Progress 01/15/21 to 01/14/25
Outputs
Target Audience:The target audience comprises plant scientists, other researchers involved in agricultural/horticultural biotechnology and breeding, as well as related stakeholders such as growers and breeders who will utilize information generated through this project to develop strategies for enhanced fruit quality, shelf life and food security. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Through this project, two postdocs a graduate student and four undergraduate students were engaged in a range of educational and training activities that are made available at Cornell, spanning lab-based experiments and field work, with an emphasis of career development in the area of crop production and food security. In addition, the postdocs and students participated in public presentation of their research at meetings and conferences, and were engaged in public outreach to early career scientists. How have the results been disseminated to communities of interest?The results have been communicated through publications in peer-reviewed journals, a webinar, and poster presentation at internation meetings, as well as a workshop with the America Fruit Growers' Association and industry stakeholders. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Identification of GDSL-hydrolase candidates in Solanum lycopersicum and biochemical characterization - Of the 107 tomato GDSL-hydrolases, 40 show a gene expression in inner and/or outer fruit epidermis, which a site of cutin deposition, and this was these genes were the focus of our studies. A phylogenetic tree of tomato GDSL-hydrolase proteins was generated with the presence of annotated GDSL-hydrolases from other species to provide insights into enzymatic function. As we previously reported (Yeats et al. 2014), the CUTIN SYNTHASE (CUS) proteins form an ancient family that is conserved among land plants. In tomato, it consists of five members with homology to evolutionary distant CUS enzymes. Investigations by other groups have revealed the involvement of several members of the GDSL-hydrolases from Arabidopsis in the synthesis of suberin, a polymer with similarities to cutin. The SUBERIN SYNTHASE (SUS) family clade in tomato has five members. Interestingly, despite its attributed role in suberin biosynthesis, AtSUS4 from Arabidopsis is a close homolog of AtCDEF1 (of the CUTIN DESTUCING FACTOR subfamily, now renamed CUSB), which acts as a cutin and suberin hydrolase. We delineated a clade of 9 proteins in the tomato CDEF family with confident bootstrap values. We completed a spatiotemporal profile of most of these genes and uncovered an unexpected gradient of expression of some of these genes down the latitudinal axis of the fruit. Our studies have shown that this correlates with cutin structure and composition, which we have now demonstrated, varies at the poles or the equator of the fruit. - We expressed a total of 14 tomato GDSL enzymes as His-tagged recombinant proteins in Nicotiana benthamiana, including CDEF and CUS subtypes) for enzymatic analyses and examined: 1) cutin polymerization activity using both MALDI-TOF and electrospray ionization (ESI) mass spectrometry to detect cutin oligomers, and a glycerol assay to detect acyltransferase activity; 2) cutin remodeling activity using a novel semi-in vivo assay that we developed using permeance of the inner fruit epidermal cuticle. We have now completed the analysis of these isoforms. - The GDSL-hydrolases that showed high expression in the epidermis were tested as priority candidates in similar in vitro experiments to characterize their interaction with the tomato cutin precursor, 2-MAG. We also expressed CUS2 and CDEF1 to characterize their functional specificity. The hydrolytic activity of AtCDEF1 toward the 2-MAG substrate was strong, with 100% conversion to 10,16-diOH-C16 acid but was absent from other enzymes with putative hydrolytic activity. However, cutin polymerization products were obtained through the activity of CUS and, surprisingly, CDEF enzymes. CDEF1 converted a comparable amount of substrate to oligomer products as CUS1; however with a higher proportion of dimers. As expected, CUS2 had polymerizing activity, although it was lower than CUS1, and no activity was observed with. The activity of CUS2, CDEF1, and CDEF1 were confirmed by a glycerol release assay following transesterification or hydrolytic activity on 2-MAG substrate. Interestingly, the result of the enzymatic incubation performed semi-in vivo (i.e. directly on a native cutin support that is the fruit inner epidermis) revealed differences between CUS1 and CDEF1. Although the reaction mixture containing CDEF1 and the 2-MAG substrate led to the production of oligomers, which are detected after incubation, no 2-MAG nor oligomers were recovered from the reaction mixture containing initially CUS1 and 2-MAG. This result suggests that 2-MAG was fully polymerized to the native cutin by CUS1, while CDEF1 may have a lower polymerization activity with the native cutin or is also able to release oligomers from it in a dynamic remodeling activity. Taken together, these results provide new insights into the relative roles of these proteins in cutin assembly and remodeling. A manuscript presenting these results, and a new model of cutin dynamics has been submitted to bioRxiv (Pulippe et al 2025). - We characterized the functions of three tomato CDEF genes (SlCDEF1, SlCDEF2 and SlCDEF3) in tomato fruit using CRISPR-Cas9 gene editing, as well as a SlCDEF1x SlCDEF2 double knockout lines and a triple knockout line, all in the M82 cultivar background. We assessed a range of fruit phenotypes: development rate, size/shape/morphology; transpirational water loss from entire fruit; permeance of isolated cuticles from different latitudinal regions of fruit from the various genotypes. The results of this study are described in the bioRxiv paper. - We performed a structure-function analysis of the tomato CUS and DSF family, using AlphaFold-based predictive modeling, comparing our observed catalytic activities with structural elements of the predicted active site. This revealed three key features that distinguish cutin synthase versus cutin hydrolase activities: the location of key amino acid residue at the active site, a predicted tunnel in the hydrolase but a cavity in the synthase, and the presences of specific amino acids in the oxyanion hole of the enzymes. This is a major step forward in determining the mechanism of cutin polymerization and degradation during fruit development, but also in all other plant organs. - We studied a candidate gene in the BAHD protein family that we hypothesized is responsible for incorporating hydroxycinnamic acids into the tomato cutin matrix, using both biochemical analysis of the recombinant protein, and gene editing to knock out its expression. We demonstrated that this protein catalyzes the incorporation of coumarate into tomato cutin in vitro and that eliminating its expression results in absence of coumarate from cutin in developing fruit. Studies of the gene edited fruit have shown no apparent effect of this deficiency on fruit growth, cuticle permeability, fruit water loss or sensitivity to UV radiation. We are now examining the effects of coumarate deficiency on susceptibility to microbial infection. With these studies we have resolved a key 'missing link' in cutin formation and a manuscript is in preparation. - As part of the objective to characterize the cuticle 'self-sealing' mechanism, we studied the phenomenon in three tomato genotypes, including a new tomato model Solanum quitoense ('Naranjilla'), which has a very high trichome density, thus facilitating visualization of the sealing process. We have also optimized a multi-stain technique to home in on the timing of the cuticle healing phenomenon in M82 wild type tomato which will provide a platform for gene expression and mechanistic studies. This has revealed the sequential deposition of callose and suberin. This staining technique has also been used in 3-D imaging of cuticle structure and deposition in wild type and cuticle mutant fruit, and has indicated a new mechanism of cuticle synthesis, involving distinct cuticle subtypes. A manuscript presenting these data is in preparation. During this analysis and screening of different fluorescent dyes, we also identified a dye that specifically highlighted the deficiency in coumarate in the BAHD gene described above, and soi identified a new tool to examine the distribution of a specific cutin type in plant tissues. This lays the groundwork for future studies of the spatiotemporal distribution and structure-function studies of cutin in the epidermis of plants, as well as the 'self-healing' mechanism as a critical process in plant organ integrity.
Publications
- Type:
Other Journal Articles
Status:
Published
Year Published:
2025
Citation:
Philippe, G., S�rensen, I., Gu�rault, A., Cross, M.J., Domozych, D.S., Clausen, M.H., Rose, J.K.C. Spatiotemporal variation in cutin polymerization and remodeling mediated by GDSL-hydrolase enzymes during tomato fruit development. bioRxiv 2025.01.09.632122; doi: https://doi.org/10.1101/2025.01.09.632122
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Progress 01/15/23 to 01/14/24
Outputs
Target Audience:The target audience comprises plant scientists, other researchers involved in agricultural/horticultural biotechnology and breeding, as well as related stakeholders such as growers and breeders who will utilize information generated through this project to develop strategies for enhanced fruit quality and shelf life. Changes/Problems:We had technical issues with tan aging MALDi-TOF mass spectrometer, and so are developing an alternate electrospray MS approach. What opportunities for training and professional development has the project provided?Through this project, two postdocs and two undergraduate students were engaged in a range of educational and training activities that are made available at Cornell. Emphasis was placed on the involvement of under-represented minorities and on considerations of broadening diversity, equity and inclusion. In addition, they have made regular presentations of their research at group meetings. How have the results been disseminated to communities of interest?The results have been communicated through publication in peer reviewed journals, a webinar and poster presentations at international meetings, as well as a workshop to the American Fruit Growers' Association and industry stakeholders What do you plan to do during the next reporting period to accomplish the goals?- Characterize the newly identified candidate gene associated with incorporation of phenolic residues in the cutin matrix - Complete the enzymatic analysis of all the targeted recombinant proteins, including assessments of additive and synergistic activities. - Use the new 3D imaging technique, in combination with transmission electron microscopy and electron tomography, to build a high spatial resolution model of cuticle deposition.
Impacts
What was accomplished under these goals?
We identified 107 tomato GDSL-hydrolases, of which 40 show expression in the inner and/or outer fruit epidermis, which correspond to the tissues producing cutin. We have now completed a spatiotemporal profile of most of these genes and uncovered an unexpected gradient of expression of some of these genes down the latitudinal axis of the fruit. Preliminary data suggest that this correlates with cutin structure and composition, which varies at the poles or the equator ofc the fruit. We continued our in vitro characterization of nine recombinant tomato GDSL proteins including CDEF and CUS subtypes) as His-tagged recombinant proteins in Nicotiana benthamiana for enzymatic analyses and examined: 1) cutin polymerization activity using both MALDI-TOF and electrospray ionization (ESI) mass spectrometry to detect cutin oligomers, and a glycerol assay to detect acyltransferase activity; 2) cutin remodeling activity using a novel semi-in vivo assay that we developed using permeance of the inner fruit epidermal cuticle. We have almost completed the analysis of these isoforms. Data obtained in a previous study indicated a polymerase activity of CUS enzymes (CUS1, AtCUS1 and PpCUS1) and hydrolytic activity of Arabidopsis AtCDEF1 toward the cutin precursor 2-MAG measured in vitro. The GDSL-hydrolases thar showed high expression in the epidermis were tested as candidates in similar in vitro experiments to evaluate their ability to interact with the tomato cutin precursor 2-MAG. We also expressed CUS2 and CDEF1 to characterize their functional specificity. The hydrolytic activity of AtCDEF1 toward the 2-MAG substrate was strong, with 100% conversion to 10,16-diOH-C16 acid but absent from other enzymes with putative hydrolytic activity. However, cutin polymerization products were obtained by the activity of CUS and, surprisingly, CDEF enzymes. CDEF1 converted a comparable amount of substrate to oligomer products as CUS1, however with a higher proportion of dimers. As expected, CUS2 had polymerizing activity, however it was lower than CUS1, and no activity was observed with. The activity of CUS2, CDEF1, and AtCDEF1 were confirmed by a glycerol release assay following transesterification or hydrolytic activity on 2-MAG substrate. Interestingly, the result of the enzymatic incubation performed semi-in vivo (i.e. directly on a native cutin support that is the fruit inner epidermis) led to differences between CUS1 and CDEF1. Although the reaction mixture containing CDEF1 and the 2-MAG substrate led to the production of oligomers, which are detected after incubation, no 2-MAG nor oligomers were recovered from the reaction mixture containing initially CUS1 and 2-MAG. This result suggests that 2-MAG was fully polymerized to the native cutin by CUS1, while CDEF1 may have a lower polymerization activity with the native cutin or is also able to release oligomers from it in a dynamic remodeling activity. Taken together, these results provide new insights into the relative roles of these proteins in cutin assembly and remodeling. A manuscript presenting these results, and a new model of cutin dynamics is in preparation. We evaluate the functions of three tomato CDEF genes (SlCDEF1, SlCDEF2 and SlCDEF3) in tomato fruit using CRISPR-Cas9 technology, as well as a SlCDEF1x SlCDEF2 double knockout lines and a triple knockout line, all in the M82 cultivar background. We assessed a range of fruit phenotypes: development rate, size/shape/morphology; transpirational water loss from entire fruit; permeance of isolated cuticles from different latitudinal regions of fruit from the various genotypes. The results of this study are described in a manuscript currently under preparation. We targeted a candidate gene that we hypothesize is responsible for integrating phenolic acids into the cutin matrix, using CRISPR. This represents a major 'missing link' in cutin formation. Recently, we determined that fruit cuticles from the CRISPR lines are deficient in coumarate, the major phenolic component. This supports out hypothesis and we are now investigating the consequences of this deficiency on cuticle properties and fruit characteristics. As part of the objective to characterize the cuticle 'self-sealing' mechanism, we have studied the phenomenon in three tomato genotypes, including a new tomato model Solanum quitoense ('Naranjilla'), which has a very high trichome density, thus facilitating visualization of the sealing process. We have also optimized a multi-stain technique to home in on the timing of the cuticle healing phenomenon in M82 wild type tomato which will provide a platform for gene expression and mechanistic studies. This has revealed the sequential deposition of callose and suberin. Importantly, this staining technique has also been used in 3-D imaging of cuticle structure and deposition in wild type and cuticle mutant fruit, and has indicated a new mechanism of cuticle synthesis, involving distinct cuticle subtypes. We have completed the analysis of tomato hypercracking 1 (hcr1) mutant, which shows extensive fruit cracking at an early developmental stage, and consequent massive deposition of suberin in the epidermis during growth and fruit dehydration. A manuscript will be submitted shortly.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
McMullen, P., Philippe, G., Day, C.T.C., Rose J.K.C., Smart, S.D. (2022) Investigating the contribution of cutin to host susceptibility of fruit. Phytopathology 112 (11) 23-23.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Rose, J.K.C. (2023) Spatiotemporal control of tomato fruit cuticle organization: a model of integrity. SOL23 International Solanaceae Genome Conference, Montreal.
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Progress 01/15/22 to 01/14/23
Outputs
Target Audience:The target audience comprises plant scientists, other researchers involved in agricultural/horticultural biotechnology and breeding, as well as related stakeholders such as growers and breeders who will utilize information generated through this project to develop strategies for enhanced fruit quality and shelf life. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Through this project, two postdocs and two undergraduate students were engaged in a range of educational and training activities that are made available at Cornell. Emphasis was placed on the involvement of under-represented minorities and on considerations of broadening diversity, equity and inclusion. In addition, they have made regular presentations of their research at group meetings. How have the results been disseminated to communities of interest?The results have been communicated through publication in peer reviewed journals, One of the postdocs, Glenn Philippe. also presented the project at an international meeting "Cutin polymerization and remodeling in tomato fruit through the coordinated action of enzymatically diverse GDSL-hydrolases" Plant Apoplastic Diffusion Barriers Meeting, Dundee, 2022. The project was also presented by the PI in several public presentations. What do you plan to do during the next reporting period to accomplish the goals?- Continue with the spatiotemporal analysis of wax and cutin deposition in the various wild type and CRISPR genotypes, including the new CUS1 x CDEF1 lines. - Apply the new biomechanical assay to fruit form these genotypes to compare with cuticle composition profiles. - Develop a cutin degradation assay using an Instron testing analyzer in conjunction with the CUS and CDEF recombinant proteins, using a fungal cutinase as a positive control. - Continue the enzymatic analysis of all the targeted recombinant proteins, including assessments of additive and synergistic activities. - Extend the studies of the new biomaterial composite ('cutan') through various biochemical and spectroscopic analyses. - Integrate the data into a spatiotemporal model of cutin dynamics associated with the various GDSL structures. - Characterize fruit from a new CRISPR line in which a candidate gene for a phenolic acid transferase has been knocked out. We hypothesize that the protein encoded by this gene is responsible for conjugating coumarate, and possibly other hydroxycinnamic acids, to cutin. This would be a major discovery if this is the case.
Impacts
What was accomplished under these goals?
- We characterized the roles of three tomato CDEF genes (SlCDEF1, SlCDEF2 and SlCDEF3) in tomato fruit using CRISPR-Cas9 technology, as well as a SlCDEF1x SlCDEF2 double knockout lines and a triple knockout line, all in the M82 cultivar background. We assessed a range of fruit phenotypes: development rate, size/shape/morphology; transpirational water loss from entire fruit; permeance of isolated cuticles from different latitudinal regions of fruit from the various genotypes. The results suggest that there is coordination of GDSL-lipase enzymes in the cutin deposition, including a novel function of cutin remodeling in response to developmental cues or environmental demands. Both SlCUS1 and SlCUS2 appear to catalyze mid-chain cross-linking and we have candidate genes that we hypothesize catalyze the formation of linear cutin chains. Significantly, as far as we are aware, this is the first example of multienzyme modification of cutin. Notably, the double knockouts also showed a loss of 'nanoridges' in multiple floral organs, indicating functional specialization of different CUS genes. We also generated a SlCDEF1 x SlCUS1 cross and are now planning to develop a segregating population. The results of this study are described in a manuscript currently under preparation, - We had previously made the surprising discovery that tomato fruit show considerable variation in cuticle composition and permeance in different regions, in a gradient from the stem to the equator to the blossom end, and have been examined this phenomenon in more detailed, considering biophysical characteristics, biochemical compositional and patterns of cuticle-related gene expression. This new spatiotemporal model is now being incorporated into our studies as we further assess cutin deposition and dynamics across the fruit and through developmental time. IN this vein we examined aspects of cuticle/cutin structure and function in a collaboration with Carmen Catala at the Boyce Thompson Institute. As part of an effort to develop a more comprehensive picture of the molecular mechanisms governing fruit development under drought, we profiled the transcriptomes of a spectrum of fruit tissues from tomato, spanning early growth through ripening and collected from plants grown under varying intensities of water stress. In addition, we compared transcriptional changes in fruit with those in leaves to highlight different and conserved transcriptome signatures in vegetative and reproductive organs. We observed extensive and diverse genetic reprogramming in different fruit tissues and leaves, each associated with a unique response to drought acclimation, including the epidermal layer and associated cuticle. Gene coexpression network analysis provided further insights into the tissue-specific regulation of distinct responses to water stress. The data highlight the spatiotemporal specificity of drought responses in tomato fruit and indicate known and unrevealed molecular regulatory mechanisms involved in drought acclimation, during both vegetative and reproductive stages of development. - We have further characterized an SlCDEF1 CRISPR line (line 7) that showed a remarkable cracking and suberization phenotype, which we are using as a valuable model to study suberization and the cracking process, which has major horticultural implications. We collaborated with Prof. Christina Nawrath, Uni Lausanne to characterize the cellular processes that mediate suberin deposition and studies of line 7 indicate are revealing new models of how suberization is initiated in fruit, and highlight a new biopolymer, which we are currently referring to as 'suberan'. Material from line 7 was provided to our collaborator Prof. Ruth Stark, for NMR analysis. - We have expressed 9 recombinant tomato GDSL genes (including CDEF and CUS subtypes) as His-tagged recombinant proteins in Nicotiana benthamiana for enzymatic analyses and examined: 1) cutin polymerization activity using by MALDI-TOF mass spectrometry to detect cutin oligomers, and a glycerol assay to detect acyltransferase activity; 2) cutin modifying (polymerizing or depolymerizing) activity using a novel semi-in vivo assay that we recently developed using permeance of the inner fruit epidermal cuticle. We have established that several CUS enzymes have polymerase activity and, and surprising CDEF1 also showed polymerizing activity bother the mass spec and semi-in vivo assays. Another unexpected observation was that the ortholog of CDEF1 from Arabidopsis does not show canonical cutinase activity, but instead released the acyl chain from the glycerol-conjugated precursor. Collectively, these results are painting a very different, and unexpectedly complex, picture of cutin dynamics than is described in current models and we will continue to use reverse genetics, biomechanics and biochemical analyses to development of new models of cutin polymerization. - As part of the objective to characterize the cuticle 'self-sealing' mechanism, we have studied the phenomenon in three tomato genotypes, including a new tomato model Solanum quitoense ('Naranjilla'), which has a very high trichome density, thus facilitating visualization of the sealing process. Removal of trichomes at harvest significantly increases fruit water loss, confirming that the trichome pores are significant routes of water loss, as in S. lycopersicum. However, isolated cuticles that have been allowed to seal prior to isolation allow less transcuticular water loss than those isolated immediately after trichome removal, indicating that pore sealing effectively inhibits this water loss through pores. Viewing these stained pores at cross section, contextualized with scanning electron microscopy (SEM) images, suggests that the sealing does not occur at the surface of the trichome scar, but rather along the base of the exposed cavity. We have developed a muti-stain technique in conjunction with confocal microscopy to home in on the timing of the cuticle healing phenomenon, which will provide a platform for gene expression and mechanistic studies. - Though our studies of cutin formation and cuticle integrity, we have uncovered evidence for a that a previously unsuspected pathways is critical for the ability to synthesize, deposit and maintain a biomechanically resilient epidermis and a hydrophobic cuticle. We identified the tomato hypercracking 1 (hcr1) mutant, which shows extensive fruit cracking at an early developmental stage, and consequent massive deposition of suberin in the epidermis during growth and fruit dehydration. Map-based cloning of the hcr1 locus and biochemical analysis suggested that the hyper-cracking phenotype is a consequence of defective sterol biosynthesis. Morphological, cytological, and molecular characterization of hcr1 fruit revealed inhibited cell expansion and division in the pericarp, leading to impaired pericarp development, resulting in uncoordinated expansion between the pericarp and the inner tissues. Cell wall analysis revealed that the hcr1 mutant has significantly lower cellulose levels in pericarp cell wall. This study suggests that sterols are important for primary cell wall deposition, and notably cellulose synthesis. Moreover, the presence of a strong cracking phenotype in fruit, but not vegetative tissues, highlights variability in the susceptibility of different organs to impaired epidermal integrity. A manuscript is in preparation. - We have developed a new biomechanical assay to measure cutin properties using an Instron instrument to test tensile properties and break strength.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Nicolas, P., Shinozaki, Y., Powell, A., Philippe, G., Snyder, S., Bao, K., Zheng, Y., Xu, Y., Courtney, L., Vrebalov, J., Casteel, C., Mueller, L., Fei, Z., Giovannoni, J.J., Rose, J.K.C., and Catal�, C. (2022) Spatiotemporal dynamics of the tomato fruit transcriptome under prolonged water stress. Plant Physiology 190: 2557-2578.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Systems for Construction and Remodeling of the Tomato Fruit Cuticle Fruit Biology Meeting. Beijing University of Agriculture, Changping, Beijing (online, 2022).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
"Cutin polymerization and remodeling in tomato fruit through the coordinated action of enzymatically diverse GDSL-hydrolases". Plant Apoplastic Diffusion Barriers Meeting, Dundee, 2022.
- Type:
Websites
Status:
Published
Year Published:
2023
Citation:
The Role of the Plant Cuticle in Providing Marketable Yield . Webinar, hosted by CropLife and American Fruit Grower.
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Progress 01/15/21 to 01/14/22
Outputs
Target Audience:The target audience comprises plant scientists, other researchers involved in agricultural/horticultural biotechnology and breeding, as well as related stakeholders such as growers and breeders who will utilize information generated through this project to develop strategies for enhanced fruit quality and shelf life. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Through this project, two postdocs and two undergraduate students were engaged in a range of educational and training activities that are made available at Cornell. Emphasis was placed on the involvement of under-represented minorities and on considerations of broadening diversity, equity and inclusion. In addition, they have made regular presentations of their research at group meetings. How have the results been disseminated to communities of interest?The results have been communicated through publication in peer reviewed journals, One of the postdocs, Glenn Philippe. also presented the project at an international meeting (International Cell Wall meeting, online due to COVID) and the project was also presented by the PI in several public presentations. What do you plan to do during the next reporting period to accomplish the goals?- Continue with the spatiotemporal mapping of wax and cutin deposition in the various wild type and CRISPR genotypes. - Perform detailed linkage analysis of the cutin profiles in the various wild type and CRISPR genotypes. - Develop a robust biomechanical assay, and potentially a cutin degradation assay, a using an Instron testing analyzer. - Perform an extensive enzymatic analysis of all the targeted recombinant proteins, including synergistic activities. - Characterize the new biomaterial composite ('cutan') through various biochemical and spectroscopic analyses. - Complete a structural model of various GDSL structures, including the various subtypes. - Integrate the data into a spatiotemporal model of cutin dynamics.
Impacts
What was accomplished under these goals?
Objective 1: Test the hypothesis that tomato CDEF enzymes act as cutin hydrolases and determine their contributions to fruit transpiration and cuticle properties. - Knocked out the expression of three tomato CDEF genes (SlCDEF1, SlCDEF2 and SlCDEF3) using CRISPR-Cas9 technology, as well as SlCDEF1x SlCDEF2 double knockouts and a triple knockout line, all in the M82 cultivar background. - Characterized a range of phenotypes: development rate, fruit size/shape/morphology; water loss from entire fruit; permeance of isolated cuticles from different latitudinal regions of fruit from the various genotypes. Outcome: made the surprising discovery that tomato fruit show considerable variation in cuticle composition and permeance in different regions, in a gradient from the stem to the equator to the blossom end. This variation was supported by qPCR analysis of multiple cuticle related genes. To our knowledge this is a previously unreported phenomenon and suggests that current views of how water loss from fruit is regulated should be revised. Based on this exciting discovery, we will carefully consider and incorporate this spatiotemporal variation as we continue with the project. - Identified an SlCDEF1 CRISPR line (line 7) that showed a remarkable cracking and suberization phenotype. It provides a valuable model to study suberization and the cracking process, which has major horticultural implications. Collaborated with Prof. Christina Nawrath, Uni Lausanne, to characterize the cellular processes that mediate suberin deposition. Preliminary studies of line 7 indicate that it will allow new models to be developed of how suberization is initiated in fruit, and highlight a new biopolymer, which we are currently referring to as 'suberan'. Material from line 7 has been provided to our collaborator Prof. Ruth Stark, for NMR analysis. - Characterized polyclonal antisera that distinguish between CDEF and CUS enzymes. These have been used to confirm gene knockouts in the CRISPR lines and will be used in immunolocalization studies. - Expressed 9 recombinant tomato GDSL genes (including CDEF and CUS subtypes) as His-tagged recombinant proteins in Nicotiana benthamiana for enzymatic analyses. Outcome: SlCUS1 and SlCDEF1 both showed strong cutin polymerizing activity using in vitro assays, with MALDI-TOF analyses to detect the formation of cutin oligomers. SlCUS2 and SlCUS3 showed weak, but detectable polymerizing activity in vitro. Surprisingly, the semi-in vivo bioassays, using tomato fruit segments, all showed polymerizing activity, but particularly for SlCUS2. Data to date suggest that are multiple cutin substrates for these enzymes in vivo, and there is some enzyme promiscuity. Other important observations are that Arabidopsis CDEF does not act as a simple cutin degrading enzyme in the bioassay, unlike the fungal cutinase control. In addition, the GDSL '440' enzyme has clear polymerizing activity in the bioassay. The bioassay and the MALDI analyses are thus complementary and give different insights. Taken together, to date the results are not consistent with a simple CUS/polymerase vs CDEF/hydrolase remodeling enzymatic system. Rather, this study is, as hoped, allowing the development of new models of cutin polymerization. Objective 2: Establish whether different SlCUS enzymes catalyze the formation of structurally distinct cutin polymers and test the hypothesis that they show synergistic or coordinated activities with SlCDEF enzymes in cutin polymer modification. - Generated crosses of SlCUS1 and SlCUS2 CRISPR lines. Quantified the amount of cutin in the resulting lines and examined the cross-linking profiles of the cutin. Outcome: Found evidence for additive effects, and a suggestion that both SlCUS1 and SlCUS2 contribute to mid-chain cross-linking. As far as we are aware, this is the first example of multienzyme modification of cutin.. The double knockouts also showed a loss of 'nanoridges' in multiple floral organs, which we are characterizing using SEM. Objective 3: Identify key structural features of CUS and CDEF proteins that define their modes of action as acyltransferases and acylhydrolases, respectively. Since the project was initiated, the release of the latest version of the Alphafold software has revolutionized the potential for structural modeling. This will be used in the next phase of the project. We are also involved in a collaboration with a colleague at Cornell to generate crystals of SlCUS1 bound to the cutin precursor, to gain direct structural information. Objective 4: Characterize a newly identified cuticle 'self-sealing' mechanism. We have validated the phenomenon with 3 tomato genotypes and adopted a new tomato model Solanum quitoense ('Naranjilla'), which has a very high trichome density. The next phase of the project will be to characterize the timing of the cuticle healing phenomenon.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Philippe, G., De Bellis, D., Rose, J.K.C. and Nawrath, C. Trafficking processes and secretion pathways underlying the formation of plant cuticles. Frontiers in Plant Science.12:786874. doi: 10.3389/fpls.2021.786874.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2021
Citation:
Philippe, G., S�rensen, I, Domozych, D.S. and Rose, J.K.C. Resolving the dynamic assembly and remodeling of plant lipidic cell walls using tomato fruit development as a model. International Plant Cell Wall Meeting 2021 (Online)
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