EYFP signals were perfectly merged in the plasma membrane (Fig. 5B–E). This result shows that LTPG2 is also localized in the plasma membrane, like other GAPs, including LTPG/LTPG1. Fig. 5 View largeDownload slide Subcellular localization (B–E) of LTPG2 and vesicular trafficking (F–I) of LTPG1 and LTPG2 in tobacco epidermal cells. (A) Schematic structures of fluorescent protein-fused LTPG/LTPG1 or LTPG2, ARF1 and dominant-negative ARF1(T31N) proteins. Asterisks indicate threonine to asparagine substitution. SP, signal peptide; GPID, GPI-anchored domain; rbsT, terminal of ribulose 1,5-biphosphate carboxylase/oxygenase small subunit from pea (Pisum sativum); EYFP, enhanced yellow fluorescent protein; mRFP, monomeric red fluorescent protein. (B–E) LTPG2:EYFP is co-localized with LTPG1:mRFP in the plasma membrane. (E) Magnification of inset box of (D). (F–I) LTPG1:mRFP and LTPG2:EYFP are co-infiltrated with ARF1 or ARF1(T31N) in tobacco epidermal cells and visualized under the confocal microscope. Arrows indicate punctate stains inside cells. Bars = 20 µm. Fig. 5 View largeDownload slide Subcellular localization (B–E) of LTPG2 and vesicular trafficking (F–I) of LTPG1 and LTPG2 in tobacco epidermal cells. (A) Schematic structures of fluorescent protein-fused LTPG/LTPG1 or LTPG2, ARF1 and dominant-negative ARF1(T31N) proteins. Asterisks indicate threonine to asparagine substitution. SP, signal peptide; GPID, GPI-anchored domain; rbsT, terminal of ribulose 1,5-biphosphate carboxylase/oxygenase small subunit from pea (Pisum sativum); EYFP, enhanced yellow fluorescent protein; mRFP, monomeric red fluorescent protein. (B–E) LTPG2:EYFP is co-localized with LTPG1:mRFP in the plasma membrane. (E) Magnification of inset box of (D). (F–I) LTPG1:mRFP and LTPG2:EYFP are co-infiltrated with ARF1 or ARF1(T31N) in tobacco epidermal cells and visualized under the confocal microscope. Arrows indicate punctate stains inside cells. Bars = 20 µm. The GAPs are sorted from the ER to the plasma membrane by an intracellular secretory pathway and vice versa by endocytic pathways (Mayor and Riezman 2004). To assess the intracellular trafficking of LTPG/LTPG1 and LTPG2, LTPG1:EYFP or LTPG2:EYFP was co-expressed with either the ARF, which plays a role in the intracellular trafficking of cargo proteins, or the GDP-binding dominant-negative mutant form, ARF1(T31N) of ARF1, which disrupts vesicular trafficking (Lee et al. 2002). The fluorescent signals from LTPG1:EYFP and LTPG2:EYFP in the co-expression of ARF1 were visualized in the plasma membrane (Fig. 5F, H). The signals were present in the intracellular space in the co-expression of ARF1(T31N) (Fig. 5G, I). Therefore, expression of ARF1(T31N) inhibited the intracellular trafficking of LTPG/LTPG1 and LTPG2, suggesting that LTPG1 and LTPG2 were targeted to the plasma membrane via the vesicular trafficking system. LTPG2 transcripts are expressed in various organs including stem epidermis and silique wall Total RNA was isolated from various organs, including the roots, young seedlings, rosette leaves, cauline leaves, stems, flowers and siliques, and then subjected to RT–PCR analysis to assess the expression of the LTPG2 transcripts. The LTPG2 transcripts were abundantly detected in all organs tested (Fig. 6A). Because the total cuticular wax content was significantly reduced in siliques of the ltpg2 mutant, quantitative real-time RT–PCR was also carried out to precisely compare the spatial expression level of LTPG2. The expression level of LTPG2 transcripts was much higher in the silique walls as well as the stem epidermal peels than in other tissues (Fig. 6B). When the levels of the LTPG/LTPG1 and LTPG2 transcripts were compared in the silique walls, they were found to be similar (Supplementary Fig. S3). Fig. 6 View largeDownload slide Spatial and temporal expression analysis of Arabidopsis LTPG2 in various organs. (A) RT–PCR analysis of LTPG2 in various Arabidopsis organs. ACTIN7 was used to determine the quantity and quality of the cDNAs. Ro, root; YS, 10-day-old seedling; RL, rosette leaf; CL, cauline leaf; St, stem; OF, open flower; Si, silique. (B) Quantitative RT–PCR analysis of LTPG2 in various Arabidopsis organs. The relative transcript abundance of eIF4a-1 and PP2A in each sample was determined and used to normalize the differences in the total RNA amount. Each value represents the means ± SE of three replicates. Ro, root; RL, rosette leaf; CL, cauline leaf; Bu, bud; OF, open flower; Si, silique; SW, silique wall; Ep, epidermal peels; St, stem. (C–N) Histochemical analysis of LTPG2 promoter:GUS expression in transgenic Arabidopsis. (C) Ten-day-old young seedling, (D) 3-week-old mature leaf, (E) 5-week-old stem, (F) cross section of (D), (G) cross section of (E), (H) open flower, (I) carpel, (J) stamen, (K) silique wall, (L) seeds, (M) sepal and (N) petal. Arrows in (C) indicate high magnification of the primary root tip (1) and lateral root tip (2); the arrow in D indicates a trichome (1). Fig. 6 View largeDownload slide Spatial and temporal expression analysis of Arabidopsis LTPG2 in various organs. (A) RT–PCR analysis of LTPG2 in various Arabidopsis organs. ACTIN7 was used to determine the quantity and quality of the cDNAs. Ro, root; YS, 10-day-old seedling; RL, rosette leaf; CL, cauline leaf; St, stem; OF, open flower; Si, silique. (B) Quantitative RT–PCR analysis of LTPG2 in various Arabidopsis organs. The relative transcript abundance of eIF4a-1 and PP2A in each sample was determined and used to normalize the differences in the total RNA amount. Each value represents the means ± SE of three replicates. Ro, root; RL, rosette leaf; CL, cauline leaf; Bu, bud; OF, open flower; Si, silique; SW, silique wall; Ep, epidermal peels; St, stem. (C–N) Histochemical analysis of LTPG2 promoter:GUS expression in transgenic Arabidopsis. (C) Ten-day-old young seedling, (D) 3-week-old mature leaf, (E) 5-week-old stem, (F) cross section of (D), (G) cross section of (E), (H) open flower, (I) carpel, (J) stamen, (K) silique wall, (L) seeds, (M) sepal and (N) petal. Arrows in (C) indicate high magnification of the primary root tip (1) and lateral root tip (2); the arrow in D indicates a trichome (1). The amount of cuticular wax was shown to be increased by treatment with salt and drought stresses (Kosma et al. 2009). In order to evaluate whether the expression of LTPG2 is regulated by abiotic stresses and abscisic acid (ABA), 10-day-old seedlings were incubated in Murashige and Skoog (MS) medium supplemented with 200 mM NaCl, 200 mM mannitol or 100 µM ABA, or exposed to dehydration stress. The RD29A gene (At5g52310), which is known to be a drought stress-inducible gene, was used as a control for the water-deficit response. Expression of the RD29A gene increased significantly in response to the treatments, whereas the level of the LTPG2 transcripts was not affected by salt and dehydration stresses or ABA (Supplementary Fig. S4). The spatial and temporal expression of the LTPG2 gene was further evaluated by introducing the β-d-glucuronidase (GUS) gene under the control of the LTPG2 promoter into Arabidopsis. Various organs from five independent transgenic lines were then stained for GUS expression. Strong GUS activity was observed in the aerial portions and root tips of the 10-day-old seedlings. When the stem and leaf tissues were cross-sectioned, the GUS gene was found to be expressed in the stem and leaf epidermis, including the trichomes, leaf mesophyll cells, and stem cortex and xylem. In addition, GUS expression was observed in the upper portion of the styles, anther filament, and veins of the sepals and petals. Furthermore, the silique walls and developing seeds were strongly stained (Fig. 6C–N). Discussion Terrestrial plants have evolved from aquatic ancestors via the development of a cuticle, which is composed of hydrophobic cutin and wax to prevent non-stomatal water loss. Although the biosynthetic pathway of cuticular lipids has been well characterized, the mechanism for the export of cuticular lipids to the plant surface through the hydrophilic cell wall layer still has not been investigated. Arabidopsis harbors 26 LTPG isoforms. Among them, the expression levels of four isoforms (LTPG/LTPG1, LTPG2, At1g55260 and At1g62790) are >2-fold higher in stem epidermal peels than in stems (Suh et al. 2005), indicating that these isoforms may be involved in the transport of cuticular wax precursors. In addition, the expression levels of two LTPG/LTPG1 and LTPG2 isoforms are approximately 3-fold higher in stem epidermal peels than those of the two other LTPG isoforms (Suh et al. 2005). Recently, LTPG/LTPG1 was reported to be involved in cuticular wax export or accumulation either directly or indirectly (Debono et al. 2009, Lee et al. 2009a). In this study, the following properties of LTPG2, which is a homolog of LTPG/LTPG1, were demonstrated. (i) The LTPG2 gene was strongly expressed in the epidermal peels of stems and silique walls, where cuticular wax biosynthesis occurs extensively. (ii) Both LTPG/LTPG1 and LTPG2 are targeted to the plasma membrane via the vesicular trafficking system. (iii) Disruption of the LTPG2 gene causes significant alterations in the cuticular wax composition and the cuticular layer structure in stems and siliques, which contain the largest amount of cuticular wax in Arabidopsis. Unlike LTPG/LTPG1, a reduction in the cuticular wax load was prominent in ltpg2 siliques, suggesting that LTPG2 might be more important to the export or accumulation of cuticular wax in siliques than in other organs. (iv) Further reduction of the total cuticular wax loads and more severe alterations of the cuticular layer were observed in the ltpg/ltpg1 ltpg2 mutant compared with each single mutant, which indicates that LTPG2 is functionally overlapped with LTPG/LTPG1 in cuticular wax export or accumulation. Previous reports have provided direct evidence that LTPG/LTPG1 is involved in a GAP family. For example, LTPG/LTPG1 was released from the Triton X-114 detergent-rich fraction of Arabidopsis callus cells when the GPI-anchoring of LTPG/LTPG1 was cleaved by treatments with phosphatidylinositol-specific phospholipase C (Borner et al. 2003). LTPG/LTPG1 was also observed in the fractions released from Arabidopsis membrane proteins after treatment with phospholipase D (Elortza et al. 2006). Furthermore, LTPG/LTPG1 was localized in the plasma membrane, but the fluorescent signals of the LTPG1Δ48:RFP were restricted in the ER of Arabidopsis protoplasts when the C-terminal hydrophobic region of LTPG1 required for the GPI-anchoring was deleted (Lee et al. 2009a). However, indirect evidence that LTPG2 could also be involved in a GAP family was provided in this study. LTPG2 contains common structural features of GAPs, such as the presence of a cleavable N-terminal hydrophobic secretion signal and a C-terminal hydrophobic region required for GPI anchoring (Orlean and Menon 2007). Additionally, the fluorescent signals of LTPG1:mRFP and LTPG2:EYFP were merged in the plasma membrane, but vesicular trafficking of LTPG2:EYFP was inhibited from the ER to the plasma membrane when LTPG2:EYFP and ARF1(T31N) were co-expressed in tobacco epidermal cells, indicating that LTPG2 is a member of a GAP family. This is supported by the fact that GAPs are known to be targeted from the ER to the plasma membrane through vesicular trafficking (Fujita et al. 2011, Mayor and Riezman 2004). According to Debono et al. (2009), LTPG/LTPG1 is highly expressed in Arabidopsis stem epidermis, where cuticular wax biosynthesis extensively occurs. The total wax load was significantly reduced on the surface of ltpg knock-down mutant stems. The cuticular wax-deficient phenotype was also observed in ltpg2 silique walls (Fig. 3, Supplementary Fig. S1), which is consistent with the predominant expression of LTPG2 gene in silique walls and a decrease in the transcript levels of LTPG1 gene in ltpg2-1 silique walls. The amount of cuticular wax was further decreased in ltpg1 ltpg2 mutants compared with either parent, indicating that LTPG/LTPG1 and LTPG2 are functionally overlapped in cuticular wax export or accumulation. In comparisons of the composition of cuticular waxes, the largest reduction of C29 alkane was observed in all ltpg, ltpg1 and ltpg2 mutants (Debono et al. 2009, Lee et al. 2009a). The reduced content was overcome by increases of the C29 secondary alcohols and C29 ketone wax loads in the ltpg1 mutant, but not in the ltpg and ltpg2 mutants. The levels of C24 and C26 fatty acids and C27 alkane in ltpg1 stems, and the level of C27 alkane in ltpg2 stems were significantly different from those in the wild-type, but no other wax components except C29 alkane were significantly altered in ltpg stems compared with the wild-type. In addition to the altered chemical phenotypes of ltpg2 and ltpg1 ltpg2 mutants, significant ultrastructural alterations in the cuticular layer in the mutant stem or silique epidermal cells were observed. Abnormalities in cuticle morphology have been frequently observed in Arabidopsis mutants, such as cer8/lacs1, lacs2, wax2/cer3, BODYGUARD (bdg) and gpat4 gpat8, which are defective in cutin and wax biosynthesis (Chen et al. 2003, Kurdyukov et al. 2006, Li et al. 2007, Lu et al. 2009, Rowland et al. 2007). Although the fine structure of the cutin polymer matrix with intracuticular waxes embedded in the matrix is still poorly understood, minor changes in the composition of cutin monomers (Supplementary Fig. S2) might cause alterations in the ultrastructure of the cuticular layer of ltpg2 and ltpg1 ltpg2 mutants, based on the fact that cutin polymer does not dissolve in the solvents used in tissue preparation for TEM. In addition, the physical and chemical properties of the altered intracuticular wax components in the ltpg2 and ltpg1 ltpg2 mutants might affect interaction with the cutin polymer in the matrix. Under drought stress conditions, the cuticular wax content in Arabidopsis significantly increases (Kosma et al. 2009), which is caused by transcriptional activation of genes involved in cuticular wax biosynthesis via the drought stress-inducible MYB96 transcription factor (Seo et al. 2011). Therefore, plants under drought stress are likely to experience an increase in wax export to efficiently transport the increased cuticular waxes. However, the LTPG/LTPG1 and LTPG2 genes were not induced under water-deficit conditions, which indicates that these genes might not be involved in elevated wax export or accumulation caused by drought stress. However, we could not exclude the possibility that the LTPG/LTPG1 and LTPG2 genes in elevated wax export or accumulation might be controlled at the post-transcriptional, translational or post-translational levels. The intracellular wax transport processes from the ER to the plasma membrane are not clear, but two hypothetical routes have been suggested: Golgi-mediated vesicular trafficking through the secretory pathway and direct molecular transfer at ER–plasma membrane contact sites (Levine and Loewen 2006, Samuels et al. 2008). According to a previous report (McFarlane et al. 2010), cuticular waxes that are not exported in abcg11 or abcg12 mutants accumulated in ER-derived inclusions, whereas the Golgi and plasma membrane morphology was unaffected in abcg11 mutants. These results suggest that secretion of cuticular wax from the ER to the plasma membrane is processed via non-vesicular lipid traffic, which is similar to the yeast lipid transport mechanism (Levine and Loewen 2006). However, LTPG1 and LTPG2 were identified to be targeted from the ER to the plasma membrane via vesicular trafficking in this study. Therefore, the functional mechanism of LTPG1 and LTPG2 in the transport of cuticular wax precursors needs to be further investigated. In this study, we attempted to better understand the role of the LTP domain in LTPG/LTPG1 and LTPG2 proteins in relation to wax export or accumulation. The LTP domain harbors eight highly conserved cysteine residues that contribute to the four disulfide bonds, which are essential for the formation of the lipid-binding cavity (Kader 1996). The xylogen ZeXYP1, harboring the non-specific LTP domain purified from a Zinnia elegans cell culture, was reported to bind specifically to stigmasterol (Motose et al. 2004). According to a previous report (Debono et al. 2009), the recombinant LTPG was shown to have the capacity to bind with the lipid probe, 2-p-toluidinonaphthalen-6-sulfonate. Therefore, it remains to be investigated whether LTPG2 can bind and transport specific or diverse wax precursors. In summary, characterization of the T-DNA-inserted ltpg2 and ltpg/ltpg1 ltpg2 mutants demonstrates that LTPG2 contributes to cuticular wax export or accumulation either directly or indirectly in Arabidopsis, and LTPG/LTPG1 and LTPG2 are functionally overlapped in the cuticular wax transport or accumulation. Materials and Methods Plant materials and growth conditions All Arabidopsis ecotype backgrounds used in this study are Columbia (Col-0). The T-DNA-tagged mutants were obtained from the Arabidopsis Biological Resource Center (http://www.arabidopsis.org). T-DNA homozygous lines were identified by using genotyping PCR. After seeds were imbibed at 4°C for 3 d, seed surfaces were sterilized in 70% (v/v) ethanol for 1 min and 20% (v/v) bleach for 5 min, followed by five washes with sterilized water. The sterilized seeds were aseptically germinated on 0.5 × MS medium supplemented with 1% (w/v) sucrose and 0.7% (w/v) agar. After 7 d, seedlings were transplanted to soil under the long day condition (16 h/8 h, light/dark) at 22°C. The double mutants were created by crossing each T-DNA homozygous line. Gene expression analysis In order to confirm the expression of LTPG2 from various Arabidopsis tissues, total RNA was isolated using the TRIzol reagent (Invitrogen) or a Nucleospin® RNA Plant Extraction Kit (Macherey-Nagel). Reverse transcription was conducted following the manufacturer’s protocols (Promega). PCR was then performed using gene-specific primers (Supplementary Table S1). For the quantitative analysis of RNA transcript levels, KAPA SYBR FAST qRT-PCR kit (KAPA Biosystem) and C1000™ thermal cycler (Bio-Rad) were utilized according to the manufacturer’s instructions. The total RNA from leaves was extracted for isolation of the T-DNA mutants and then RT–PCR was performed using gene-specific primers (Supplementary Table S1). GUS expression assay To isolate the promoter region of LTPG2, a region upstream (1152 bp) from the translation start site was amplified from Arabidopsis genomic DNA using a specific primer containing HindIII and SalI restriction enzyme sites. The specific primers are described in Supplementary Table S1. The DNA fragment of the LTPG2 promoter was inserted into the pBI101 binary vector and then this construct was introduced into the wild-type by Agrobacterium tumefaciens-mediated transformation using the vacuum infiltration method described by Bechtold et al. (1993). Seeds that had been bulk harvested from each pot were then sterilized and germinated on 0.5 × MS agar medium supplemented with 50 µg ml−1 kanamycin and 100 µg ml−1 carbenicillin. The surviving T1 seedlings were subsequently transplanted into soil and used for further analyses. Young seedlings, rosette leaves, stems, flowers and developing siliques were incubated in GUS staining solution [100 mM sodium phosphate (pH 7.0), 1 mM 5-bromo-4-chloro-3-indolyl-β-d-glucuronide, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, 10 mM Na2EDTA and 0.1% (v/v) Triton X-100] at 37°C for 16 h. The chlorophyll of stained tissues was then removed using graded ethanol. Images were acquired using a Leica L2 microscope (Leica). To visualize the cross sections of stems and leaves, dehydrated samples were embedded in acrylic resin (LR White Resin, London Resin Company) and then 15–20-µm-thick sections were cut using a MT990 microtome (RMC). The tissue sections were observed and photographed under light microscopy (Leica L2). Subcellular localization and vesicular trafficking assays To produce a LTPG/LTPG1 coding sequence (CDS) fused with mRFP and a LTPG2 CDS fused with EYFP (LTPG2:EYFP), a triple template PCR was performed following the method described by Tian et al. (2004). The ARF1 and ARF1(T31N) CDS were amplified using specific primers, ARF1-F and ARF1-R, from former transient constructs (Lee et al. 2002). The LTPG1:mRFP and LTPG2:EYFP or ARF1 and ARF1(T31N) fragments were inserted into the pFAST vector (Lee et al. 2009a) through digestion with SmaI/BamHI or SacI/SacI restriction enzymes. The LTPG1:mRFP, LTPG2:EYFP, ARF1 and ARF1(T31N) binary vectors were introduced into the A. tumefaciens strain GV3101 by the freeze–thaw method (An 1987). To investigate the localization of LTPG/LTPG1 and LTPG2, Agrobacterium harboring the LTPG2:EYFP or LTPG1:mRFP vectors were co-injected at a concentration of OD600 = 0.8 in the abaxial side of Nicotiana benthamiana leaves. To examine trafficking in plant cells, Agrobacterium harboring the LTPG1:EYFP or LTPG2:EYFP vectors were co-infected with Agrobacterium harboring the ARF1 or ARF1(T31N) vectors in the abaxial side of N. benthamiana leaves. YFP and RFP fluorescence were analyzed using a TCS SP5 AOBS/tandem laser confocal scanning microscope (Leica) at 36–48 h after injection. For YFP and RFP, the excitation wavelengths were 514 and 561 nm, respectively, and the emitted fluorescence was collected at 518–553 and 573–616 nm, respectively. Analysis of cuticular wax The cuticular waxes were extracted by immersing 15-cm-inflorescence stems or leaves in 5 ml of chloroform at room temperature for 30 s. n-Octacosane, docosanoic acid and 1-tricosanol were added as internal standards. The extracts were then dried by heating at 40°C under a gentle stream of nitrogen. The wax extracts were dissolved in bis-N,N-trimethylsilyl trifluoroacetamide (Sigma):pyridine (1:1, v/v) and then all of the hydroxyl-containing compounds were transformed into their corresponding trimethylsilyl derivatives at 90°C for 30 min. The samples were evaporated under nitrogen and dissolved in heptane:toluene (1:1, v/v). The qualitative composition was then evaluated by capillary GC–MS (GCMS-QP2010, Shimadzu, Japan; column 60 m HP-5, 0.32 mm i.d., df = 0.25 µm, Agilent) using a He carrier gas inlet pressure of 1.0 ml min−1 and a mass spectrometric detector (GCMS-QP2010, Shimadzu). The GC–MS protocol was as follows: injection at 220°C, maintenance of the temperature at 220°C for 4.5 min followed by an increase to 290°C at a rate of 3°C min−1. The temperature was maintained at 290°C for 10 min, after which it was raised to 300°C at a rate of 2°C min−1 and held for 10 min. Quantitative analysis of the mixtures was performed using capillary GC with a flame ionization detector under the same GC conditions described above. Single compounds were quantified against the internal standard by automatically integrating the peak areas. TEM analysis The upper part of 10-cm-inflorescence stems from 4- to 5-week-old plants was used for TEM analysis. Arabidopsis wild-type and ltpg1, ltpg2 and ltpg1 ltpg2 mutant stems and siliques were fixed in a fixation solution [2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M sodium carcodylate buffer (pH 7.4)] for 16 h at 4°C. They were then rinsed in 0.1 M sodium carcodylate buffer (pH 7.4) and further fixed in 1% (w/v) osmium tetroxide (OsO4) for 16 h at 4°C. After rinsing with 0.1 M sodium carcodylate buffer, the samples were dehydrated with graded ethanol and embedded in LR White Resin (London Resin Company). Thin sections (50–60-nm thicknesses) were then prepared using an ultramicrotome (RMC MT X) and collected on nickel grids (1-GN, 150 mesh). Next, these sections were stained with uranyl acetate and lead citrate, and examined under a transmission electron microscope (Tecnai 12, Philips). Funding This work was supported by grants from the World Class University Project (R31-2009-000-20025-0), the National Research Foundation of Korea and the Next-Generation BioGreen 21 Program (no. PJ008203) and 15 Agenda Program (PJ007441201008), the Rural Development Administration, Republic of Korea. 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TI - Characterization of Glycosylphosphatidylinositol-Anchored Lipid Transfer Protein 2 (LTPG2) and Overlapping Function between LTPG/LTPG1 and LTPG2 in Cuticular Wax Export or Accumulation in Arabidopsis thaliana
JF - Plant and Cell Physiology
DO - 10.1093/pcp/pcs083
DA - 2012-08-11
UR - https://www.deepdyve.com/lp/oxford-university-press/characterization-of-glycosylphosphatidylinositol-anchored-lipid-eHq2PoN20Y
SP - 1391
EP - 1403
VL - 53
IS - 8
DP - DeepDyve
ER -