Cuticular Wax Accumulation Research Articles

Wheat CHD3 protein TaCHR729 regulates the cuticular wax biosynthesis required for stimulating germination of Blumeria graminis f.sp. tritici.

Powdery mildew caused by Blumeria graminis f.sp. tritici (Bgt) is a detrimental disease of bread wheat (Triticum aestivum). Bgt infection initiates with the germination of its conidia, which is stimulated by plant cuticle-derived wax signals. Here, we identified wheat 3-KETOACYL-CoA SYNTHASE (TaKCS6), a homolog of barley HvKCS6, as a key enzyme in the biosynthesis of wheat cuticular wax. We found that both cuticular wax accumulation and Bgt germination were impeded on leaves of TaKCS6-knockdown plants. The TaKCS6 promoter-associated bHLH type transcription factor 1 (TaKPAB1) binds to the TaKCS6 promoters and recruits the CHD3 protein TaCHR729 to them via physical association. Knockdown of TaCHR729 results in decreased trimethylation of histone H3 Lys 4 (H3K4me3) at the TaKCS6 promoters and down-regulation of TaKCS6 transcription, leading to a reduction of cuticular wax accumulation and Bgt germination on leaves. We further identified very-long-chain aldehydes with a chain length above C24 as the signals regulated by the TaCHR729-TaKPAB1-TaKCS6 pathway for stimulating Bgt germination. Our study thus reveals that the transcription factor-mediated recruitment of chromatin remodeling machinery is essential for regulating the biosynthesis of cuticular wax that is required for stimulating Bgt germination in bread wheat.

Isolation and functional identification of an apple MdCER1 gene

The outermost layer of the plant epidermis is covered by cuticular wax, which resists UV radiation, insects, and pathogens, and protects the plant from various environmental stressors. We cloned MdCER1 to further study the molecular regulatory pathway of cuticular wax in apple (Malus × domestica Borkh.). The results showed that MdCER1 is a homolog of Arabidopsis CER1, which is essential for wax synthesis. A subcellular localization study revealed that MdCER1 was localized on the endoplasmic reticulum. We examined the expression pattern of MdCER1 in different tissues and found that it was constitutively expressed in roots, stems, leaves, flowers, and fruits, with the highest expression in leaves. Ectopic expression of MdCER1 promoted the accumulation of cuticular wax by changing the permeability of the epidermis and the response to abscisic acid, as well as by improving drought resistance in Arabidopsis.

Characterization of increased cuticular wax mutant and analysis of genes involved in wax biosynthesis in Dianthus spiculifolius

Cuticular wax formation on the surface of plant leaves is associated with drought-stress tolerance. The identification of wax biosynthesis-related genes will contribute to the genetic improvement of drought resistance in plants. In this study, we characterize a novel Dianthus spiculifolius mutant with increased cuticular wax. The mutant exhibited stronger drought resistance as indicated by less leaf wilting and death, higher leaf relative water content and water retention capacity, and slower water loss and chlorophyll extraction than did the wild type during drought treatment. In the mutant leaves, 2 730 upregulated and 2 151 downregulated differentially expressed genes (DEGs) were identified by transcriptome sequencing. A wax biosynthesis pathway of the identified DEGs was significantly enriched. Finally, three key genes (DsCER1, DsMAH1, and DsWSD1) involved in wax biosynthesis were identified and verified by qPCR. These results suggest that differential expression of DEGs involved in wax biosynthesis may be associated with the increase in cuticular wax in the mutant. Taken together, our results help elucidate wax formation patterns in D. spiculifolius. Furthermore, the DEGs involved in wax biosynthesis identified here may be valuable genetic resources for improving plant stress tolerance through increased accumulation of cuticular wax.

GCN5 contributes to stem cuticular wax biosynthesis by histone acetylation of CER3 in Arabidopsis.

Cuticular wax is a major component of the surface cuticle of plants, which performs crucial functions in optimizing plant growth. Histone acetylation regulates gene expression in diverse biological processes, but its role in cuticular wax synthesis is not well understood. In this study, we observed that mutations of the Arabidopsis thaliana histone acetyltransferase GENERAL CONTROL NON-REPRESSED PROTEIN5 (GCN5) impaired the accumulation of stem cuticular wax. Three target genes of GCN5, ECERIFERUM3 (CER3), CER26, and CER1-LIKE1 (CER1-L1), were identified by RNA-seq and ChIP assays. H3K9/14 acetylation levels at the promoter regions of CER3, CER26, and CER1-L1 were consistently and significantly decreased in the gcn5-2 mutant as compared to the wild-type. Notably, overexpression of CER3 in the gcn5-2 mutant rescued the defect in stem cuticular wax biosynthesis. Collectively, these data demonstrate that GCN5 is involved in stem cuticular wax accumulation by modulating CER3 expression via H3K9/14 acetylation, which underlines the important role of histone acetylation in cuticular wax biosynthesis.

Three Fatty Acyl-Coenzyme A Reductases, BdFAR1, BdFAR2 and BdFAR3, are Involved in Cuticular Wax Primary Alcohol Biosynthesis in Brachypodium distachyon.

Plant cuticular wax is a heterogeneous mixture of very long chain fatty acids (VLCFAs) and their derivatives. Primary alcohols are the dominant wax components throughout leaf development of Brachypodium distachyon (Brachypodium). However, the genes involved in primary alcohol biosynthesis have not been investigated and their exact biological function remains unclear in Brachypodium to date. Here, we monitored the leaf wax profile and crystal morphology during Brachypodium leaf morphogenesis, and isolated three Brachypodium fatty acyl-CoA reductase (FAR) genes, named BdFAR1, BdFAR2 and BdFAR3, then analyzed their biochemical activities, substrate specificities, expression patterns, subcellular localization and stress induction. Transgenic expression of BdFAR genes in yeast (Saccharomyces cerevisiae), tomato (Solanum lycopersicum), Arabidopsis (Arabidopsis thaliana) and Brachypodium increased the production of primary alcohols. The three BdFAR genes were preferentially expressed in Brachypodium aerial tissues, consistent with known sites of wax primary alcohol deposition, and localized in the endoplasmic reticulum (ER) in Arabidopsis protoplasts. Finally, expression of the BdFAR genes was induced by drought, cold and ABA treatments, and drought stress significantly increased cuticular wax accumulation in Brachypodium. Taken together, these results indicate that the three BdFAR genes encode active FARs involved in the biosynthesis of Brachypodium wax primary alcohols and respond to abiotic stresses.

The MIEL1 E3 Ubiquitin Ligase Negatively Regulates Cuticular Wax Biosynthesis in Arabidopsis Stems

Cuticular wax is an important hydrophobic layer that covers the plant aerial surface. Cuticular wax biosynthesis is shaped by multiple layers of regulation. In particular, a pair of R2R3-type MYB transcription factors, MYB96 and MYB30, are known to be the main participants in cuticular wax accumulation. Here, we report that the MYB30-INTERACTING E3 LIGASE 1 (MIEL1) E3 ubiquitin ligase controls the protein stability of the two MYB transcription factors and thereby wax biosynthesis in Arabidopsis. MIEL1-deficient miel1 mutants exhibit increased wax accumulation in stems, with up-regulation of wax biosynthetic genes targeted by MYB96 and MYB30. Genetic analysis reveals that wax accumulation of the miel1 mutant is compromised by myb96 or myb30 mutation, but MYB96 is mainly epistatic to MIEL1, playing a predominant role in cuticular wax deposition. These observations indicate that the MIEL1-MYB96 module is important for balanced cuticular wax biosynthesis in developing inflorescence stems.

Wax Crystal-Sparse Leaf 4, encoding a β-ketoacyl-coenzyme A synthase 6, is involved in rice cuticular wax accumulation.

WSL4 encodes a KCS6 protein which is required for cuticular wax accumulation in rice. Very long chain fatty acids (VLCFAs) are essential precursors for cuticular wax biosynthesis. VLCFA biosynthesis occurs in the endoplasmic reticulum and requires the fatty acid elongase (FAE) complex. The β-ketoacyl-coenzyme A synthase (KCS) catalyzes the first step of FAE-mediated VLCFA elongation. Here we characterized the Wax Crystal-Sparse Leaf 4 (WSL4) gene involved in leaf cuticular wax accumulation in rice. The wsl4 mutant displayed a pleiotropic phenotype including dwarfism, less tiller numbers and reduced surface wax load. Map-based cloning and nucleotide sequencing results revealed that wsl4 carried a single nucleotide substitution in the second exon of a putative KCS6 gene, encoding one subunit of the FAE complex for VLCFAs. Genetic complementation confirmed that the mutation in WSL4 was responsible for the phenotype of wsl4. WSL4 was constitutively expressed in various rice tissues and localized in the endoplasmic reticulum. Both WSL4-RNAi transgenic lines and WSL4 knocked-out mutants exhibited wax-deficient phenotypes similar to the wsl4 mutant. These data indicate that WSL4 is required for cuticular wax accumulation in rice.

Control of Plant Water Use by ABA Induction of Senescence and Dormancy: An Overlooked Lesson from Evolution

Drought stress is a condition that in specific climate contexts results in insufficient water availability and often limits plant productivity through perturbing development and reducing plant growth and survival. Plants use senescence of old leaves and dormancy of buds and seeds to survive extreme environmental conditions. The plant hormone ABA accumulates after drought stress, and increases plant survival by inducing quick responses such as stomatal closure, and long-term responses such as extended growth inhibition, osmotic regulation, accumulation of cuticular wax, senescence, abscission and dormancy. Here we focus on how the long-term ABA responses contribute to plant survival during severe drought stress. Leaf senescence and abscission of older leaves reduce total plant transpirational water loss and increase the transfer of nutrients to meristems and to some storage tissues. Osmotic regulation favors water consumption in sink tissues, and accumulation of cuticular wax helps to seal the plant surface and limits non-stomatal water loss.

The MIEL1 E3 Ubiquitin Ligase Negatively Regulates Cuticular Wax Biosynthesis in Arabidopsis Stems.

Cuticular wax is an important hydrophobic layer that covers the plant aerial surface. Cuticular wax biosynthesis is shaped by multiple layers of regulation. In particular, a pair of R2R3-type MYB transcription factors, MYB96 and MYB30, are known to be the main participants in cuticular wax accumulation. Here, we report that the MYB30-INTERACTING E3 LIGASE 1 (MIEL1) E3 ubiquitin ligase controls the protein stability of the two MYB transcription factors and thereby wax biosynthesis in Arabidopsis. MIEL1-deficient miel1 mutants exhibit increased wax accumulation in stems, with up-regulation of wax biosynthetic genes targeted by MYB96 and MYB30. Genetic analysis reveals that wax accumulation of the miel1 mutant is compromised by myb96 or myb30 mutation, but MYB96 is mainly epistatic to MIEL1, playing a predominant role in cuticular wax deposition. These observations indicate that the MIEL1-MYB96 module is important for balanced cuticular wax biosynthesis in developing inflorescence stems.

Ethephon improved drought tolerance in maize seedlings by modulating cuticular wax biosynthesis and membrane stability

Cuticular wax is the outermost thin hydrophobic layer covering the surface of aerial plant parts, which provides a primary waterproof barrier and protection against different environmental stresses. The aim of the present study was to investigate the role of ethephon, as an ethylene-releasing compound, in counteracting drought stress by modulating cuticular wax biosynthesis, water balance, and antioxidant regulation in maize seedlings. Our results showed that ethephon significantly increased the ethylene evolution rate, regulate the expression of cuticular wax synthesis regulatory gene ZmERE and the wax biosynthetic genes ZmGL1, ZmGL15, ZmFDH1, and ZmFAE1, and promote cuticular wax accumulation in maize seedlings under normal or drought stress conditions. Moreover, ethephon was shown to might markedly reduce water loss and chlorophyll leaching in leaves, and maintain higher relative water content and leaf water potential under drought stress. Ethephon significantly decreased malondialdehyde and hydrogen peroxide concentrations and electrolyte leakage, but increased the accumulation of proline and the activities of SOD, POD, and CAT. In addition, ethephon resulted in an increase in the ratio of root and shoot under drought stress. These results indicated that ethephon could improve maize performance under drought stress by modulating cuticular wax synthesis to maintain water status and membrane stability for plant growth.

Cuticular Wax Accumulation Is Associated with Drought Tolerance in Wheat Near-Isogenic Lines.

Previous studies have shown that wheat grain yield is seriously affected by drought stress, and leaf cuticular wax is reportedly associated with drought tolerance. However, most studies have focused on cuticular wax biosynthesis and model species. The effects of cuticular wax on wheat drought tolerance have rarely been studied. The aims of the current study were to study the effects of leaf cuticular wax on wheat grain yield under drought stress using the above-mentioned wheat NILs and to discuss the possible physiological mechanism of cuticular wax on high grain yield under drought stress. Compared to water-irrigated (WI) conditions, the cuticular wax content (CWC) in glaucous and non-glaucous NILs under drought-stress (DS) conditions both increased; mean increase values were 151.1 and 114.4%, respectively, which was corroborated by scanning electronic microscopy images of large wax particles loaded on the surfaces of flag leaves. The average yield of glaucous NILs was higher than that of non-glaucous NILs under DS conditions in 2014 and 2015; mean values were 7368.37 kg·ha−1 and 7103.51 kg·ha−1. This suggested that glaucous NILs were more drought-tolerant than non-glaucous NILs (P = 0.05), which was supported by the findings of drought tolerance indices TOL and SSI in both years, the relatively high water potential and relative water content, and the low ELWL. Furthermore, the photosynthesis rate (Pn) of glaucous and non-glaucous wheat NILs under DS conditions decreased by 7.5 and 9.8%, respectively; however, glaucous NILs still had higher mean values of Pn than those of non-glaucous NILs, which perhaps resulted in the higher yield of glaucous NILs. This could be explained by the fact that glaucous NILs had a smaller Fv/Fm reduction, a smaller PI reduction and a greater ABS/RC increase than non-glaucous NILs under DS conditions. This is the first report to show that wheat cuticular wax accumulation is associated with drought tolerance. Moreover, the leaf CWC can be an effective selection criterion in the development of drought-tolerant wheat cultivars.

Kelch-motif containing acyl-CoA binding proteins AtACBP4 and AtACBP5 are differentially expressed and function in floral lipid metabolism

We herein demonstrated two of the Arabidopsis acyl-CoA-binding proteins (ACBPs), AtACBP4 and AtACBP5, both function in floral lipid metabolism and they may possibly play complementary roles in Arabidopsis microspore-to-pollen development. Histological analysis on transgenic Arabidopsis expressing β-glucuronidase driven from the AtACBP4 and AtACBP5 promoters, as well as, qRTPCR analysis revealed that AtACBP4 was expressed at stages 11-14 in the mature pollen, while AtACBP5 was expressed at stages 7-10 in the microspores and tapetal cells. Immunoelectron microscopy using AtACBP4- or AtACBP5-specific antibodies further showed that AtACBP4 and AtACBP5 were localized in the cytoplasm. Chemical analysis of bud wax and cutin using gas chromatographyflame ionization detector and GC-mass spectrometry analyses revealed the accumulation of cuticular waxes and cutin monomers in acbp4, acbp5 and acbp4acbp5 buds in comparison to the wild type (Col-0). Fatty acid profiling demonstrated a decline in stearic acid and an increase in linolenic acid in acbp4 and acbp4acbp5 buds, respectively, over Col-0. Analysis of inflorescences from acbp4 and acbp5 revealed that there was an increase of AtACBP5 expression in acbp4, and an increase of AtACBP4 expression in acbp5. Deletion analysis of the AtACBP4 and AtACBP5 5'-flanking regions indicated the minimal promoter activity for AtACBP4 (-145/+103) and AtACBP5 (-181/+81). Electrophoretic mobility shift assays identified a pollen-specific cis-acting element POLLEN1 (AGAAA) mapped at AtACBP4 (-157/-153) which interacted with nuclear proteins from flower and this was substantiated by DNase I footprinting. In Arabidopsisthaliana, six acyl-CoA-binding proteins (ACBPs), designated as AtACBP1 to AtACBP6, have been identified to function in plant stress and development. AtACBP4 and AtACBP5 represent the two largest proteins in the AtACBP family. Despite having kelch-motifs and sharing a common cytosolic subcellular localization, AtACBP4 and AtACBP5 differ in spatial and temporal expression. Histological analysis on transgenic Arabidopsis expressing β-glucuronidase driven from the respective AtACBP4 and AtACBP5 promoters, as well as, qRT-PCR analysis revealed that AtACBP4 was expressed at stages 11-14 in mature pollen, while AtACBP5 was expressed at stages 7-10 in the microspores and tapetal cells. Immunoelectron microscopy using AtACBP4- or AtACBP5-specific antibodies further showed that AtACBP4 and AtACBP5 were localized in the cytoplasm. Chemical analysis of bud wax and cutin using gas chromatography-flame ionization detector and GC-mass spectrometry analyses revealed the accumulation of cuticular waxes and cutin monomers in acbp4, acbp5 and acbp4acbp5 buds, in comparison to the wild type. Analysis of inflorescences from acbp4 and acbp5 revealed that there was an increase of AtACBP5 expression in acbp4, and an increase of AtACBP4 expression in acbp5. Deletion analysis of the AtACBP4 and AtACBP5 5'-flanking regions indicated the minimal promoter region for AtACBP4 (-145/+103) and AtACBP5 (-181/+81). Electrophoretic mobility shift assays identified a pollen-specific cis-acting element POLLEN1 (AGAAA) within AtACBP4 (-157/-153) which interacted with nuclear proteins from flower and this was substantiated by DNase I footprinting. These results suggest that AtACBP4 and AtACBP5 both function in floral lipidicmetabolism and they may play complementary roles in Arabidopsis microspore-to-pollen development.

OsGL1-3 is involved in cuticular wax biosynthesis and tolerance to water deficit in rice.

Cuticular wax covers aerial organs of plants and functions as the outermost barrier against non-stomatal water loss. We reported here the functional characterization of the Glossy1(GL1)-homologous gene OsGL1-3 in rice using overexpression and RNAi transgenic rice plants. OsGL1-3 gene was ubiquitously expressed at different level in rice plants except root and its expression was up-regulated under ABA and PEG treatments. The transient expression of OsGL1-3–GFP fusion protein indicated that OsGL1-3 is mainly localized in the plasma membrane. Compared to the wild type, overexpression rice plants exhibited stunted growth, more wax crystallization on leaf surface, and significantly increased total cuticular wax load due to the prominent changes of C30–C32 aldehydes and C30 primary alcohols. While the RNAi knockdown mutant of OsGL1-3 exhibited no significant difference in plant height, but less wax crystallization and decreased total cuticular wax accumulation on leaf surface. All these evidences, together with the effects of OsGL1-3 on the expression of some wax synthesis related genes, suggest that OsGL1-3 is involved in cuticular wax biosynthesis. Overexpression of OsGL1-3 decreased chlorophyll leaching and water loss rate whereas increased tolerance to water deficit at both seedling and late-tillering stages, suggesting an important role of OsGL1-3 in drought tolerance.

Differentiated cuticular wax content and expression patterns of cuticular wax biosynthetic genes in bloomed and bloomless broccoli (Brassica oleracea var. italica)

The aerial surfaces of plants are covered with a wax layer that serves the essential functions of limiting non-stomatal water loss and acting as protective barrier against environmental stresses. We selected two broccoli lines, bloomed (MC91) and bloomless (MC117), and analyzed their phenotypes related to cuticular wax accumulation. The total wax amount was 1.93-fold higher in MC91 leaves compared to MC117 leaves. All of the studied cuticular wax compounds were 1.07–3.79-fold higher in MC91 plants compared to MC117 plants except for the C31 alkane. The wax compositions did not essentially different between the two broccoli lines, but some compounds were found at significantly higher levels in MC91 plants compared to MC117 plants, mainly reflecting differences in C29 alkanes, C29 secondary alcohols and C29 ketones. To investigate gene regulation by bloom phenotype, we analyzed the mRNA expression patterns of various cuticular wax biosynthetic genes. Our results revealed that LACS1, KCS1, KCR1, ECR, CER3 and MAH1 were expressed more in MC91 plants compared to MC117 plants at both 3 and 10 weeks. The expression levels of the studied cuticular wax biosynthetic genes were significantly induced by drought stress, which is known to induce cuticular wax deposition. Together, these results show that the cuticular wax accumulation of broccoli is regulated by cuticular wax biosynthetic gene expression and can be affected by environmental signals.

Over-expression of SlSHN1 gene improves drought tolerance by increasing cuticular wax accumulation in tomato.

Increasing cuticular wax accumulation in plants has been associated with improving drought tolerance in plants. In this study, a cDNA clone encoding the SlSHN1 transcription factor, the closest ortholog to WIN/SHN1 gene in Arabidopsis, was isolated from tomato plant. Expression analysis of SlSHN1 indicated that it is induced in response to drought conditions. The over-expression of SlSHN1 in tomato under the control of the constitutive CaMV 35S promoter produced plants that showed mild growth retardation phenotype with shiny and dark green leaves. Scanning electron microscopy showed that the over-expression of SlSHN1 in tomato resulted in higher cuticular wax deposition on leaf epidermial tissue when compared to non-transformed plants. Expression analysis in transgenic lines over-expressing SlSHN1 indicated that several wax-related synthesis genes were induced. Transgenic tomato plants over-expressing SlSHN1 showed higher drought tolerance when compared with wild type plants; this was reflected in delayed wilting of transgenic lines, improved water status and reduced water loss rate when compared with wild type plants. In conclusion, the SlSHN1 gene can modulate wax accumulation and could be utilized to enhance drought tolerance in tomato plant.

Cuticular wax biosynthesis is up-regulated by the MYB94 transcription factor in Arabidopsis.

The aerial parts of all land plants are covered with hydrophobic cuticular wax layers that act as the first barrier against the environment. The MYB94 transcription factor gene is expressed in abundance in aerial organs and shows a higher expression in the stem epidermis than within the stem. When seedlings were subjected to various treatments, the expression of the MYB94 transcription factor gene was observed to increase approximately 9-fold under drought, 8-fold for ABA treatment and 4-fold for separate NaCl and mannitol treatments. MYB94 harbors the transcriptional activation domain at its C-terminus, and fluorescent signals from MYB94:enhanced yellow fluorescent protein (eYFP) were observed in the nucleus of tobacco epidermis and in transgenic Arabidopsis roots. The total wax loads increased by approximately 2-fold in the leaves of the MYB94-overexpressing (MYB94 OX) lines, as compared with those of the wild type (WT). MYB94 activates the expression of WSD1, KCS2/DAISY, CER2, FAR3 and ECR genes by binding directly to their gene promoters. An increase in the accumulation of cuticular wax was observed to reduce the rate of cuticular transpiration in the leaves of MYB94 OX lines, under drought stress conditions. Taken together, a R2R3-type MYB94 transcription factor activates Arabidopsis cuticular wax biosynthesis and might be important in plant response to environmental stress, including drought.

Enhanced expression of EsWAX1 improves drought tolerance with increased accumulation of cuticular wax and ascorbic acid in transgenic Arabidopsis

Drought can activate several stress responses in plants, such as stomatal closure, accumulation of cuticular wax and ascorbic acid (AsA), which have been correlated with improvement of drought tolerance. In this study, a novel MYB gene, designed as EsWAX1, was isolated and characterized from Eutrema salsugineum. EsWAX1 contained a full-length open reading frame (ORF) of 1068 bp, which encoding 355 amino acids. Transcript levels of EsWAX1 were quickly inducible by drought stress and ABA treatment, indicating that EsWAX1 may act as a positive regulator in response to drought stress. Ectopic expression of EsWAX1 increased accumulation of cuticular wax via modulating the expression of several wax-related genes, such as CER1, KCS2 and KCR1. Scanning electron microscopy further revealed higher densities of wax crystalline structures on the adaxial surfaces of leaves in transgenic Arabidopsis plants. In addition, the expression of several AsA biosynthetic genes (VTC1, GLDH and MIOX4) was significantly up-regulated in EsWAX1-overexpressing lines and these transgenic plants have approximately 23–27% more total AsA content than WT plants. However, the high-level expression of EsWAX1 severely disrupted plant normal growth and development. To reduce negative effects of EsWAX1 over-expression on plant growth, we generated transgenic Arabidopsis plants expressing EsWAX1 driven by the stress-inducible RD29A promoter. Our data indicated the RD29A::EsWAX1 transgenic plants had greater tolerance to drought stress than wild-type plants. Taken together, the EsWAX1 gene is a potential regulator that may be utilized to improve plant drought tolerance by genetic manipulation.

An ATP Binding Cassette Transporter Is Required for Cuticular Wax Deposition and Desiccation Tolerance in the Moss Physcomitrella patens

The plant cuticle is thought to be a critical evolutionary adaptation that allowed the first plants to colonize land, because of its key roles in regulating plant water status and providing protection from biotic and abiotic stresses. Much has been learned about cuticle composition and structure through genetic and biochemical studies of angiosperms, as well as underlying genetic pathways, but little is known about the cuticles of early diverging plant lineages. Here, we demonstrate that the moss Physcomitrella patens, an extant relative of the earliest terrestrial plants, has a cuticle that is analogous in both structure and chemical composition to those of angiosperms. To test whether the underlying cuticle biosynthetic pathways were also shared among distant plant lineages, we generated a genetic knockout of the moss ATP binding cassette subfamily G (ABCG) transporter Pp-ABCG7, a putative ortholog of Arabidopsis thaliana ABCG transporters involved in cuticle precursor trafficking. We show that this mutant is severely deficient in cuticular wax accumulation and has a reduced tolerance of desiccation stress compared with the wild type. This work provides evidence that the cuticle was an adaptive feature present in the first terrestrial plants and that the genes involved in their formation have been functionally conserved for over 450 million years.

Effect of Stresses on Leaf Cuticular Wax Accumulation and Its Relationship to Expression of <I>OsGL1</I>-Homologous Genes in Rice

植物角质层蜡质在抵抗各种生物和非生物胁迫中起着非常重要的作用。本试验以水稻( Oryza sativa L.)幼苗为材料, 分别以200 mmol L -1 NaCl、12% PEG、1.0% H 2 O 2 、40℃高温和8℃低温为逆境, 研究叶角质层蜡质的积累情况以及其与水稻蜡质合成相关基因 OsGL1 表达的关系。扫描电镜观察以及叶角质层蜡质总量测定结果表明, 12% PEG、1.0% H 2 O 2 和8℃低温处理下水稻幼苗叶角质层蜡质的积累明显增加, 而200 mmol L -1 NaCl和40℃高温处理下叶角质层蜡质覆盖量略有下降。RT-PCR分析显示, 逆境处理下水稻蜡质合成相关基因 OsGL1 的表达量变化与水稻幼苗叶角质层蜡质的积累存在相关性。

Coexpression patterns indicate that GPI-anchored non-specific lipid transfer proteins are involved in accumulation of cuticular wax, suberin and sporopollenin

The non-specific lipid transfer proteins (nsLTP) are unique to land plants. The nsLTPs are characterized by a compact structure with a central hydrophobic cavity and can be classified to different types based on sequence similarity, intron position or spacing between the cysteine residues. The type G nsLTPs (LTPGs) have a GPI-anchor in the C-terminal region which attaches the protein to the exterior side of the plasma membrane. The function of these proteins, which are encoded by large gene families, has not been systematically investigated so far. In this study we have explored microarray data to investigate the expression pattern of the LTPGs in Arabidopsis and rice. We identified that the LTPG genes in each plant can be arranged in three expression modules with significant coexpression within the modules. According to expression patterns and module sizes, the Arabidopsis module AtI is functionally equivalent to the rice module OsI, AtII corresponds to OsII and AtIII is functionally comparable to OsIII. Starting from modules AtI, AtII and AtIII we generated extended networks with Arabidopsis genes coexpressed with the modules. Gene ontology analyses of the obtained networks suggest roles for LTPGs in the synthesis or deposition of cuticular waxes, suberin and sporopollenin. The AtI-module is primarily involved with cuticular wax, the AtII-module with suberin and the AtIII-module with sporopollenin. Further transcript analysis revealed that several transcript forms exist for several of the LTPG genes in both Arabidopsis and rice. The data suggests that the GPI-anchor attachment and localization of LTPGs may be controlled to some extent by alternative splicing.