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Plant cuticular waxes serve as highly responsive adaptations to variable environments 1-7 . Aliphatic waxes consist of very-long-chain (VLC) compounds produced from 1-alcohol- or alkane-forming pathways 5,8 . The existing variation in 1-alcohols and alkanes across Arabidopsis accessions revealed that 1-alcohol amounts are negatively correlated with aridity factors, whereas alkanes display the opposite behaviour. How carbon resources are allocated between the 1-alcohol and alkane pathways responding to environmental stimuli is still largely unknown. Here, in Arabidopsis, we report a novel 1-alcohol biosynthesis pathway in which VLC acyl-CoAs are first reduced to aldehydes by CER3 and then converted into 1-alcohols via a newly identified putative aldehyde reductase SOH1. CER3, previously shown to interact with CER1 in alkane synthesis, is identified to interact with SOH1 as well, channelling wax precursors into either alcohol- or alkane-forming pathways, and the directional shunting of these precursors is tightly regulated by the SOH1-CER3-CER1 module in response to environmental conditions., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature Limited.)

Wax biosynthesis is closely controlled by many regulators under different environmental conditions. We have previously shown that the module miR156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE9 (SPL9)-DEWAX is involved in the diurnal regulation of wax production; however, it was not determined whether other SPLs are also involved in wax synthesis. Here, we report that SPL13 also regulates drought-induced wax production, by directly and indirectly affecting the expression of the two wax biosynthesis genes ECERIFERUM1 (CER1) and CER4, respectively. In addition, we show that SPL13 together with SPL9 redundantly regulates wax accumulation under both normal and drought stress conditions, and that simultaneous mutation of both genes additively increases cuticle permeability and decreases drought tolerance. However, in contrast to SPL9, SPL13 does not seem to participate in the DEWAX-mediated diurnal regulation of wax production., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Stevia rebaudiana (stevia) is a plant in the Asteraceae that contains several biologically active compounds including the antidiabetic diterpene glycosides (e.g. stevioside, rebaudioside and dulcoside) that can serve as zero-calorie sugar alternatives. In this study, an elicitation strategy was applied using 5% polyethylene glycol (PEG), sodium chloride (NaCl; 50 and 100 mM) and gibberellic acid (2.0 and 4.0 mg/L GA 3 ) to investigate their effect on shoot morphogenesis, and the production of phenolics, flavonoids, total soluble sugars, proline and stevioside, as well as antioxidant activity, in shoot cultures of S. rebaudiana. Herewith, the media supplemented with 2 mg/L and 4 mg/L GA 3 exhibited the highest shooting response (87% and 80%). The augmentation of lower concentrations of GA 3 (2 mg/L) in combination with 6-benzylaminopurine (BAP) resulted in the maximum mean shoot length (11.1 cm). The addition of 100 mM NaCl salts to the media led to the highest observed total phenolics content (TPC; 4.11 mg/g-DW compared to the control 0.52 mg/g-DW), total flavonoids content (TFC; 1.26 mg/g-DW) and polyphenolics concentration (5.39 mg/g-DW) in shoots cultured. However, the maximum antioxidant activity (81.8%) was observed in shoots raised in media treated with 50 mM NaCl. The application of 2 mg/L of GA 3 resulted in the highest accumulation of proline (0.99 μg/mL) as compared to controls (0.37 μg/mL). Maximum stevioside content (71 µL/mL) was observed in cultures supplemented with 100 mM NaCl and 5% PEG, followed by the 4 mg/L GA 3 treatment (70 µL/mL) as compared to control (60 µL/mL). Positive correlation was observed between GA 3 and stevioside content. Notably, these two compounds are derived from a shared biochemical pathway. These results suggest that elicitation is an effective option to enhance the accumulation of steviosides and other metabolites and provides the groundwork for future industrial scale production using bioreactors., (© 2024. The Author(s).)

The plant cuticle is composed of cuticular wax and cutin polymers and plays an essential role in plant tolerance to diverse abiotic and biotic stresses. Several stresses, including water deficit and salinity, regulate the synthesis of cuticular wax and cutin monomers. However, the effect of wounding on wax and cutin monomer production and the associated molecular mechanisms remain unclear. In this study, we determined that the accumulation of wax and cutin monomers in Arabidopsis leaves is positively regulated by wounding primarily through the jasmonic acid (JA) signaling pathway. Moreover, we observed that a wound- and JA-responsive gene (CYP96A4) encoding an ER-localized cytochrome P450 enzyme was highly expressed in leaves. Further analyses indicated that wound-induced wax and cutin monomer production was severely inhibited in the cyp96a4 mutant. Furthermore, CYP96A4 interacted with CER1 and CER3, the core enzymes in the alkane-forming pathway associated with wax biosynthesis, and modulated CER3 activity to influence aldehyde production in wax synthesis. In addition, transcripts of MYC2 and JAZ1, key genes in JA signaling pathway, were significantly reduced in cyp96a4 mutant. Collectively, these findings demonstrate that CYP96A4 functions as a cofactor of the alkane synthesis complex or participates in JA signaling pathway that contributes to cuticular wax biosynthesis and cutin monomer formation in response to wounding., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Cuticular waxes of plants impart tolerance to many forms of environmental stress and help shed dangerous human pathogens on edible plant parts. Although the chemical composition of waxes on a wide variety of important crops has been described, a detailed wax compositional analysis has yet to be reported for lettuce ( Lactuca sativa L.), one of the most widely consumed vegetables. We present herein the leaf wax content and composition of 12 genetically diverse lettuce cultivars sampled across five time points during their vegetative growth phase in the field. Mean total leaf wax amounts across all cultivars varied little over 28 days of vegetative growth, except for a notable decrease in total waxes following a major precipitation event, presumably due to wax degradation from wind and rain. All lettuce cultivars were found to contain a unique wax composition highly enriched in 22- and 24-carbon length 1-alcohols (docosanol and tetracosanol, respectively). In our report, the dominance of these shorter chain length 1-alcohols as wax constituents represents a relatively rare phenotype in plants. The ecological significance of these dominant and relatively short 1-alcohols is still unknown. Although waxes have been a target for improvement of various crops, no such work has been reported for lettuce. This study lays the groundwork for future research that aims to integrate cuticular wax characteristics of field grown plants into the larger context of lettuce breeding and cultivar development., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

The plant cuticle, a structure primarily composed of wax and cutin, forms a continuous coating over most aerial plant surfaces. The cuticle plays important roles in plant tolerance to environmental stress, including stress imposed by drought. Some members of the 3-KETOACYL-COA SYNTHASE (KCS) family are known to act as metabolic enzymes involved in cuticular wax production. Here we report that Arabidopsis (Arabidopsis thaliana) KCS3, which was previously shown to lack canonical catalytic activity, instead functions as a negative regulator of wax metabolism by reducing the enzymatic activity of KCS6, a key KCS involved in wax production. We demonstrate that the role of KCS3 in regulating KCS6 activity involves physical interactions between specific subunits of the fatty acid elongation complex and is essential for maintaining wax homeostasis. We also show that the role of the KCS3-KCS6 module in regulating wax synthesis is highly conserved across diverse plant taxa from Arabidopsis to the moss Physcomitrium patens, pointing to a critical ancient and basal function of this module in finely regulating wax synthesis., Competing Interests: Conflict of interest statement. The authors declare no conflict of interest., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Genetic signature of climate adaptation has been widely recognized across the genome of many organisms; however, the eco-physiological basis for linking genomic polymorphisms with local adaptations remains largely unexplored. Using a panel of 218 world-wide Arabidopsis accessions, we characterized the natural variation in root suberization by quantifying 16 suberin monomers. We explored the associations between suberization traits and 126 climate variables. We conducted genome-wide association analysis and integrated previous genotype-environment association (GEA) to identify the genetic bases underlying suberization variation and their involvements in climate adaptation. Root suberin content displays extensive variation across Arabidopsis populations and significantly correlates with local moisture gradients and soil characteristics. Specifically, enhanced suberization is associated with drier environments, higher soil cation-exchange capacity, and lower soil pH; higher proportional levels of very-long-chain suberin is negatively correlated with moisture availability, lower soil gravel content, and higher soil silt fraction. We identified 94 putative causal loci and experimentally proved that GPAT6 is involved in C16 suberin biosynthesis. Highly significant associations between the putative genes and environmental variables were observed. Roots appear highly responsive to environmental heterogeneity via regulation of suberization, especially the suberin composition. The patterns of suberization-environment correlation and the suberin-related GEA fit the expectations of local adaptation for the polygenic suberization trait., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Iso-branched wax compounds are well known in plants, but their biosynthetic pathways are still mostly unknown. It has been speculated that branched waxes are derived from branched-chain amino acid (BCAA) catabolism, but the evidence for this is very limited. Gas chromatography-flame ionisation detection (GC-FID) analysis revealed that mutations in two subunits of the branched-chain ketoacid dehydrogenase (BCKDH) complex, a key enzyme complex in the degradation of BCAAs, significantly decreased the amounts of branched wax compounds, indicating that BCAA degradation may be integral to the synthesis of iso-branched wax. Substrate feeding studies further revealed that the metabolic precursor of iso-branched wax compounds is isobutyric acid (iBA), which is derived from valine degradation in Arabidopsis. We also isolated a novel mutant and found that its branched wax deficient phenotype could not be rescued by iBA. Map-based cloning together with complementation analysis revealed that mutation in ACYL-ACTIVATING ENZYME 9 (AAE9) is responsible for this phenotype. Genetic and enzyme activity analysis demonstrated that AAE9 is located downstream of the BCAA degradation pathway, and that it activates iBA to isobutyryl-CoA for use on branched wax synthesis. Taken together, our study demonstrates that AAE9 is a key factor connecting BCAA catabolism with branched wax biosynthesis., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

Water deficit stress negatively affects wheat growth, physiology, and yield. In lab and hydroponic experiments, osmotic stress levels (control, -2, -4, -6 and -8 Bars) created by PEG-6000, caused a significant decline in germination, mean germination time, root, shoot, and coleoptile length in both wheat genotypes examined. Germination was inhibited more in Wafaq-2001 than in Chakwal-50. Wafaq-2001 showed a higher susceptibility index based on root and shoot dry weight than did Chakwal-50. Wheat plants exhibited osmotic adjustment through the accumulation of proline, soluble sugars, soluble proteins, and free amino acids, and increased antioxidation activities of superoxide dismutase, peroxidase, catalase, and malondialdehyde. Increasing water deficit stress caused a linear decline in chlorophyll contents, leaf membrane stability, and relative water content in all wheat plants, with Wafaq-2001 showing a more severe negative impact on these parameters with increasing stress levels. The results suggest the possibility of utilizing some of these parameters as quantitative indicators of water stress tolerance in plants. Gas exchange measurements (photosynthesis, transpiration, stomatal conductance), leaf osmotic potential, water potential, and yield attributes decreased more abruptly with increasing water deficit, whereas leaf cuticular wax content increased in both genotypes, with more severe impacts on Wagaq-2001. More reduction in biochemical, physiological, and yield attributes was observed in Wafaq-2001 than was observed in Chakwal-50. Based on these results, we can conclude that Chakwal-50 is a more drought-tolerant genotype, and has excellent potential for future use in breeding programs to improve wheat drought tolerance., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

The aerial surfaces of land plants have a protective layer of cuticular wax. Alkanes are common components of these waxes, and their abundance is affected by a range of stresses. The CER16 protein has been implicated in alkane biosynthesis in the cuticular wax of Arabidopsis ( Arabidopsis thaliana ). Here, we identified two new mutant alleles of CER16 in Arabidopsis resulting in production of less wax with dramatically fewer alkanes than the wild type. Map-based cloning with genetic analysis revealed that the cer16 phenotype was caused by complete loss of AT5G44150 , encoding a protein with no known domains or motifs. Comparative transcriptomic analysis revealed that transcripts of CER3 , previously shown to play a principal role in alkane production, were markedly reduced in the cer16 mutants. To define the relationship between CER3 and CER16, we transformed the full CER3 gene into a cer16 mutant. Transgenic CER3 expression was silenced, and levels of small interfering RNAs targeting CER3 were significantly increased. Mutating two major components of the RNA-silencing machinery in a cer16 genetic background restored CER3 transcript levels to wild-type levels, with the stems restored to wild-type glaucousness. We suggest that CER16 deficiency induces post-transcriptional gene silencing of both endogenous and exogenous expression of CER3 ., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Plant surface waxes form an outer barrier that protects the plant from many forms of environmental stress. The deposition of cuticular waxes on the plant surface is regulated by external environmental changes, including light and dark cycles. However, the underlying molecular mechanisms controlling light regulation of wax production are still poorly understood, especially at the posttranscriptional level. In this paper, we report the regulation of cuticular wax production by the miR156-SPL9 (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9) module in Arabidopsis ( Arabidopsis thaliana ). When compared with wild-type plants, miR156 and SPL9 mutants showed significantly altered cuticular wax amounts in both stems and leaves. Furthermore, it was found that SPL9 positively regulates gene expression of the alkane-forming enzyme ECERIFERUM1 ( CER1 ), as well as the primary (1-) alcohol-forming enzyme ECERIFERUM4 ( CER4 ), to enhance alkane and 1-alcohol synthesis, respectively. Our results indicate that complex formation of SPL9 with a negative regulator of wax synthesis, DEWAX, will hamper SPL9 DNA binding ability, possibly by interfering with SPL9 homodimerization. Combined with their diurnal gene and protein expressions, this dynamic repression-activation transcriptional module defines a dynamic mechanism that may allow plants to optimize wax synthesis during daily cycles. These findings provide a regulatory framework for environmental signal integration in the regulation of wax synthesis., (© 2019 American Society of Plant Biologists. All rights reserved.)

The cuticle plays a critical role as barrier between plant and environment. Here, cuticular wax morphology, cuticular wax and cutin monomer composition, and expression of associated genes in five above ground organs were examined in model extremophyte Thellungiella salsugineum. Alkanes, ketones, and 2-alcohols were the predominant wax constitutes in rosette leaves, inflorescence stem leaves, stems, and siliques, whereas alkanes and acids were the predominant cuticular lipids in whole flowers. Unsubstituted acids were the most abundant cutin monomers in vegetative organs, especially C18:2 dioic acids, which reached the highest levels in stems. Hydroxy fatty acids were the predominant cutin monomers in flowers, especially 16-OH C16:0 and diOH C16:0. High-throughput RNA-Seq analysis using the Hiseq4000 platform was performed on these five above organs of T. salsugineum, and the differentially expressed lipid-associated genes and their associated metabolic pathways were identified. Expression of genes associated in previous reports to cuticle production, including those having roles in cuticle lipid biosynthesis, transport, and regulation were examined. The association of cuticle lipid composition and gene expression within different organs of T. salsugineum, and potential relationships between T. salsugineum's extreme cuticle and its adaptation to extreme environments is discussed., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The plant cuticle is a complex structure composed primarily of wax and cutin, but also contains cutan, glycerolipids, phenolics, polysaccharides and proteins. The cuticle plays an important protective role as barrier between plants and their environment. In this paper, 4-week-old leaves produced either on the rosette or on the inflorescence stem of the model extremophyte Thellungiella salsuginea were examined using scanning electron microscopy, cuticle permeability assays and chemical composition analysis. Results showed that stem leaves (SL) had more abundant cuticle lipids and lower cuticle permeability than rosette leaves (RL). SL were dominated by alkanes, especially the C29 and C31 homologs, whereas in RL the most abundant wax class was free very long-chain acids. The major cutin monomers for both leaf types were C18:2 dioic acids and 18-OH C18:2 acids. We performed Illumina high-throughput sequencing for SL and RL, and 3577 differentially expressed genes were identified. Sixty-five genes possibly involved in cuticular lipid biosynthesis, transport, or regulation was selected for further analysis. Many cuticle-associated genes exhibited differential expression levels that could be associated with compositional differences between these two leaf types. Furthermore, transcription factors and other regulatory proteins previously associated with cuticle production were expressed at higher levels in SL than in RL. The associations between gene expression and characteristics of this extremophile's leaf cuticles sheds new light on cuticle as an adaptive trait in extreme environments, and contributes new information that may guide efforts to modify crop cuticles for improved stress tolerance., (© 2018 Scandinavian Plant Physiology Society.)

Main Conclusion: Identification of NnCER2 and NnCER2-LIKE from Nelumbo nucifera, which are required for the very-long-chain fatty acid elongation, provides new evidence that CER2 proteins are evolutionarily conserved across the eudicots. CER2-LIKE family proteins have been described as core components of the fatty acid elongase complex in Arabidopsis, maize, and rice, having specific function in synthesis of the C 30 to C 34 fatty acyl-CoA precursors of cuticular waxes. Little is known about the functional conservation in this gene family across species. In this study, two CER2-LIKE family proteins, NnCER2 and NnCER2-LIKE, were characterized from sacred lotus (Nelumbo nucifera), which is an ancient basal eudicot. The transcriptional expression of NnCER2 and NnCER2-LIKE was found in floating leaf blades, emergent petioles and vertical leaves, petals, and anthers. The NnCER2 and NnCER2-LIKE proteins were localized to the endoplasmic reticulum and nucleus. Overexpressing NnCER2 and NnCER2-LIKE in Arabidopsis led to alteration of cuticle wax structure in inflorescence stems, and this was associated with elevated 30, 32, and 34 carbon length wax compounds, and their derivatives. The different substrate specificities of NnCER2 and NnCER2-LIKE were explored using co-expression with AtCER6 in yeast cells. These findings provide clear evidence that the function of CER2 family proteins in producing VLCFAs is highly conserved across the eudicots.

To mitigate the effects of heat and drought stress, a better understanding of the genetic control of physiological responses to these environmental conditions is needed. To this end, we evaluated an upland cotton ( Gossypium hirsutum L.) mapping population under water-limited and well-watered conditions in a hot, arid environment. The elemental concentrations (ionome) of seed samples from the population were profiled in addition to those of soil samples taken from throughout the field site to better model environmental variation. The elements profiled in seeds exhibited moderate to high heritabilities, as well as strong phenotypic and genotypic correlations between elements that were not altered by the imposed irrigation regimes. Quantitative trait loci (QTL) mapping results from a Bayesian classification method identified multiple genomic regions where QTL for individual elements colocalized, suggesting that genetic control of the ionome is highly interrelated. To more fully explore this genetic architecture, multivariate QTL mapping was implemented among groups of biochemically related elements. This analysis revealed both additional and pleiotropic QTL responsible for coordinated control of phenotypic variation for elemental accumulation. Machine learning algorithms that utilized only ionomic data predicted the irrigation regime under which genotypes were evaluated with very high accuracy. Taken together, these results demonstrate the extent to which the seed ionome is genetically interrelated and predictive of plant physiological responses to adverse environmental conditions., (Copyright © 2018 Pauli et al.)

Key Message: Molecular markers linked to QTLs controlling post-harvest fruit water loss in pepper may be utilized to accelerate breeding for improved shelf life and inhibit over-ripening before harvest. Bell pepper (Capsicum annuum L.) is an important vegetable crop world-wide. However, marketing is limited by the relatively short shelf life of the fruit due to water loss and decay that occur during prolonged storage. Towards breeding pepper with reduced fruit post-harvest water loss (PWL), we studied the genetic, physiological and biochemical basis for natural variation of PWL. We performed quantitative trait locus (QTL) mapping of fruit PWL in multiple generations of an interspecific cross of pepper, which resulted in the identification of two linked QTLs on chromosome 10 that control the trait. We further developed near-isogenic lines (NILs) for characterization of the QTL effects. Transcriptome analysis of the NILs allowed the identification of candidate genes associated with fruit PWL-associated traits such as cuticle biosynthesis, cell wall metabolism and fruit ripening. Significant differences in PWL between the NILs in the immature fruit stage, differentially expressed cuticle-associated genes and differences in the content of specific chemical constituents of the fruit cuticle, indicated a likely influence of cuticle composition on the trait. Reduced PWL in the NILs was associated with delayed over-ripening before harvest, low total soluble solids before storage, and reduced fruit softening after storage. Our study enabled a better understanding of the genetic and biological processes controlling natural variation in fruit PWL in pepper. Furthermore, the genetic materials and molecular markers developed in this study may be utilized to breed peppers with improved shelf life and inhibited over-ripening before harvest.

We report n-6 monounsaturated primary alcohols (C 26:1 , C 28:1 , and C 30:1 homologs) in the cuticular waxes of Arabidopsis (Arabidopsis thaliana) inflorescence stem, a class of wax not previously reported in Arabidopsis. The Arabidopsis cer17 mutant was completely deficient in these monounsaturated alcohols, and CER17 was found to encode a predicted ACYL-COENZYME A DESATURASE LIKE4 (ADS4). Studies of the Arabidopsis cer4 mutant and yeast variously expressing CER4 (a predicted fatty acyl-CoA reductase) with CER17/ADS4, demonstrated CER4's principal role in synthesis of these monounsaturated alcohols. Besides unsaturated alcohol deficiency, cer17 mutants exhibited a thickened and irregular cuticle ultrastructure and increased amounts of cutin monomers. Although unsaturated alcohols were absent throughout the cer17 stem, the mutation's effects on cutin monomers and cuticle ultrastructure were much more severe in distal than basal stems, consistent with observations that the CER17/ADS4 transcript was much more abundant in distal than basal stems. Furthermore, distal but not basal stems of a double mutant deficient for both CER17/ADS4 and LONG-CHAIN ACYL-COA SYNTHETASE1 produced even more cutin monomers and a thicker and more disorganized cuticle ultrastructure and higher cuticle permeability than observed for wild type or either mutant parent, indicating a dramatic genetic interaction on conversion of very long chain acyl-CoA precursors. These results provide evidence that CER17/ADS4 performs n-6 desaturation of very long chain acyl-CoAs in both distal and basal stems and has a major function associated with governing cutin monomer amounts primarily in the distal segments of the inflorescence stem., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Populus euphratica is an important native tree found in arid regions from North Africa and South Europe to China, and is known to tolerate many forms of environmental stress, including drought. We describe cuticle waxes, cutin and cuticle permeability for the heteromorphic leaves of P. euphratica growing in two riparian habitats that differ in available soil moisture. Scanning electron microscopy revealed variation in epicuticular wax crystallization associated with leaf type and site. P. euphratica leaves are dominated by cuticular wax alkanes, primary-alcohols and fatty acids. The major cutin monomers were 10,16-diOH C 16 :0 acids. Broad-ovate leaves (associated with adult phase growth) produced 1.3- and 1.6-fold more waxes, and 2.1- and 0.9-fold more cutin monomers, than lanceolate leaves (associated with juvenile phase growth) at the wetter site and drier site, respectively. The alkane-synthesis-associated ECERIFERUM1 (CER1), as well as ABC transporter- and elongase-associated genes, were expressed at much higher levels at the drier than wetter sites, indicating their potential function in elevating leaf cuticle lipids in the dry site conditions. Higher cuticle lipid amounts were closely associated with lower cuticle permeability (both chlorophyll efflux and water loss). Our results implicate cuticle lipids as among the xeromorphic traits associated with P. euphratica adult-phase broad-ovate leaves. Results here provide useful information for protecting natural populations of P. euphratica and their associated ecosystems, and shed new light on the functional interaction of cuticle and leaf heterophylly in adaptation to more arid, limited-moisture environments., (© 2016 Scandinavian Plant Physiology Society.)

The allotetraploid species Brassica napus L. is a global crop of major economic importance, providing canola oil (seed) and vegetables for human consumption and fodder and meal for livestock feed. Characterizing the genetic diversity present in the extant germplasm pool of B. napus is fundamental to better conserve, manage and utilize the genetic resources of this species. We used sequence-based genotyping to identify and genotype 30,881 SNPs in a diversity panel of 782 B. napus accessions, representing samples of winter and spring growth habits originating from 33 countries across Europe, Asia, and America. We detected strong population structure broadly concordant with growth habit and geography, and identified three major genetic groups: spring (SP), winter Europe (WE), and winter Asia (WA). Subpopulation-specific polymorphism patterns suggest enriched genetic diversity within the WA group and a smaller effective breeding population for the SP group compared to WE. Interestingly, the two subgenomes of B. napus appear to have different geographic origins, with phylogenetic analysis placing WE and WA as basal clades for the other subpopulations in the C and A subgenomes, respectively. Finally, we identified 16 genomic regions where the patterns of diversity differed markedly from the genome-wide average, several of which are suggestive of genomic inversions. The results obtained in this study constitute a valuable resource for worldwide breeding efforts and the genetic dissection and prediction of complex B. napus traits.

Background: The majority of commercial cotton varieties planted worldwide are derived from Gossypium hirsutum, which is a naturally occurring allotetraploid produced by interspecific hybridization of A- and D-genome diploid progenitor species. While most cotton species are adapted to warm, semi-arid tropical and subtropical regions, and thus perform well in these geographical areas, cotton seedlings are sensitive to cold temperature, which can significantly reduce crop yields. One of the common biochemical responses of plants to cold temperatures is an increase in omega-3 fatty acids, which protects cellular function by maintaining membrane integrity. The purpose of our study was to identify and characterize the omega-3 fatty acid desaturase (FAD) gene family in G. hirsutum, with an emphasis on identifying omega-3 FADs involved in cold temperature adaptation., Results: Eleven omega-3 FAD genes were identified in G. hirsutum, and characterization of the gene family in extant A and D diploid species (G. herbaceum and G. raimondii, respectively) allowed for unambiguous genome assignment of all homoeologs in tetraploid G. hirsutum. The omega-3 FAD family of cotton includes five distinct genes, two of which encode endoplasmic reticulum-type enzymes (FAD3-1 and FAD3-2) and three that encode chloroplast-type enzymes (FAD7/8-1, FAD7/8-2, and FAD7/8-3). The FAD3-2 gene was duplicated in the A genome progenitor species after the evolutionary split from the D progenitor, but before the interspecific hybridization event that gave rise to modern tetraploid cotton. RNA-seq analysis revealed conserved, gene-specific expression patterns in various organs and cell types and semi-quantitative RT-PCR further revealed that FAD7/8-1 was specifically induced during cold temperature treatment of G. hirsutum seedlings., Conclusions: The omega-3 FAD gene family in cotton was characterized at the genome-wide level in three species, showing relatively ancient establishment of the gene family prior to the split of A and D diploid progenitor species. The FAD genes are differentially expressed in various organs and cell types, including fiber, and expression of the FAD7/8-1 gene was induced by cold temperature. Collectively, these data define the genetic and functional genomic properties of this important gene family in cotton and provide a foundation for future efforts to improve cotton abiotic stress tolerance through molecular breeding approaches.

The pathogenesis of malaria is strongly correlated with secretion of the micronemes, the apical organelles which contain the adhesins required for invasion of Plasmodium falciparum into human erythrocytes. A critical event in P. falciparum erythrocyte invasion is the production of calcium transients. After entering the cell, Ca(2+) binds to soluble Ca(2+)-binding proteins, such as the double C2 domains (DOC2). Recently, deletion of a P. falciparum DOC2 protein, PfDOC2, was shown to cause impairment in microneme secretion. However, PfDOC2 remains poorly characterized. Here, we report that PfDOC2 is expressed throughout the erythrocytic cycle and demonstrate that it is associated with membrane fractions and binds to calcium when it is part of these membranous structures. In summary, we show that PfDOC2 is a calcium lipid-binding protein of the protein kinase C type of DOC2 proteins., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The impact of water-deficit stress on leaf cuticular waxes and cutin monomers, and traits associated with cuticle permeability were examined in Shandong and Yukon ecotypes of Eutrema salsugineum (syn. Thellungiella salsuginea). Although Shandong exhibits glaucous leaves, and Yukon is non-glaucous, wax amounts on non-stressed Yukon leaves were 4.6-fold higher than on Shandong, due mainly to Yukon's eightfold higher wax fatty acids, especially the C22 and C24 acid homologues. Water deficit caused a 26.9% increase in total waxes on Shandong leaves, due mainly to increased C22 and C24 acids; and caused 10.2% more wax on Yukon, due mainly to an increase in wax alkanes. Total cutin monomers on non-stressed leaves of Yukon were 58.3% higher than on Shandong. Water deficit caused a 28.2% increase in total cutin monomers on Shandong, whereas total cutin monomers were not induced on Yukon. With or without stress, more abundant cuticle lipids were generally associated with lower water loss rates, lower chlorophyll efflux rates and an extended time before water deficit-induced wilting. In response to water deficit, Shandong showed elevated transcription of genes encoding elongase subunits, consistent with the higher stress induction of acids by Shandong. Yukon's higher induction of CER1 and CER3 transcripts may explain why alkanes increased most on Yukon after water deficit. Eutrema, with its diverse cuticle lipids and responsiveness, provides a valuable genetic resource for identifying new genes and alleles effecting cuticle metabolism, and lays groundwork for studies of the cuticle's role in extreme stress tolerance., (Published 2013. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

The ECERIFERUM9 (CER9) gene encodes a putative E3 ubiquitin ligase that functions in cuticle biosynthesis and the maintenance of plant water status. Here, we found that CER9 is also involved in abscisic acid (ABA) signaling in seeds and young seedlings of Arabidopsis (Arabidopsis thaliana). The germinated embryos of the mutants exhibited enhanced sensitivity to ABA during the transition from reversible dormancy to determinate seedling growth. Expression of the CER9 gene is closely related to ABA levels and displays a similar pattern to that of ABSCISIC ACID-INSENSITIVE5 (ABI5), which encodes a positive regulator of ABA responses in seeds. cer9 mutant seeds exhibited delayed germination that is independent of seed coat permeability. Quantitative proteomic analyses showed that cer9 seeds had a protein profile similar to that of the wild type treated with ABA. Transcriptomics analyses revealed that genes involved in ABA biosynthesis or signaling pathways were differentially regulated in cer9 seeds. Consistent with this, high levels of ABA were detected in dry seeds of cer9. Blocking ABA biosynthesis by fluridone treatment or by combining an ABA-deficient mutation with cer9 attenuated the phenotypes of cer9. Whereas introduction of the abi1-1, abi3-1, or abi4-103 mutation could completely eliminate the ABA hypersensitivity of cer9, introduction of abi5 resulted only in partial suppression. These results indicate that CER9 is a novel negative regulator of ABA biosynthesis and the ABA signaling pathway during seed germination., (© 2014 American Society of Plant Biologists. All Rights Reserved.)

Pepper (Capsicum spp.) fruits are covered by a relatively thick coating of cuticle that limits fruit water loss, a trait previously associated with maintenance of postharvest fruit quality during commercial marketing. To shed light on the chemical-compositional diversity of cuticles in pepper, the fruit cuticles from 50 diverse pepper genotypes from a world collection were screened for both wax and cutin monomer amount and composition. These same genotypes were also screened for fruit water loss rate and this was tested for associations with cuticle composition. Our results revealed an unexpectedly large amount of variation for the fruit cuticle lipids, with a more than 14-fold range for total wax amounts and a more than 16-fold range for cutin monomer amounts between the most extreme accessions. Within the major wax constituents fatty acids varied from 1 to 46%, primary alcohols from 2 to 19%, n-alkanes from 13 to 74% and triterpenoids and sterols from 10 to 77%. Within the cutin monomers, total hexadecanoic acids ranged from 54 to 87%, total octadecanoic acids ranged from 10 to 38% and coumaric acids ranged from 0.2 to 8% of the total. We also observed considerable differences in water loss among the accessions, and unique correlations between water loss and cuticle constituents. The resources described here will be valuable for future studies of the physiological function of fruit cuticle, for the identification of genes and QTLs associated with fruit cuticle synthesis in pepper fruit, and as a starting point for breeding improved fruit quality in pepper., (© 2013 Scandinavian Plant Physiology Society.)

To understand the role of fruit cuticle lipid composition in fruit water loss, an advanced backcross population, the BC(2)F(2) , was created between the Capsicum annuum (PI1154) and the Capsicum chinense (USDA162), which have high and low post-harvest water loss rates, respectively. Besides dramatic differences in fruit water loss, preliminary studies also revealed that these parents exhibited significant differences in both the amount and composition of their fruit cuticle. Cuticle analysis of the BC(2)F(2) fruit revealed that although water loss rate was not strongly associated with the total surface wax amount, there were significant correlations between water loss rate and cuticle composition. We found a positive correlation between water loss rate and the amount of total triterpenoid plus sterol compounds, and negative correlations between water loss and the alkane to triterpenoid plus sterol ratio. We also report negative correlations between water loss rate and the proportion of both alkanes and aliphatics to total surface wax amount. For the first time, we report significant correlations between water loss and cutin monomer composition. We found positive associations of water loss rate with the total cutin, total C(16) monomers and 16-dihydroxy hexadecanoic acid. Our results support the hypothesis that simple straight-chain aliphatic cuticle constituents form more impermeable cuticular barriers than more complex isoprenoid-based compounds. These results shed new light on the biochemical basis for cuticle involvement in fruit water loss., (Copyright © Physiologia Plantarum 2012.)

Mutation of the ECERIFERUM9 (CER9) gene in Arabidopsis (Arabidopsis thaliana) causes elevated amounts of 18-carbon-length cutin monomers and a dramatic shift in the cuticular wax profile (especially on leaves) toward the very-long-chain free fatty acids tetracosanoic acid (C₂₄) and hexacosanoic acid (C₂₆). Relative to the wild type, cer9 mutants exhibit elevated cuticle membrane thickness over epidermal cells and cuticular ledges with increased occlusion of the stomatal pore. The cuticular phenotypes of cer9 are associated with delayed onset of wilting in plants experiencing water deficit, lower transpiration rates, and improved water use efficiency measured as carbon isotope discrimination. The CER9 protein thus encodes a novel determinant of plant drought tolerance-associated traits, one whose deficiency elevates cutin synthesis, redistributes wax composition, and suppresses transpiration. Map-based cloning identified CER9, and sequence analysis predicted that it encodes an E3 ubiquitin ligase homologous to yeast Doa10 (previously shown to target endoplasmic reticulum proteins for proteasomal degradation). To further elucidate CER9 function, the impact of CER9 deficiency on interactions with other genes was examined using double mutant and transcriptome analyses. For both wax and cutin, cer9 showed mostly additive effects with cer6, long-chain acyl-CoA synthetase1 (lacs1), and lacs2 and revealed its role in early steps of both wax and cutin synthetic pathways. Transcriptome analysis revealed that the cer9 mutation affected diverse cellular processes, with primary impact on genes associated with diverse stress responses. The discovery of CER9 lays new groundwork for developing novel cuticle-based strategies for improving the drought tolerance and water use efficiency of crop plants.

A novel mutant of Arabidopsis (Arabidopsis thaliana), having highly glossy inflorescence stems, postgenital fusion in floral organs, and reduced fertility, was isolated from an ethyl methanesulfonate-mutagenized population and designated glossyhead1 (gsd1). The gsd1 locus was mapped to chromosome 1, and the causal gene was identified as a new allele of Acetyl-Coenzyme A Carboxylase1 (ACC1), a gene encoding the main enzyme in cytosolic malonyl-coenzyme A synthesis. This, to our knowledge, is the first mutant allele of ACC1 that does not cause lethality at the seed or early germination stage, allowing for the first time a detailed analysis of ACC1 function in mature tissues. Broad lipid profiling of mature gsd1 organs revealed a primary role for ACC1 in the biosynthesis of the very-long-chain fatty acids (C(20:0) or longer) associated with cuticular waxes and triacylglycerols. Unexpectedly, transcriptome analysis revealed that gsd1 has limited impact on any lipid metabolic networks but instead has a large effect on environmental stress-responsive pathways, especially senescence and ethylene synthesis determinants, indicating a possible role for the cytosolic malonyl-coenzyme A-derived lipids in stress response signaling.

Mulberry leaves are the sole source of food for silkworms (Bombyx mori), and moisture content of the detached leaves fed to silkworms determines silkworm growth and cocoon yield. Since leaf dehydration in commercial sericulture is a serious problem, development of new methods that minimize post-harvest water loss are greatly needed. In the present study, variability in moisture retention capacity (MRC, measured as leaf relative water content after one to 5 h of air-drying) was examined by screening 290 diverse mulberry accessions and the relationship between MRC and leaf surface (cuticular) wax amount was determined. Leaf MRC varied significantly among accessions, and was found to correlate strongly with leaf wax amount. Scanning electron microscopic analysis indicated that leaves having crystalline surface waxes of increased facet size and density were associated with high MRC accessions. Leaf MRC at 5 h after harvest was not related to other parameters such as specific leaf weight, and stomatal frequency and index. This study suggests that mulberry accessions having elevated leaf surface wax amount and crystal size and density exhibit reduced leaf post-harvest water loss, and could provide the foundation for selective breeding of improved cultivars., (Copyright 2010 Elsevier Masson SAS. All rights reserved.)

Infestation of wheat by Hessian fly larvae causes a variety of physical and biochemical modifications of the host plant. Changes occur in cuticle permeability, lipid composition and gene transcript abundance, and these responses differ substantially between resistant and susceptible wheat lines. Staining assays revealed that susceptible plants exhibited a generalized increase in leaf sheath epidermal permeability during infestation; whereas, epidermal permeability was only minimally affected in resistant plants. Furthermore, temporal profiling using gas chromatographic methods revealed that changes in cuticle lipid (wax and cutin) composition correlated well with differing levels of epidermal permeability in susceptible and resistant plants. Temporal analysis of cuticle-associated gene mRNA levels, by quantitative real-time PCR, indicated a relationship between transcript abundance and changes in cuticle lipid profiles of resistant and susceptible plants. Results suggest that conserving cuticle integrity via induction of specific wax constituents and maintenance of cutin amounts, determined by the accumulation of cuticle-associated transcripts, could be important components of wheat resistance to Hessian fly larvae.

Recent studies suggest that fruit cuticle is an important contributing factor to tomato (Solanum lycopersicum) fruit shelf life and storability. Moreover, it has been hypothesized that variation in fruit cuticle composition may underlie differences in traits such as fruit resistance to desiccation and microbial infection. To gain a better understanding of cuticle lipid composition diversity during fruit ontogeny and to assess if there are common features that correlate with ripening, we examined developmental changes in fruit cuticle wax and cutin monomer composition of delayed-ripening tomato fruit mutants, ripening inhibitor (rin) and non-ripening (nor) and delayed-ripening landrace Alcobaça. Previous reports show that fruit ripening processes such as climacteric ethylene production, cell wall degradation and color change are significantly delayed, or do not occur, in these lines. In the study presented here, however, we show that fruits from rin, nor and Alcobaça have cuticle lipid compositions that differ significantly from normal fruits of Ailsa Craig (AC) even at very early stages in fruit development, with continuing impacts throughout ripening. Moreover, rin, nor and the Alcobaça lines show quite different wax profiles from AC and each other throughout fruit development. Although cutin monomer composition differed much less than wax composition among the genotypes, all delayed-ripening lines possessed higher relative amounts of C(18) monomers than AC. Together, these results reveal new genetic associations between cuticle and fruit development processes and define valuable genetic resources to further explore the importance of cuticle in fruit shelf life.

In this study, we report isolation of a phosphatase gene designated GhHL1 from cotton and its functional characterization. GhHL1 transcripts were detected in all cotton tissues examined. Southern blotting analysis indicated that it exists in multiple-copies. Biochemical analysis showed that GhHL1 was Mg2+-dependent and cation-sensitive. Purified recombinant GhHL1 protein dephosphorylated both 3',5'-bisphosphate nucleotide and inositol 1,4-bisphosphate, demonstrating dual 3',5'-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase activities. Overexpression of GhHL1 complemented yeast hal2 mutant and enhanced yeast growth under elevated NaCl or LiCl, showing a role in salt tolerance associated with ionic stress response. Taken together, these results show that GhHL1 is a functional and good candidate gene, which might be used to improve salt tolerance in plants.

Arabidopsis (Arabidopsis thaliana) plants subjected to water deficit, sodium chloride (NaCl), or abscisic acid treatments were shown to exhibit a significant increase in the amount of leaf cuticular lipids. These stress treatments led to increases in cuticular wax amount per unit area of 32% to 80%, due primarily to 29% to 98% increases in wax alkanes. Of these treatments, only water deficit increased the total cutin monomer amount (by 65%), whereas both water deficit and NaCl altered the proportional amounts of cutin monomers. Abscisic acid had little effect on cutin composition. Water deficit, but not NaCl, increased leaf cuticle thickness (by 49%). Electron micrographs revealed that both water-deprived and NaCl-treated plants had elevated osmium accumulation in their cuticles. The abundance of cuticle-associated gene transcripts in leaves was altered by all treatments, including those performed in both pot-grown and in vitro conditions. Notably, the abundance of the ECERIFERUM1 gene transcript, predicted to function in alkane synthesis, was highly induced by all treatments, results consistent with the elevated alkane amounts observed in all treatments. Further, this induction of cuticle lipids was associated with reduced cuticle permeability and may be important for plant acclimation to subsequent water-limited conditions. Taken together, these results show that Arabidopsis provides an excellent model system to study the role of the cuticle in plant response to drought and related stresses, and its associated genetic and cellular regulation.

Plant cuticles are broadly composed of two major components: polymeric cutin and a mixture of waxes, which infiltrate the cutin matrix and also accumulate on the surface, forming an epicuticular layer. Although cuticles are thought to play a number of important physiological roles, with the most important being to restrict water loss from aerial plant organs, the relative contributions of cutin and waxes to cuticle function are still not well understood. Tomato (Solanum lycopersicum) fruits provide an attractive experimental system to address this question as, unlike other model plants such as Arabidopsis, they have a relatively thick astomatous cuticle, providing a poreless uniform material that is easy to isolate and handle. We identified three tomato mutants, cutin deficient 1 (cd1), cd2 and cd3, the fruit cuticles of which have a dramatic (95-98%) reduction in cutin content and substantially altered, but distinctly different, architectures. This cutin deficiency resulted in an increase in cuticle surface stiffness, and in the proportions of both hydrophilic and multiply bonded polymeric constituents. Furthermore, our data suggested that there is no correlation between the amount of cutin and the permeability of the cuticle to water, but that cutin plays an important role in protecting tissues from microbial infection. The three cd mutations were mapped to different loci, and the cloning of CD2 revealed it to encode a homeodomain protein, which we propose acts as a key regulator of cutin biosynthesis in tomato fruit.

We report a role for the Arabidopsis (Arabidopsis thaliana) RESURRECTION1 (RST1) gene in plant defense. The rst1 mutant exhibits enhanced susceptibility to the biotrophic fungal pathogen Erysiphe cichoracearum but enhanced resistance to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola. RST1 encodes a novel protein that localizes to the plasma membrane and is predicted to contain 11 transmembrane domains. Disease responses in rst1 correlate with higher levels of jasmonic acid (JA) and increased basal and B. cinerea-induced expression of the plant defensin PDF1.2 gene but reduced E. cichoracearum-inducible salicylic acid levels and expression of pathogenesis-related genes PR1 and PR2. These results are consistent with rst1's varied resistance and susceptibility to pathogens of different life styles. Cuticular lipids, both cutin monomers and cuticular waxes, on rst1 leaves were significantly elevated, indicating a role for RST1 in the suppression of leaf cuticle lipid synthesis. The rst1 cuticle exhibits normal permeability, however, indicating that the disease responses of rst1 are not due to changes in this cuticle property. Double mutant analysis revealed that the coi1 mutation (causing defective JA signaling) is completely epistatic to rst1, whereas the ein2 mutation (causing defective ethylene signaling) is partially epistatic to rst1, for resistance to B. cinerea. The rst1 mutation thus defines a unique combination of disease responses to biotrophic and necrotrophic fungi in that it antagonizes salicylic acid-dependent defense and enhances JA-mediated defense through a mechanism that also controls cuticle synthesis.

Plant cuticle is an extracellular lipid-based matrix of cutin and waxes, which covers aerial organs and protects them from many forms of environmental stress. We report here the characterization of CER8/LACS1, one of nine Arabidopsis long-chain acyl-CoA synthetases thought to activate acyl chains. Mutations in LACS1 reduced the amount of wax in all chemical classes on the stem and leaf, except in the very long-chain fatty acid (VLCFA) class wherein acids longer than 24 carbons (C(24)) were elevated more than 155%. The C(16) cutin monomers on lacs1 were reduced by 37% and 22%, whereas the C(18) monomers were increased by 28% and 20% on stem and leaf, respectively. Amounts of wax and cutin on a lacs1-1 lacs2-3 double mutant were much lower than on either parent, and lacs1-1 lacs2-3 had much higher cuticular permeability than either parent. These additive effects indicate that LACS1 and LACS2 have overlapping functions in both wax and cutin synthesis. We demonstrated that LACS1 has synthetase activity for VLCFAs C(20)-C(30), with highest activity for C(30) acids. LACS1 thus appears to function as a very long-chain acyl-CoA synthetase in wax metabolism. Since C(16) but not C(18) cutin monomers are reduced in lacs1, and C(16) acids are the next most preferred acid (behind C(30)) by LACS1 in our assays, LACS1 also appears to be important for the incorporation of C(16) monomers into cutin polyester. As such, LACS1 defines a functionally novel acyl-CoA synthetase that preferentially modifies both VLCFAs for wax synthesis and long-chain (C(16)) fatty acids for cutin synthesis.

Sesame (Sesamum indicum L.) is one of the most important oilseed crops, having seeds and oil that are highly valued as a traditional health food. The objective of this study was to evaluate leaf cuticular wax constituents across a diverse selection of sesame cultivars, and the responses of these waxes to drought-induced wilting. Water-deficit was imposed on 18 sesame cultivars by withholding irrigation for 15d during the post-flowering stage, and the effect on seed yield and leaf waxes compared with a well-watered control. Leaf cuticular waxes were dominated by alkanes (59% of total wax), with aldehydes being the next-most abundant class. Compared to well-irrigated plants, drought treatment caused an increase in wax amount on most cultivars, with only three cultivars having a notable reduction. When expressed as an average across all cultivars, drought treatment caused a 30% increase in total wax amount, with a 34% increase in total alkanes, a 13% increase in aldehydes, and a 28% increase in the total of unknowns. In all cultivars, the major alkane constituents were the C27, C29, C31, C33, and C35 homologs, whereas the major aldehydes were the C30, C32, and C34 homologs, and drought exposure had only minor effects on the chain length distribution within these and other wax classes. Drought treatments caused a large decrease in seed yield per plant, but did not affect the mean weight of individual seeds, showing that sesame responds to post-flowering drought by reducing seed numbers, but not seed size. Seed yield was inversely correlated with the total wax amount (-0.466*), indicating that drought induction of leaf wax deposition does not contribute directly to seed set. Further studies are needed to elucidate the ecological role for induction of the alkane metabolic pathway by drought in regulating sesame plant survival and seed development in water-limiting environments.

The softening of fleshy fruits, such as tomato (Solanum lycopersicum), during ripening is generally reported to result principally from disassembly of the primary cell wall and middle lamella. However, unsuccessful attempts to prolong fruit firmness by suppressing the expression of a range of wall-modifying proteins in transgenic tomato fruits do not support such a simple model. 'Delayed Fruit Deterioration' (DFD) is a previously unreported tomato cultivar that provides a unique opportunity to assess the contribution of wall metabolism to fruit firmness, since DFD fruits exhibit minimal softening but undergo otherwise normal ripening, unlike all known nonsoftening tomato mutants reported to date. Wall disassembly, reduced intercellular adhesion, and the expression of genes associated with wall degradation were similar in DFD fruit and those of the normally softening 'Ailsa Craig'. However, ripening DFD fruit showed minimal transpirational water loss and substantially elevated cellular turgor. This allowed an evaluation of the relative contribution and timing of wall disassembly and water loss to fruit softening, which suggested that both processes have a critical influence. Biochemical and biomechanical analyses identified several unusual features of DFD cuticles and the data indicate that, as with wall metabolism, changes in cuticle composition and architecture are an integral and regulated part of the ripening program. A model is proposed in which the cuticle affects the softening of intact tomato fruit both directly, by providing a physical support, and indirectly, by regulating water status.

Insertional mutagenesis of Arabidopsis (Arabidopsis thaliana) was used to identify a novel recessive mutant, designated resurrection1 (rst1), which possesses a dramatic alteration in its cuticular waxes and produces shrunken nonviable seeds due to arrested embryo development. The RST1 gene sequence associated with these phenotypes was verified by three independent, allelic, insertion mutants, designated rst1-1, rst1-2, and rst1-3, with inserts in the first exon, 12th intron, and fourth exon, respectively. These three rst1 allelic mutants have nearly identical alterations in their wax profiles and embryo development. Compared to wild type, the wax on rst1 inflorescence stems is reduced nearly 60% in total amount, has a proportional reduction in aldehydes and aldehyde metabolites, and has a proportional increase in acids, primary alcohols, and esters. Compared to wild type, the C(29) alkanes on rst1 are nearly 6-fold lower, and the C(30) primary alcohols are 4-fold higher. These results indicate that rst1 causes shunting of most wax precursors away from alkane synthesis and into the primary-alcohol-producing branch of the pathway. In contrast to stems, the wax on rst1 mutant leaves increased roughly 43% in amount relative to the wild type, with the major increase occurring in the C(31) and C(33) alkanes. Unique among known wax mutants, approximately 70% of rst1 seeds are shrunken and nonviable, with these being randomly distributed within both inflorescence and silique. Viable seeds of rst1 are slightly larger than those of wild type, and although the viable rst1 seeds contain more total triacylglycerol-derived fatty acids, the proportions of these fatty acids are not significantly different from wild type. Shrunken seeds contain 34% of the fatty acids of wild-type seeds, with proportionally more palmitic, stearic, and oleic acids, and less of the longer and more desaturated homologs. Histological analysis of aborted rst1 seeds revealed that embryo development terminates at the approximate heart-shaped stage, whereas viable rst1 and wild-type embryos develop similarly. The RST1 gene encodes a predicted 1,841-amino acid novel protein with a molecular mass of 203.6 kD and a theoretical pI of 6.21. The RST1 transcript was found in all tissues examined including leaves, flowers, roots, stems, and siliques, but accumulation levels were not correlated with the degree to which different organs appeared affected by the mutation. The new RST1 gene reveals a novel genetic connection between lipid synthesis and embryo development; however, RST1's exact role is still quite unknown. The degree to which RST1 is associated with lipid signaling in development is an important focus of ongoing studies.

To shed new light on gene involvement in plant cuticular-wax production, 11 eceriferum (cer) mutants of Arabidopsis having dramatic alterations in wax composition of inflorescence stems were used to create 14 double cer mutants each with two homozygous recessive cer loci. A comprehensive analysis of stem waxes on these double mutants revealed unexpected CER gene interactions and new ideas about individual CER gene functions. Five of the 14 double cer mutants produced significantly more total wax than one of their respective cer parents, indicating from a genetic standpoint a partial bypassing (or complementation) of one cer mutation by the other. Eight of the 14 double cer mutants had alkane amounts lower than both respective cer parents, suggesting that most of these CER gene products play a major additive role in alkane synthesis. Other results suggested that some CER genes function in more than one step of the wax pathway, including those associated with sequential steps in acyl-CoA elongation. Surprisingly, complete epistasis was not observed for any of the cer gene combinations tested. Significant overlap or redundancy of genetic operations thus appears to be a central feature of wax metabolism. Future studies of CER gene product function, as well as the utilization of CER genes for crop improvement, must now account for the complex gene interactions described here.

Pseudomonas syringae relies on type III secretion system to deliver effector proteins into the host cell for parasitism. Type III genes are induced in planta, but host factors affecting the induction are poorly understood. Here we report on the identification of an Arabidopsis mutant, att1 (for aberrant induction of type three genes), that greatly enhances the expression of bacterial type III genes avrPto and hrpL. att1 plants display enhanced disease severity to a virulent strain of P. syringae, suggesting a role of ATT1 in disease resistance. ATT1 encodes CYP86A2, a cytochrome P450 monooxygenase catalyzing fatty acid oxidation. The cutin content is reduced to 30% in att1, indicating that CYP86A2 plays a major role in the biosynthesis of extracellular lipids. att1 has a loose cuticle membrane ultrastructure and shows increased permeability to water vapor, demonstrating the importance of the cuticle membrane in controlling water loss. The enhanced avrPto-luc expression is specific to att1, but not another cuticle mutant, wax2. The results suggest that certain cutin-related fatty acids synthesized by CYP86A2 may repress bacterial type III gene expression in the intercellular spaces.

Salt cress (Thellungiella halophila) is a small winter annual crucifer with a short life cycle. It has a small genome (about 2 x Arabidopsis) with high sequence identity (average 92%) with Arabidopsis, and can be genetically transformed by the simple floral dip procedure. It is capable of copious seed production. Salt cress is an extremophile native to harsh environments and can reproduce after exposure to extreme salinity (500 mm NaCl) or cold to -15 degrees C. It is a typical halophyte that accumulates NaCl at controlled rates and also dramatic levels of Pro (>150 mm) during exposure to high salinity. Stomata of salt cress are distributed on the leaf surface at higher density, but are less open than the stomata of Arabidopsis and respond to salt stress by closing more tightly. Leaves of salt cress are more succulent-like, have a second layer of palisade mesophyll cells, and are frequently shed during extreme salt stress. Roots of salt cress develop both an extra endodermis and cortex cell layer compared to Arabidopsis. Salt cress, although salt and cold tolerant, is not exceptionally tolerant of soil desiccation. We have isolated several ethyl methanesulfonate mutants of salt cress that have reduced salinity tolerance, which provide evidence that salt tolerance in this halophyte can be significantly affected by individual genetic loci. Analysis of salt cress expressed sequence tags provides evidence for the presence of paralogs, missing in the Arabidopsis genome, and for genes with abiotic stress-relevant functions. Hybridizations of salt cress RNA targets to an Arabidopsis whole-genome oligonucleotide array indicate that commonly stress-associated transcripts are expressed at a noticeably higher level in unstressed salt cress plants and are induced rapidly under stress. Efficient transformation of salt cress allows for simple gene exchange between Arabidopsis and salt cress. In addition, the generation of T-DNA-tagged mutant collections of salt cress, already in progress, will open the door to a new era of forward and reverse genetic studies of extremophile plant biology.

We conducted a novel non-visual screen for cuticular wax mutants in Arabidopsis thaliana (L.) Heynh. Using gas chromatography we screened over 1,200 ethyl methane sulfonate (EMS)-mutagenized lines for alterations in the major A. thaliana wild-type stem cuticular chemicals. Five lines showed distinct differences from the wild type and were further analyzed by gas chromatography and scanning electron microscopy. The five mutants were mapped to specific chromosome locations and tested for allelism with other wax mutant loci mapping to the same region. Toward this end, the mapping of the cuticular wax ( cer) mutants cer10 to cer20 was conducted to allow more efficient allelism tests with newly identified lines. From these five lines, we have identified three mutants defining novel genes that have been designated CER22, CER23, and CER24. Detailed stem and leaf chemistry has allowed us to place these novel mutants in specific steps of the cuticular wax biosynthetic pathway and to make hypotheses about the function of their gene products.

Insertional mutagenesis of Arabidopsis ecotype C24 was used to identify a novel mutant, designated wax2, that had alterations in both cuticle membrane and cuticular waxes. Arabidopsis mutants with altered cuticle membrane have not been reported previously. Compared with the wild type, the cuticle membrane of wax2 stems weighed 20.2% less, and when viewed using electron microscopy, it was 36.4% thicker, less opaque, and structurally disorganized. The total wax amount on wax2 leaves and stems was reduced by >78% and showed proportional deficiencies in the aldehydes, alkanes, secondary alcohols, and ketones, with increased acids, primary alcohols, and esters. Besides altered cuticle membranes, wax2 displayed postgenital fusion between aerial organs (especially in flower buds), reduced fertility under low humidity, increased epidermal permeability, and a reduction in stomatal index on adaxial and abaxial leaf surfaces. Thus, wax2 reveals a potential role for the cuticle as a suppressor of postgenital fusion and epidermal diffusion and as a mediator of both fertility and the development of epidermal architecture (via effects on stomatal index). The cloned WAX2 gene (verified by three independent allelic insertion mutants with identical phenotypes) codes for a predicted 632-amino acid integral membrane protein with a molecular mass of 72.3 kD and a theoretical pI of 8.78. WAX2 has six transmembrane domains, a His-rich diiron binding region at the N-terminal region, and a large soluble C-terminal domain. The N-terminal portion of WAX2 is homologous with members of the sterol desaturase family, whereas the C terminus of WAX2 is most similar to members of the short-chain dehydrogenase/reductase family. WAX2 has 32% identity to CER1, a protein required for wax production but not for cuticle membrane production. Based on these analyses, we predict that WAX2 has a metabolic function associated with both cuticle membrane and wax synthesis. These studies provide new insight into the genetics and biochemistry of plant cuticle production and elucidate new associations between the cuticle and diverse aspects of plant development.

Floral volatiles, which are small and generally water-insoluble, must move from their intracellular sites of synthesis through the outermost cuticle membrane before release from the flower surface. To determine whether petal cuticle might influence volatile emissions, we performed the first analysis of petal cuticle development and its association with the emission of flower volatiles using Antirrhinum majus L. (snapdragon) as a model system. Petal cuticular wax amount and composition, cuticle thickness and ultrastructure, and the amounts of internal and emitted methylbenzoate (the major snapdragon floral scent compound) were examined during 12 days, from flower opening to senescence. Normal (n-) alkanes were found to be the major wax class of snapdragon petals (29.0% to 34.3%) throughout the 12 days examined. Besides n-alkanes, snapdragon petals possessed significant amounts of methyl branched alkanes (23.6-27.8%) and hydroxy esters (12.0-14.0%). Hydroxy esters have not been previously reported in plants. Changes in amount of methylbenzoate inside the petals followed closely with levels of methylbenzoate emission, suggesting that snapdragon petal cuticle may provide little diffusive resistance to volatile emissions. Moreover, clear associations did not exist between methylbenzoate emission and the cuticle properties examined during development. Nevertheless, the unique wax composition of snapdragon petal cuticles shows similarities with those of other highly permeable cuticles, suggesting an adaptation that could permit rapid volatile emission by scented flowers.

We present cuticular wax chemical profiles for the leaves and stems of Arabidopsis wildtype Landsberg erecta and eleven isogenic eceriferum mutants: cer5, cer10 to cer15, and cer17 to cer20. These cer mutants have wax profiles that are different from those of wildtype in chemical chain length distribution, amount per chemical class, and/or total wax load. Analyses of detailed leaf and stem wax profiles for these cer mutants have allowed us to place some of these mutants at specific steps in wax production. The cer13 gene is predicted to affect release of the 30 carbon fatty acid from the elongation complex or the reduction of C30 fatty acid to C30 aldehyde. The CER19 gene product is predicted to be involved in C28 to C30 fatty acyl-CoA elongation. The CER20 gene is predicted to affect the oxidation of C29 alkane to C29 secondary alcohol. Several predicted gene products affect only stem specific steps in the wax pathway.

The present work was carried out to evaluate how plant growth and cultural practices influence the amount and composition of cuticular waxes on leaves of rose cultivars. The total amount of cuticular wax per leaf area was higher for rose cultivar Apollo Parade than for Charming Parade. Both cultivars had waxes dominated by alkanes, with the major alkanes being the C31 and C33 homologues. Primary alcohols were the next most abundant constituent class, with C26 as the dominant homologue. Compared with Charming Parade, Apollo Parade had higher proportions of its total wax load as primary alcohols but lower acids and aldehydes. The proportion of alkanes in the total load on these cultivars was similar. Commercially produced roses are routinely treated with paclobutrazol (PBZ) to retard growth. PBZ treatments caused a 10% increase in total wax load and changes in the proportions of certain wax constituents within 11 days of application. Notable was an increase in the total proportion of acids in the total load 25 days after PBZ application, primarily because of increased C28 acids. An alternative method of retarding plant growth is production of roses under limited water availability. When Apollo Parade roses experienced periods of moderate drought stress during production, the wax load per leaf area increased 14 and 8% above control levels at 24 and 38 days after imposition of drought, respectively. Drought caused similar changes in the proportions of individual wax constituents as did PBZ application.

Gas chromatography/mass spectrometry (GC/MS), with an ion trap mass analyzer, was used to examine the very-long-chain cuticular acid and certain non-acid wax constituents on the leaf sheath surface of Sorghum bicolor before and during 36 hours of light exposure. The mass spectra of the trimethylsilylated acids and alcohols did not match any of those published in searchable mass spectral libraries. The observed differences can be related to the interaction between water and the trimethylsilylated acids and alcohols. Understanding the observed mass spectra of the very-long-chain plant waxes is critical for studies that employ GC/MS with the ion trap mass analyzer to elucidate cuticular wax compositions on plants., (Copyright 2001 John Wiley & Sons, Ltd.)

Leaf sheath cuticular waxes on wild-type Sorghum bicolor were approximately 96% free fatty acids, with the C28 and C30 acids being 77 and 20% of these acids, respectively. Twelve mutants with markedly reduced wax load were characterized for chemical composition. In all of the 12 mutants, reduction in the amount of C28 and C30 acids accounted for essentially all of the reduction in total wax load relative to wildtype. The bm2 mutation caused a 99% reduction in total waxes. The bm4, bm5, bm6, bm7 and h10 mutations caused more than 91% reduction in total waxes, whereas the remaining six mutants, bm9, bm11, h7, h11, h12 and h13, caused between 35 and 78% reduction in total wax load. Relative to wild-type, bm4 caused a large increase in the absolute amount of C22, C24 and C26 acids, and reduction in the C28 and longer acids, suggesting that bm4 may suppress elongation of C26, acyl-CoA primarily. The h10 mutation increased the absolute amounts of the longest chain length acids, but reduced shorter acids, suggesting that h10 may suppress termination of acyl-CoA elongation. The bm6, bm9, bm11, h7, h11, h12 and h13 mutations increased the relative amounts, but not absolute amounts, of longer chain acids. Based on chemical composition alone, it is still uncertain which genes and their products were altered by these mutations. Nevertheless, these Sorghum cuticular wax mutants should provide a valuable resource for future studies to elucidate gene involvement in the biosynthesis of cuticular waxes, in particular, the very-long-chain fatty acids.