Progress 04/15/12 to 01/14/16
Outputs
Target Audience:The research focused on elucidating the biosynthesis and regulation of cuticle polymer formation and its importance in tomato fruit development, ripening and postharvest quality traits. We made substantial advances in identifying key cuticle related genes and regulatory processes that influence fruit developments and quality. The target audience is therefore broad and includes basic research scientists as well as breeders and growers, who will benefit from new insights into providing consumers with consistently high quality crops through to enhancing profitability. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Julian DeLellis-Mitch- phenotyping of abscisic acid mutant tomato fruits and characterization of the cd2 mutant. Graduate students: Nick Segerson- Biochemical characterization of CD1. Generation of transgenic tomato lines. Laetitia Martin- characterization of the cd2 mutant and evaluation of the relationship between cuticle and ABA signaling How have the results been disseminated to communities of interest?1) Published manuscripts, as described elsewhere in this report. 2) Oral/ Poster Presentations: "The formation and function of plant cuticles: tomato fruit as a model" Shanghai Normal University, Shanghai, China (2014). "Plant biotechnology: current and future needs and the challenges of the 21st century" Shanghai Jiao Tong University, Shanghai, China (2014). "Insights into the formation and functions of the plant cuticle using functional genomic technologies" Shanghai Jiao Tong University, Shanghai, China (2014). 'High Spatial Resolution Functional Genomics of Fleshy Fruit Development and Environmental Responses", NSF Awardee meeting, Washington DC (2014). "The Tomato Expression Atlas: a new database for interrogating tomato gene expression and co-expression at the cell-type level of resolution". International SOL Meeting, Porto Seguro, Brazil (2014). "A Systems Biology Analysis of the Formation, Function and Evolutionary Origins of Plant Cuticles" University of Georgia, Athens, GA (2015) "A New Platform and Database for Interrogating Tomato Gene Expression and Co-expression with Cell-type Resolution" Plant Genomics Congress, London, U.K. (2015) "Probing the Assembly and Functions of Plant Cell Wall Structural Lipids" Plant Cell Wall Gordon Conference, Bentley University, Waltham, MA (2015) "The Formation, Function and Evolution of the Plant Cuticle: Views through the Lens ofCell Type-Specific Genomics" Wake Forest University (2015) "The Structure, Formation and Evolutionary Origins of Plant Cuticles" University of British Columbia (2016) What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This project aims to understand the formation and functions of plant 'skins', or cuticles, and more specifically the roles of fruit cuticles in resisting desiccation, spoilage and infection by microbes. The results of the research can be used to develop strategies to improve fruit quality and post-harvest handling, storage and processing. This would be achieved firstly through the generation of fruit with substantially enhanced longevity, thereby potentially extending the ripening time on the vine and elevating crop nutritional status. The potential impact of the research is broad and should extend from providing individual consumers with consistently high quality crops through to enhancing profitability for breeders and growers. Much remains to be learnt about cuticle structure and formation, and the basis of the apparent regulatory relationship with abiotic stresses and the plant hormone abscisic acid (ABA). This project has lead to a more comprehensive understanding of these crucial aspects of fruit biology, using tomato fruit as a model system, and has helped understand how plants respond to, and tolerate, biotic and abiotic stresses. Using two tomato mutants that have drastic (>90%) reductions in levels of the polymer cutin in their fruit cuticles (cutin deficient 1 and -2; cd1 and cd2) as a centerpiece, the research provided new insights into the biosynthesis and regulation of cutin formation and the importance of this structure in tomato fruit development, ripening and postharvest quality traits. Specifically, the research lead to the discovery of a key enzyme that is responsible for cutin formation and it was shown that this same class of enzymes also make cuticles in other plants, including crops, and were likely essential for land plant evolution. This by itself has great implications for helping researchers understand how plants originally colonized land and can tolerate dry habitats. In addition, the research identified two critical regulators of for cuticle formation and revealed that, unexpectedly, plants synthesize cuticles on their leaves and fruits in different ways. Collectively these results have moved the field forward substantially such that we are now closer to being able to 'custom-design' plant skins for crops to help improve quality, shelf life and drought tolerance. Specific project objectives: (A) Characterize the newly discovered tomato cutin synthase CD1 and a putative paralog. We previously reported that cutin polymerization in tomato (Solanum lycopersicum) fruit occurs via transesterification of hydroxyacylglycerol precursors, catalyzed by the GDSL-motif lipase/hydrolase family protein (GDSL) Cutin Deficient 1 (CD1). Our results reveal a conserved mechanism of cutin polyester synthesis in land plants, and suggest that elaborations of the linear polymer, such as branching or crosslinking, may require additional, as yet unknown, factors. We have now shown that a paralog of CUS1, previously called CD1B, and now renamed CUS2, also has cutin polymerase activity that appears to show some differences from that of CUS1. We have generated predicted 3D protein structural models of the CUS proteins, as well as a structurally related cutinase, and have developed a mechanistic model of how cutin is polymerized, as well as the first structure-function model that differentiates cutin assembly and depolymerization. We have generated transgenic tomato lines with suppressed expression of CUS2, which have reduced levels of polymeric cutin, and although the fruit phenotype is not as severe as that of CUS1 mutant fruit. We have generated a model explaining the roles of different CUS genes in regulating the formation of cutin over the plant surface. (B) Test the hypothesis that the tomato gene CD2 encodes a transcriptional regulator of cuticle biosynthesis, mediated through interaction with abscisic acid (ABA); An important goal of this project was to identify key regulatory factors including transcription factors (TFs), and phytohormones, which regulate cuticle formation. We previously demonstrated that the TF CD2 is required form cutin formation. We used laser capture microdissection (LCM) coupled with RNA-seq profiling to contrast the epidermal transcriptomes of wild type and the cd2 mutant fruits and identified suites of differentially expressed genes, of which including many are already known to be involved in lipid biosynthesis. We also showed that CD2 is predominantly expressed in the outer and inner epidermis of the tomato pericarp, and at the subcellular level localizes to the nucleus; consistent with a role in regulating cuticle formation. We used CHIP-Seq analysis to identify CD2 regulated genes and associated CD2-binding motifs, as well as a co-immunoprecipitation analysis to uncover CD2-interacting proteins. A manuscript presenting those results is in preparation. As well as the CD2 analysis, we also evaluated the potential function of the phytohormone abscisic acid (ABA) in regulating cuticle formation. ABA has been shown to trigger an increase of cuticular waxes in response to drought. However, it is not known if ABA regulates the expression of genes involved in cuticle formation during plant development. To answer this question, the impact of ABA deficiency on cuticle formation was investigated by using three ABA-deficient tomato mutants. Microscopy and biochemical analyses revealed that the polyester cutin was reduced in the leaf of the mutants and was prone to structural abnormalities.Application of ABA partially rescued these phenotype, confirming that the ABA deficiency was responsible for the observed phenotype. Additionally, transcript expression analyses of genes involved in cutin and wax biosynthesis, transport and regulation, suggested a general down-regulation of this pathway in the ABA mutants. Taken together, these data suggest a role for ABA in leaf cuticle development independent of the effects of osmotic stress and show for the first time an action of ABA on cutin formation. A manuscript presenting those results was positively reviewed by Plant Physiology and a revised version is about to be submitted. (C) Analyze the structure, composition and function of the previously uncharacterized tomato fruit inner epidermal cuticle; We showed that the inner epidermal fruit cells form a thin cuticle with a cutin composition that differs from that of the far thicker outer epidermal cuticle. The inner epidermis cutin, shown by FTIR spectroscopy to exhibit a typical polyester structure, in that it is primarily composed of 16-hydroxypalmitic acid, and the outer epidermis is rich in 10,16-dihydroxypalmitic acid. This difference is likely attributed to the relative expression of a midchain hydroxylase of the CYP77A subfamily, the transcript levels of which are 40 times greater in the outer than inner epidermis. Without a secondary hydroxyl, the inner epidermis cutin matrix is likely more linear and more extensible. The developmental or physiological function of the inner cuticle is still unclear, but will be addressed in proposed future studies through spatially targeted gene knockouts. (D) Assess the consequences of cuticle defects on the transcriptome landscape of tomato fruit and test the hypothesis that the cuticle plays multiple roles in stress responses. This objective was addressed in part through the analyses outlined in Objective B, through RNA-Seq profiling of CD2 fruits. This analysis has uncovered a previously unexpected relationship between the synthesis of cuticle components, cell wall polysaccharides and a range of phenylpropanoids. An exciting outcome is the indication or potential mechanisms by which the cuticle is anchored to the underling polysaccharide cell wall. We have identified candidate proteins that perform this cross-linking, and possible restructuring, and we are assessing their function at present.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2016
Citation:
Martin, L.B.B., Fich, E.A., Domozych, D.S. and Rose, J.K.C. (2015) Cuticle biosynthesis is developmentally regulated by abscisic acid in tomato leaves but not in fruit. Plant Physiology.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Fich, E.A., Segerson, N.A. and Rose, J.K.C. (2016) The plant polyester cutin: biosynthesis, structure and biological roles. Annual Plant Reviews.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Chatterjee, S., Matas, A.J., Isaacson, T., Kehlet, C., Rose, J.K.C. and Stark, R.E. (2016) Solid-state 13C NMR delineates the architectural design of biopolyesters in native and genetically altered tomato fruit cuticles. Biomacromolecules 17: 215-24.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2016
Citation:
Martin, L.B.B., Sherwood, R.W., Nicklay, J.J., Yang, Y., Muratore-Schroeder, T.L., Anderson, E.T., Thannhauser, T.W, Rose, J.K.C. and Zhang, S. (2015) Application of wide selected-ion monitoring (WiSIM)-data independent acquisition (DIA) to identify tomato fruit proteins regulated by the transcription factor CUTIN DEFICIENT-2. IN press, Proteomics.
|
Progress 04/15/13 to 04/14/14
Outputs
Target Audience: The proposed research is helping elucidate the biosynthesis and regulation of cuticle polymer formation and its importance in tomato fruit development, ripening and postharvest quality traits. We have already made significant advances in identifying key cuticle related genes and regulatory processes that influence fruit developments and quality. The target audience is therefore broad and includes basic research scientists as well as breeders and growers, who will benefit from new insights into providing consumers with consistently high quality crops through to enhancing profitability. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Undergraduate students Julian DeLellis-Mitch- phenotyping of abscisic acid mutant tomato fruits and characterization of the cd2 mutant. Graduate students: Nick Segerson- Biochemical characterization of CD1. Generation of transgenic tomato lines. Laetitia Martin- characterization of the cd2 mutant and evaluation of the relationship between cuticle and ABA signaling Eric Fich- Generation and characterization of transgenic tomato lines. How have the results been disseminated to communities of interest? 1) Published manuscripts, as described elsewhere in this report. 2) Oral/ Poster Presentations: “Peeling open the structure, function and evolutionary origins of the plant cuticle” Department of Plant Biology seminar, Cornell University, NY (2013). “The structure, functions and biosynthetic pathway of the tomato fruit cuticle”, JSOL meeting, Tsukuba, Japan (2013). “Peeling apart the structure, functions and diversity of fruit cuticles” Annual meeting Society for Experimental Biology, Valencia, Spain (2013). “Understanding the molecular basis of fruit softening: historical perspectives and future directions” Centro de Investigación Científica de Yucatán A.C, Mexico (2013). “Peeling apart the structures, functions and diversity of fruit cuticles: what, where, how and when? Plant Apoplastic Diffusion Barriers (PADIBA) meeting, Lausanne, Switzerland (2013). “Dissecting the structure, functions and diversity of tomato fruit cuticles” SOL 2013 Meeting, Beijing, China (2013). “The formation and function of plant cuticles: tomato fruit as a model” Institute of Plant Physiology and Ecology, Shanghai, China (2013). “Multilayered strategies for attack, defense and counter-defense in the plant cell wall”. University of Copenhagen (2013). “The formation and function of plant cuticles: tomato fruit as a model” Shanghai Normal University, Shanghai, China (2014). “Insights into the formation and functions of the plant cuticle using functional genomic technologies” Shanghai Jiao Tong University, Shanghai, China (2014). What do you plan to do during the next reporting period to accomplish the goals? See section above "What was accomplished under these goals?"
Impacts
What was accomplished under these goals?
(1) We previously reported that cutin polymerization in tomato (Solanum lycopersicum) fruit occurs via transesterification of hydroxyacylglycerol precursors, catalyzed by the GDSL-motif lipase/hydrolase family protein (GDSL) Cutin Deficient 1 (CD1). We undertook additional biochemical characterization of CD1 and putative orthologs from Arabidopsis thaliana and the moss Physcomitrella patens, revealing a distinct clade of cutin synthases within the large GDSL super-family. We have demonstrated that members of this ancient and conserved family of cutin synthase-like (CUS) proteins act as polyester synthases with negligible hydrolytic activity. Moreover, solution-state NMR analysis indicates that CD1 (renamed CUS1) catalyzes the formation of primarily linear cutin oligomeric products in vitro. These results reveal a conserved mechanism of cutin polyester synthesis in land plants, and suggest that elaborations of the linear polymer, such as branching or cross-linking, may require additional, as yet unknown, factors. These results have been published in The Plant Journal. (2) We have also recently determined that a paralog of CUS1, previously called CD1B and now renamed CUS2, also has cutin polymerase activity. We have generated transgenic tomato lines with suppressed expression of CUS2 and a preliminary analysis of fruits from the T1 lines indicates that they have reduced levels of polymeric cutin, although the phenotype is not as severe as that of CUS1 mutant fruit. We are currently testing the hypothesis that CUS1 and CUS2 act synergistically to catalyze cutin polymerization in vivo. (3) An important goal of this project is to identify key regulatory factors including transcription factors (TFs), and phytohormones, which regulate cuticle formation. We previously demonstrated that the TF CD2 is required form cutin formation. We have now used laser capture microdissection (LCM) coupled with RNA-seq profiling to contrast the epidermal transcriptomes of wild type and the cd2 mutant fruits and identified suites of differentially expressed genes, of which including many are already known to be involved in lipid biosynthesis. These genes encode proteins that collectively span many stages of cuticle formation, including early lipid biosynthesis in the chloroplast, subsequent modification of cuticle precursors in the cytosol and endoplasmic reticulum, lipid trafficking and secretion to the apoplast and cuticle assembly. Additional genes of unknown function showed substantial downregulation and represent candidates for new components of the cuticle biosynthetic pathway. We have also shown that CD2 is predominantly expressed in the outer and inner epidermis of the tomato pericarp, and at the subcellular level localizes to the nucleus; consistent with a role in regulating cuticle formation. More recently we have performed as CHIP-Seq analysis to identify CD2 regulated genes and associated CD2-binding motifs, as well as a co-immunoprecipitation analysis to uncover CD2-interacting proteins. As well as the CD2 analysis, we have been evaluating the potential function of the phytohormone abscisic acid (ABA) in regulating cuticle formation. Results to date indicate that ABA indeed plays a role in cuticle formation in both leaves and fruit, as ABA deficient tomato mutants have abnormal cuticle structures. However, the relationship appears to be more complex than originally anticipated as the mutant leaf cuticles are thinner than wild type (WT) leaves, while those of fruits are considerably thicker. We have determined that ABA levels in fruits of the cd2 mutant are significantly reduced during later fruit maturation and ripening, but not in the early stages of fruit. We have also determined that numerous genes implicated in biotic and abiotic stresses are differentially expressed between cd2 and its corresponding WT and interestingly, genes involved in ABA responses are up-regulated in cd2, suggesting a modulation of ABA biosynthesis and downstream signaling. The underlying mechanisms are currently under investigation. (4) As part of this project, we reported in a paper in The Plant Journal that fruit cuticles of wild tomato species show considerable diversity in waxes and cutin. In the last year we have studied the leaf and fruit cuticles of the stress tolerant wild tomato species S. pennellii, as part of an international genome sequencing consortium. We observed a three-fold greater abundance of waxes, primarily very long chain alkanes, and higher levels of triterpenoids in S.pennellii. These two features are, with increasing frequency, correlated with greater resistance to water flux across the cuticle. Additionally, the phenylpropanoid component of S.pennellii cutin was reduced to ~20% of that found in S. lycopersicum. We thus compared the expression of orthologs from known cuticle biosynthesis related genes between the two species. The expression of CER1, an ortholog of a key regulator of alkane levels in Arabidopsis thaliana, was substantially higher in S. pennellii, consistent with the greater abundance of alkanes. In contrast, a consistent differential expression pattern was not evident for the genes associated with the biosynthesis of aliphatic cutin components, in agreement with the absence of major differences in cutin biochemical data. Lastly, the abundance of cutin phenylpropanoids in S. lycopersicum correlates with the much higher expression of two feruloyltransferase homologs and PAL1, the phenylpropanoid pathway gateway enzyme. This comparative analysis of the genome sequences of S. pennellii and S.lycopersicum, together with the gene expression studies, demonstrates the great potential for elucidating the ecological constraints and adaptations underlying traits of great agronomic importance. These results are presented in a manuscript that has just been published online in Nature Genetics. Additional related accomplishments citing this grant: (5) As part of a study of the role and diversity of cutin synthases, we have determined that the moss Physcomitrella patens, an extant relative of the earliest terrestrial plants, has a cuticle that is similar in structure and chemical composition to those of tomato and other angiosperms. To test whether the underlying cuticle biosynthetic pathways were also shared amongst distant plant lineages, we generated a genetic knockout of the moss ABCG transporter PpABCG7, a putative ortholog of A. thaliana ABCG transporters involved in cuticle precursor trafficking. We have shown that this mutant is severely deficient in cuticular wax accumulation and has a reduced tolerance of desiccation stress compared to 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 over 450 million years. A manuscript presenting these data was published in The Plant Cell. (6) In collaboration with a research group in Mexico, we have been studying cuticle biosynthesis in mango (Mangifera indica) fruit, using a similar LCM/RNA-seq approach to identify epidermal specific genes. This fruit synthesizes large amounts of waxes during postharvest storage, and so it is anticipated that this study will provide insights into not only the cutin and wax biosynthetic pathways, and their degree of conservation with analogous pathways in tomato, but also suggest strategies for enhancing fruit shelf life.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Yeats, T.H., Buda, G.J., Wang, Z., Moyle, L.C., Jetter, R., Schaffer, A.A. and Rose, J.K.C. (2012) The fruit cuticles of wild tomato species exhibit architectural and chemical diversity, providing a new model for studying the evolution of cuticle function. The Plant Journal 69: 655-666.
- Type:
Book Chapters
Status:
Published
Year Published:
2013
Citation:
Ruiz-May, E. and Rose, J.K.C. (2013) Cell wall architecture and metabolism in ripening fruit and the complex relationship with softening. In The Molecular Biology and Biochemistry of Fruit Ripening. Eds. J.J. Giovannoni, M. Poole, G. B. Seymour and G.A. Tucker: Pub. Wiley pp. 163-187.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Yeats, T.H. and Rose, J.K.C. (2013) The formation and function of plant cuticles. Plant Physiology 163: 5-20.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Buda, G.J., Barnes, W.J., Fich, E.A., Park, S., Yeats, T.H., Zhao, L., Domozych, D. and Rose, J.K.C. (2013) An ABCG transporter is required for cuticular wax deposition and desiccation tolerance in the moss Physcomitrella patens. The Plant Cell 25: 4,000-4013.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Yeats, T.H., Huang, W., Chatterjee, S., Viart, H. M.-F., Clausen, M.H., Stark, R.E. and Rose, J.K.C. (2014) Tomato cutin deficient 1 (CD1) and putative orthologs comprise an ancient family of cutin synthase-like (CUS) proteins that are conserved among land plants. The Plant Journal 77: 667-675.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Bolger, A., Scossa, F., Bolger, M.E., Lanz, C., Maumus, F., Tohge, T., Quesneville, H., Alseekh, S., S�rensen, I., Lichenstein, G., Fich, E.A., Conte, M., Keller, H., Schneeberger, K., Schwacke, R., Ofner, I., Vrebalov, J., Xu, Y., Osorio, S., Aflitos, S.A., Schijlen, E., Jim�nez-Gom�z, J.M., Ryngajllo, M., Kimura, S., Kumar, R., Koenig, D., Headland, L.R., Maloof, J.N., Sinha, N., van Ham, R.C.H.J., Lankhorst, R.K., Mao, L., Vogel, A., Arsova, B., Panstruga, R., Fei, Z., Rose, J.K.C., Zamir, D., Carrari, F., Giovannoni, J.J., Weigel, D., Usadel, B. and Fernie, A.R. (2014) The genome of the highly stress tolerant wild tomato species Solanum pennellii (Nature Genetics Published online July 27 2014doi:10.1038/ng.3046).
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Martin, L.B.B. and Rose, J.K.C. (2014) Theres more than one way to skin a fruit: formation and functions of fruit cuticles. Journal of Experimental Botany published online July 15, 2014, doi: 10.1093/jxb/eru301.
|
Progress 04/15/12 to 04/14/13
Outputs
Target Audience: The proposed research is helping elucidate the biosynthesis and regulation of cuticle polymer formation and its importance in tomato fruit development, ripening and postharvest quality traits. We have already made significant advances in identifying key cuticle related genes and regulatory processes that influence fruti developments and quality. The target audience is therefore broad and includes both basic research scientists as well as breeders and growers, who will benefit from new insights into providing consumers with consistently high quality crops through to enhancing profitability. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Undergraduate students: William Barnes- analysis of tomato mutant fruit cuticle composition and effects on development. Julian DeLellis-Mitch- phenotyping of abscisic acid mutant tomato fruits and characterization of the cd2 mutant. Graduate students: Trevor Yeats- Biochemical characterization of CD1. Generation of transgenic tomato lines. Laetitia Martin- characterization of the cd2 mutant and evaluation of the relationship between cuticle and ABA signaling Eric Fich- Generation and characterization of transgenic tomato lines. How have the results been disseminated to communities of interest? 1) Published manuscripts, as described eslewhere in this report. 2) Oral/ Poster Presentations: “Tissue and cell type specific transcriptome profiling of tomato fruit provides insights into metabolic and regulatory specialization?” Advanced Technology Seminar Series, Cornell (2012). “Peeling apart the structural and functional complexity of the cuticularized plant cell wall” Genomic Science Contractors-Grantees Meeting, Washington DC (2012). “The structure, function and origins of plant cell wall polymers: a fruit perspective” Beijing Agricultural University, Beijing, China (2012). “The structure, function and origins of plant cell wall polymers: a fruit perspective” Shanghai Jiao Tong University, Shanghai, China (2012). “The identification and in vitro activity of a plant cutin synthase” NYU-Poly, New York (2012) "The regulation of cutin synthesis in tomato fruit" Plant Cell Walls Gordon Research Conference and Plant Cell Walls Gordon Research Seminar, Colby College (2012) What do you plan to do during the next reporting period to accomplish the goals? (A) Characterize the newly discovered tomato cutin synthase CD1 and a putative paralog CD1B. We hypothesize that a closely related tomato CUS protein SlCUS2 (formerly CD1B), is responsible for polymerization of a subset of cutin in the outer epidermal cuticle, possibly in a developmentally distinct pattern from SlCUS1. We have now generated transgenic tomato lines with suppressed expression of SlCUS2 and will next characterize fruits from those lines to test this hypothesis. (B) Test the hypothesis that CD2 encodes a transcriptional regulator of cuticle biosynthesis, mediated through interaction with ABA. Results to date suggest that CD2 in indeed a transcription factor that both regulates cutin biosynthesis pathway and is also implicated in the lipid homeostasis of the cell. Future experiments will investigate the interaction with ABA signaling and identify putative DNA binding motifs of the promoters of the CD2 regulated genes. (C) Analyze the structure, composition and function of the previously uncharacterized tomato fruit inner epidermal cuticle. We have now identified a suit of cutin biosynthesis genes that are predominantly expressed in the inner epidermis and that likely contribute to the synthesis of this structure. Three of these genes have been targeted for suppression using an RNAi strategy and the primary transformants have been generated. (D) Assess the consequences of cuticle or ABA defects on the transcriptome of the tomato fruit pericarp and test the hypothesis that the cuticle plays multiple roles in stress responses. A strong cuticle defective phenotype is already known to exist in leaves of the sitiens tomato mutant but our preliminary histological data suggest that the fruit cuticle of all the ABA mutant lines shows no major structural perturbation. Similarly, while it is known that the leaves of the ABA mutants wilt faster than their WT equivalents, we have determined that the permeability of their fruit cuticles is not significantly different. This suggests that ABA is not a key regulator of cuticle formation during normal growth and development in tomato fruits. Future experiments will examine the differences between the leaf and fruit cuticle phenotypes of the ABA deficient lines in more detail.
Impacts
What was accomplished under these goals?
(A) Characterize the newly discovered tomato cutin synthase CD1 and a putative paralog CD1B. We have recently completed a more detailed biochemical study of the enzymatic features of the CD1 protein (now renamed SlCUS1) and show that it exclusively catalyzes the formation of linear, and not branched, oligomeric products. This provides the first detailed insights into the mechanisms of cutin polymer assembly and suggests that there are additional factors required for the formation of branched or cross-linked cutin. We have also shown that homologous enzymes with the same activity are present across the taxonomic spectrum of extant land plants, indicating that a conserved cutin biosynthesis pathway evolved prior to the divergence of bryophytes and tracheophytes. Furthermore, we have determined through phylogenetic analysis that these proteins form a distinct clade within the GDSL superfamily, members of which we now designate as cutin synthase (CUS). A manuscript describing these resultsis currently under review. (B) Test the hypothesis that CD2 encodes a transcriptional regulator of cuticle biosynthesis, mediated through interaction with ABA. The tomato CD2 gene encodes a putative transcription factor (TF), a mutation in which results in severe reduction in polymeric cutin. An important goal of this project is to address the hypothesis that CD2 regulates core suites of cuticle biosynthetic genes and other key regulatory factors including other TFs, and potentially ABA. To identify genes regulated by CD2, we used laser capture microdissection coupled with RNA-seq profiling to contrast the epidermal transcriptomes (of wild type and the cd2 mutant fruits at 15 days after anthesis, a developmental stage of maximal cuticle biosynthesis). Approximately 60 genes were differentially expressed, of which more than half are already known to be involved in lipid biosynthesis. These genes encode proteins that collectively span many stages of cuticle formation, including early lipid biosynthesis in the chloroplast, subsequent modification of cuticle precursors in the cytosol and endoplasmic reticulum, lipid trafficking and secretion to the apoplast and cuticle assembly. Additional genes of unknown function showed substantial downregulation and represent candidates for new components of the cuticle biosynthetic pathway. Furthermore, CD2 fused to a fluorescent marker protein localizes in the nucleus when expressed transiently in onion epidermal cells and GUS reporter lines showed that CD2 is expressed mainly in the outer and inner epidermis of the tomato pericarp, tissues responsible for cuticle biosynthesis. (C) Analyze the structure, composition and function of the previously uncharacterized tomato fruit inner epidermal cuticle. During our microscopic studies of the tomato fruit outer epidermal cuticle, we identified an ‘internal’ cuticle: a lipid rich layer covering the inner epidermis, adjacent to the locule, which defines the inner boundary of the pericarp. As far as we know, this macromolecular structure has never before been characterized and we have found only two references to its possible existence in the literature. This exciting discovery raises many questions related to whether the inner and outer epidermal cuticles have the same composition, when the inner is synthesized, its role in growth and development, and whether the associated biosynthetic genes are the same as those of the outer epidermal cuticle. We have shown by FTIR spectroscopy that the inner epidermal cuticle exhibits a typical polyester structure, is primarily composed of 16-hydroxypalmitic acid, while the outer epidermal has higher levels of 10,16-dihydroxypalmitic acid. This difference is likely attributed to the relative expression of a mid-chain hydroxylase of the CYP77A subfamily, the transcript levels of which are 40 times greater in the outer than inner epidermis. In addition, comparative NMR analyses of dewaxed cuticles revealed that the ratio of carboxyls to long-chain aliphatic moieties was comparable to, and slightly greater for, inner versus outer dewaxed cuticles. A similar trend was found for the ratio of total oxygenated aliphatic carbons (CHO and CH2O) to long-chain aliphatic groups. This finding further emphasizes the cutin-like compositional similarity. However, the (CHO/CH2O) ratio was twofold lower for the inner cuticle, which is consistent with a diminished proportion of cross-linkable structural elements in the inner cuticle. Taken together, these data suggest that the cutin polymers of the inner epidermal cuticle are less cross linked than those of the outer cuticle and that the polyester matrix is more pliable. (D) Assess the consequences of cuticle or ABA defects on the transcriptome of the tomato fruit pericarp and test the hypothesis that the cuticle plays multiple roles in stress responses. Numerous lines of evidence suggest that ABA signaling and cuticle biosynthesis are closely related, and indeed both are involved in limiting water loss. Two hypotheses that will be tested in this Objective are: 1) ABA is a key regulator of cuticle formation; and 2) cuticle composition and integrity are key factors that influence water relations in the fruit and that there is an associated feedback mechanism that alters ABA levels. We have determined that ABA levels in fruits of the cd2 mutant are significantly reduced during later fruit maturation and ripening, but not in the early stages of fruit. We have also determined that numerous genes implicated in biotic and abiotic stresses are differentially expressed between cd2 and its corresponding WT and interestingly, genes involved in ABA responses are up-regulated in cd2, suggesting a modulation of ABA biosynthesis and downstream signaling. In parallel, we have been testing the hypothesis that the cuticle is impaired in the ABA deficient tomato mutants sitiens, flacca and notabilis. Additional accomplishments; As part of a study of the role and diversity of cutin synthases, we have determined 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 tomato and other angiosperms.To test whether the underlying cuticle biosynthetic pathways were also shared amongst distant plant lineages, we generated a genetic knockout of the moss ABCG transporter PpABCG7, a putative ortholog of Arabidopsis thaliana ABCG transporters involved in cuticle precursor trafficking. We have shown that this mutant is severely deficient in cuticular wax accumulation and has a reduced tolerance of desiccation stress compared to 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 over 450 million years. A manuscript presenting these data will be submitted in the near future.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Yeats, T.H., Martin, L.B.B., Viart, H. M-F., Isaacson, T., He, Y., Zhao, L., Matas, A.J., Buda, G., Domozych, D.S., Clausen, M.H. and Rose, J.K.C. (2012) The identification of cutin synthase: formation of the plant polyester cutin. Nature Chemical Biology 8: 609-611.
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2013
Citation:
Ruiz-May, E. and Rose, J.K.C. (2013) Cell wall architecture and metabolism in ripening fruit and the complex relationship with softening. In The Molecular Biology and Biochemistry of Fruit Ripening. Eds. J.J. Giovannoni, M. Poole, G. B. Seymour and G.A. Tucker: Pub. Wiley (In press).
- Type:
Journal Articles
Status:
Under Review
Year Published:
2013
Citation:
Yeats, T.H. and Rose, J.K.C. (2013) The formation and function of plant cuticles. Plant Physiology.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2013
Citation:
Yeats, T.H., Huang, W., Chatterjee, S., Viart, H. M.-F., Clausen, M.H., Stark, R.E. and Rose, J.K.C. (2013) Biochemical characterization of an ancient family of cutin synthase (CUS) proteins that are conserved among land plants. The Journal of Biological Chemistry.
|