Progress 10/01/08 to 09/30/13
Outputs
Target Audience: The work on the interactions of ripening tomato fruits with the gray mold (Botrytis cinerea) has been reported in scientific papers (below) and at postharvest biology and technology research conferences. It was the topic of my Inaugural Presentation at the Postharvest Unlimited V conference in Cyprus (June, 2014). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Graduate student Barbara Blanco-Ulate completed their Ph.D. program in the UC Davis Plant Biology Graduate Program. Dr. Blanco-Ulate now works as a postdoctoral researcher in the UC Davis Viticulture and Enology Department How have the results been disseminated to communities of interest? Some of these results have been presented at research conferences and to relevant industry members. Published reports are available to all interested parties. What do you plan to do during the next reporting period to accomplish the goals? Aspects of each of the areas of research mentioned will be developed further.
Impacts
What was accomplished under these goals?
For several years the work on our fruit postharvest biology-related projects has been reported through a regional research project. This year that work is reported here. These projects focus on (1) the fruit softening that accompanies tomato fruit softening, (2) the increasing pathogen susceptibility of ripening tomatoes and other fruits, and (3) the mechanistic and genetic linkages of fruit softening and the increased susceptibility of ripened fruits. Several related aspects of cell wall change accompany and contribute to the ripening fruit's softening. These include partial digestion and solubilization of pectin polysaccharides, cooperative action of different fruit wall-digesting proteins (i.e., polygalacturonase and expansin) as pectins are broken down, the generation of pectin oligosaccharides that can influence the tomato fruit's production of ethylene and the overall rate of ripening processes, and the ripening fruit's maintenance of its capacity to synthesize new polysaccharides even though it is acting to break down many of those polysaccharides. Work reported in the citations below addressed all of these aspects of fruit cell wall metabolism. The reports also featured work done by Ms. Blanco-Ulate as she completed her Ph.D. program. Dr. Blanco-Ulate identified the many tomato fruit and Botrytis cinerea (gray mold) genes that encode cell wall digesting enzymes that are expressed as the fruit ripens and the pathogen establishes infections on the fruit. Important conclusions from her work are related to (1) the fact that both the fruit and the pathogen express genes encoding enzymes that target the same fruit wall polysaccharides and (2) the apparent assistance in cell wall modification that the ripening fruit provides in support of the pathogen's lesion development.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Blanco-Ulate B, Morales-Cruz A, Amrine KCH, Labavitch JM, Powell ALT and Cantu D. 2014. Genome-wide transcriptional profiling of Botrytis cinerea genes targeting plant cell walls during infections of different hosts. Frontiers in Plant Sciences 5: Article 435.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2015
Citation:
Blanco-Ulate B, Cantu D, Vicente AR. Powell ALT, Greve LC and Labavitch JM. 2014. Cell wall metabolism: The Yin and Yang of fruit postharvest biology. This was presented as the keynote address at the Postharvest Unlimited V conference (June, 2014 in Cyprus). A paper with the same title and authors has been submitted and will be published in a volume of Acta Horticulturae.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: For several years, we (Dr. Ann Powell, Prof. Alan Bennett, I and our younger UC Davis colleagues) have been studying the factors (genetic and biochemical) that contribute to the increasing pathogen susceptibility of ripening tomato fruits. Our studies focus on the details of the growth of Botrytis cinerea on wild-type 'Ailsa Craig' (AC) mature green (MG) and red ripe (RR) stage tomato fruit and on AC fruit that are homozygous for the rin, nor and Cnr ripening mutations (all are mutations in transcription factor-encoding genes). These genotypes are useful because the fruit do not ripen normally and the ripe fruit show dramatic differences in their susceptibility to B. cinerea relative to the AC ripe fruit. Thus, an analysis of the genes whose expression patterns are influenced by Cnr, RIN and NOR should help us to identify genes that link fruit ripening, in general, to the ripening-associated increase in fruit pathogen susceptibility. Ms. Barbara Blanco-Ulate, a Ph.D. candidate in the Plant Biology Graduate Group, is the primary bench scientist on this project. She is now examining fruit pathogen susceptibility in fruits with pairs of the ripening gene mutations and is using an extensive set of monoclonal antibodies to different tomato cell wall polysaccharides ("glycomic" analysis) to describe the changes in cell wall integrity that accompany fruit infection by B. cinerea. We (Dr. Powell, I and our Israeli colleagues) completed a one-year BARD feasibility study to determine whether PGIPs (polygalacturonase-inhibiting proteins) expressed in rootstocks used for grafted tomatoes can be exported to scions and will then protect scion tissues and the fruits they bear against rotting pathogens. A full proposal submitted to BARD in 2011 received positive reviews but was not supported because of budget restrictions at BARD. The proposal was resubmitted in Sept., 2012. To support that work, we started work to create a transgenic tomato rootstock of the "cuatomate" type that expresses the pear PGIP (pPGIP)-encoding gene. Seeds from the original transgenic plants were collected and plants from these seeds were screened for presence of the pPGIP DNA and PGIP activity. Seeds collected from the pPGIP-expressing cuatomate lines will be grown out and the plants will be screened to identify plants homozygous for the pPGIP gene. These plants will be used in tests of the ability of a tomato rootstock to export PGIP to scion tissues. Several years ago we obtained pectin-derived oligosaccharides (PDOs) from Breaker stage tomato fruit and subsequent PDO structural analyses indicated that these oligosaccharides had been digested from cell wall pectins by enzymes with a lyase rather than hydrolase mode of action. This indicated that pectin or pectate lyase enzymes (PLs) were active in ripening tomatoes. We have begun using available tomato fruit transcriptomic data (our own and that of others) and bioinformatics information to clone tomato fruit PL-encoding genes. Our longer term plan is to generate tomato lines with suppressed PL activity and, eventually, suppressed expression of PL, PG and Expansin in one line. PARTICIPANTS: UC Davis researchers Ann L.T. Powell, Alan Bennett and John Labavitch, graduate students Barbara Blanco-Ulate and Ka Lai Lam Cheng, visiting researchers Javier Lopez-Balthazar (Mexico) and Hakan Aktas (Turkey), professional researcher Helen Chang and undergraduate researcher Lucas McKinnon all contributed to the work reported. Our Israeli colleagues, Drs. Eli Fallik and Menahem Edelstein of the Volcani Institute are involved in the work on the "rootstock" project. TARGET AUDIENCES: The results from the fruit ripening/pathogen susceptibility studies will be of immediate interest to seed companies that would like to improve the pathogen resistance of tomato cultivars. Depending on the specific outcomes of our studies, eventually the information that we develop will be of interest to tomato growers and growers of grafted fruits like tomatoes and melons. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Processes controlled by the NOR transcription factor, but not by RIN and Cnr, are associated with the tomato fruit's ripening-associated increase in pathogen susceptibility. This provides a focus for our effort to identify the link between ripening and pathogen susceptibility. In the longer term, we hope to manipulate the fruit ripening program so that increasing pathogen susceptibility is no longer linked to fruit ripening. Our analysis of (1) the cell wall-degrading enzymes expressed by B. cinerea as it infects tomato fruits and of (2) the changes in tomato fruit cell walls that are caused by pathogen enzyme action is designed to help us identify key steps in pathogen infection of fruit tissues. Our work to determine whether rootstock-produced factors like PGIPs can provide pathogen protection for grafted tomato scions could provide a model for improving pathogen and pest protection for a variety of grafted annual fruit crops. Work our group reported earlier has indicated that fruit PG and Expansin proteins combine to alter fruit cell walls during ripening. However, it is clear that these two enzymes are not the only contributors to fruit cell wall change during ripening. Our work to identify the tomato PL-encoding genes is designed to add important information to enhance efforts to improve postharvest management of fruit ripening and softening. Because the softening of fruits during their ripening contributes to the fruit's pathogen susceptibility, the work on PLs may also support future work to manage the pathogen problems of ripened fruits more effectively.
Publications
- No publications reported this period
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: For several years, we (Dr. Ann Powell, Prof. Alan Bennett, I and our younger UC Davis colleagues) have been studying the factors (genetic and biochemical) that contribute to the increasing pathogen susceptibility of ripening tomato fruits. Our studies focus on the details of the growth of Botrytis cinerea on wild-type 'Ailsa Craig' (AC) mature green (MG) and red ripe (RR) stage tomato fruit and on AC fruit that are homozygous for the rin, nor and Cnr ripening mutations (all are mutations in transcription factor-encoding genes). These genotypes are useful because the fruit do not ripen normally and the ripe fruit show dramatic differences in their susceptibility to B. cinerea relative to the AC ripe fruit. Thus, an analysis of the genes whose expression patterns are influenced by Cnr, RIN and NOR should help us to identify genes that link fruit ripening, in general, to the ripening-associated increase in fruit pathogen susceptibility. At present, the main bench scientist on this project is Ms. Barbara Blanco-Ulate, a graduate student in the Plant Biology Graduate Group. Barbara has been very productive. However, she spent several months in the Spring and Summer preparing for her Ph.D. qualifying oral exam and that reduced the amount of progress that she was able to make this year. Nevertheless, she has been preparing and screening the tomato crosses that will be used to determine the impact of one ripening mutation on the expression another. These studies will determine the developmental priorities of the RIN, NOR and Cnr transcription factors and prove to be useful for a number of fruit pathogen susceptibility tests that are planned. We (Dr. Powell, I and our UCD colleagues) completed a one-year BARD feasibility study to determine whether PGIPs expressed in rootstocks used for grafted tomatoes can be exported to scions and will then protect scion tissues and the fruits they bear against rotting pathogens. The project is shared with researchers from the Volcani Center in Israel (Drs. Menahem Edelstein and Eli Fallik). Together with our colleagues we have shown that (1) the pear PGIP (pPGIP) produced in a transgenic tomato line that was used as a rootstock is transported across the graft union into tomato scions that do not make pPGIP and that (2) the pathogen defenses of wild-type tomato scions are enhanced by grafting them to pPGIP-expressing rootstocks. These results must be confirmed in additional trials before a manuscript can be prepared. A full BARD proposal based on the work in the feasibility study has been submitted. In preparation for that full (3-year) project we designed a gene construct to develop a new set of pPGIP expressing tomato lines and the UC Davis Plant Transformation Facility is now preparing true tomato rootstock lines that express the pPGIP. We will learn about the success of the full BARD proposal in May/June, 2012. PARTICIPANTS: UC Davis researchers Ann L.T. Powell and Prof. Alan Bennett, graduate student Barbara Blanco-Ulate, visiting researchers Javier Lopez-Balthazar (Mexico), and undergraduate researchers Jo Odias (US) and Thy Thy Nguyen (Thailand) all contributed to the work reported. As indicated in the report, Drs. Eli Fallik and Menahem Edelstein of the Volcani Institute in Israel are involved in the work of the "rootstock" project. TARGET AUDIENCES: The results from the fruit ripening/pathogen susceptibility studies will be of immediate interest to seed companies interested in improving the pathogen resistance of tomato cultivars. Depending on the specific outcomes of our studies, eventually the information that we develop will be of interest to tomato growers and growers of grafted fruits like tomatoes and melons. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Processes controlled by the NOR transcription factor, but not by RIN and Cnr, are associated with the tomato fruit's ripening-associated increase in pathogen susceptibility. This provides a focus for our effort to identify the link between ripening and pathogen susceptibility. In the longer term, we hope to manipulate the fruit ripening program so that increasing pathogen susceptibility is no longer linked to fruit ripening. Our analysis of (1) the cell wall-degrading enzymes expressed by B. cinerea as it infects tomato fruits and of (2) the changes in tomato fruit cell walls that are caused by pathogen enzyme action is designed to help us identify key steps in pathogen infection of fruit tissues. Our work to determine whether rootstock-produced factors like PGIPs can provide pathogen protection for grafted tomato scions could provide a model for improving pathogen and pest protection for a variety of grafted annual fruit crops.
Publications
- Haroldsen VM, Szczerba MW, Aktas H, Lopez-Balthazar J, Odias MJ, Chi-Ham CL, Labavitch JM, Bennett AB, Powell ALT. 2012. Mobility of transgenic nucleic acids and proteins within grafted rootstocks for agricultural improvement. Frontiers in Plant Science (in press)
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: We have continued our studies of the details of the growth of Botrytis cinerea on wild-type 'Ailsa Craig' (AC) mature green (MG) and red ripe (RR) stage tomato fruit and on AC fruit that are homozygous for the rin, nor and Cnr ripening mutations. These genotypes are useful because the fruit do not ripen normally and the ripe fruit show dramatic differences in their susceptibility to B. cinerea relative to the AC ripe fruit. Cnr fruit are more susceptible than RR AC fruit and the MG Cnr fruit are very susceptible to colonization by the gray mold. While rin fruit do not ripen normally the "RR" stage rin fruit, like the RR AC fruit, become susceptible to B. cinerea. In contrast to the RR AC fruit, "RR" stage nor fruit remain resistant to B. cinerea. The Cnr, RIN and NOR genes encode transcription factors that participate in controlling normal ripening. Thus, an analysis of the genes influenced by Cnr, RIN and NOR should help us to identify genes that link fruit ripening, in general, to the ripening-associated increase in fruit pathogen susceptibility. Because the fruit of different genotypes are differentially attacked by B. cinerea we are now engaged in a comparison of the metabolism of fruit cell walls that occurs when the pathogen grows on fruit of the different genotypes. We are performing a series of aqueous solvent wall extractions; eventually what is solubilized by each extractant will be characterized biochemically in terms of the neutral sugar and uronic acid contents of extracted polymers. We also have begun an interaction with Prof. Michael Hahn (Complex Carbohydrate Research Center, U. of Georgia) who will use a series of ELISA assays involving his series of cell wall polymer-targeting monoclonal antibodies to identify the polysaccharide epitopes in each extract. Our colleague Jan van Kan (Wageningen U., the Netherlands) has provided probes for detecting the specific mRNAs of each of B. cinerea's polygalacturonase (PG) genes. These have been used to determine whether the pathogen expresses each of its PGs when it grows on MG and RR tomatoes of the WT and Cnr, rin and nor genotypes. We found that not all PGs are expressed on all fruit ripening stages or genotypes. The data indicate that B. cinerea's expression of PGs 5 and 6 is crucial for a successful infection of tomato fruits. We are now working with Drs. Menahem Edelstein and Eli Fallik (Volcani Center, Israel) in a study to determine whether PGIPs expressed in rootstocks used for grafted tomatoes can be exported to scions and will then protect scion tissues and the fruits they bear against rotting pathogens. We are in the middle of a one-year feasibility study. We now know that the pear fruit PGIP, a PGIP we have worked with extensively, is moved in the xylem from a pear PGIP expressing rootstock into grafted scion tissues. We are now working to learn if enough PGIP is moved from the rootstock into the scion to provide pathogen protection. PARTICIPANTS: UC Davis researchers Ann L.T. Powell, graduate student Barbara Blanco-Ulate, visiting researchers Kalai Lam (Costa Rica) and Javier Lopez (Mexico), and undergraduate researchers Jo Odias and Thy Thy Nguyen all contributed to the work reported. As indicated in the report, Drs. Eli Fallik and Menahem Edelstein of the Volcani Institute in Israel are involved in the work of the "rootstock" project. TARGET AUDIENCES: Depending on the outcome of our studies, eventually the information that we develop will be of interest to tomato growers and growers of grafted fruits like tomatoes and melons PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Processes controlled by the NOR transcription factor, but not by RIN and Cnr, are associated with the tomato fruit's ripening-associated increase in pathogen susceptibility. This provides a focus for our effort to identify the link between ripening and pathogen susceptibility. In the longer term, we hope to manipulate the fruit ripening program so that increasing pathogen susceptibility is no longer linked to fruit ripening. Our analysis of (1) the cell wall-degrading enzymes expressed by B. cinerea as it infects tomato fruits and of (2) the changes in tomato fruit cell walls that are caused by pathogen enzyme action is designed to help us identify key steps in pathogen infection of fruit tissues. Our work to determine whether rootstock-produced factors like PGIPs can provide pathogen protection for grafted tomato scions could provide a model for improving pathogen and pest protection for a variety of grafted annual fruit crops.
Publications
- No publications reported this period
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: We have been studying the increase in susceptibility to pathogens that accompanies the ripening of fruit; the interaction of the gray mold pathogen Botrytis cinerea with ripening tomatoes is our experimental system, in large part because there are many genetic variants of tomato that have modified ripening programs because of mutations in transcription factors (e.g., NOR, RIN, CNR) or because we have engineered changes in the expression of cell wall modifying enzymes by the ripening fruit (e.g., suppression of polygalacturonase [PG] and expansin [EXP] expression). Affymetrix Tomato GeneChips were used to follow gene expression in healthy ripening fruits and in mature green (MG) and red ripe (RR) fruits 3 days after they were inoculated with B. cinerea. A comprehensive analysis of probe sets that were either up- or down-regulated during ripening, as a result of inoculation, and in fruits that were ripening as the pathogen infections spread. While infections spread only slowly on MG fruit that have been inoculated, it is clear that the infection led to increased expression of many genes whose expression normally increases as ripening progresses, an observation that is consistent with the idea that fruit ripening includes processes that support pathogen development. MG fruit from a line mutated in the RIN transcription factor showed substantially slowed ripening but were about as susceptible to B. cinerea as inoculated wild-type fruit, indicating that the ripening-associated increase in fruit pathogen susceptibility is not dependent on ripening processes that are regulated by RIN. In contrast, while MG fruit from a line mutated in the NOR transcription factor also show delayed ripening, they are largely resistant to the gray mold, indicating that some thus far unidentified ripening process that is controlled by NOR is associated with the ripening-associated increase in fruit pathogen susceptibility. Some of our studies of infected, ripening tomato fruit require that we analyze the changes in fruit cell wall composition. These analysess have been compromised by the fact that the isolation of fruit cell walls also isolates cell walls of B. cinerea. Therefore we carried out an analysis of the pathogen's cell walls, starting with cultures of B. cinerea. After homogenizing the mycelium and preparing hot alcohol-insoluble cell wall preparations, we fractionated the cell wall preparations into alkali-soluble and -insoluble fractions and measured their content of chitin (low) and several neutral sugars (high for glucose, much lower for galactose and arabinose, but other neutral sugars were also present). PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The observation that ripening processes controlled by the NOR transcription factor, but not by RIN, are associated with the ripening-associated increase in fruit pathogen susceptibility provides a focus for our effort to identify the link between ripening and pathogen susceptibility. In the longer term, we hope to manipulate the fruit ripening program so that increasing pathogen susceptibility is no longer linked to the ripening process. Our analysis of the B. cinerea cell wall has identified some wall composition markers that we can use to determine how much of a "fruit" cell wall preparation was of pathogen origin.
Publications
- Cantu, D., Blanco-Ulate, B., Yang, L., Labavitch, J.M., Bennett, A.B. and Powell, A.L.T. 2009. Ripening-regulated susceptibility of tomato fruit to Botrytis cinerea requires NOR but not RIN or ethylene. Plant Physiology 150:1434-1459.
- Cantu, D., Greve, L.C., Labavitch, J.M. and Powell, A.L.T. 2009. Characterization of the cell wall of the ubiquitous plant pathogen Botrytis cinerea. Mycological Research 113: 1396-1403.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: For many years, an important component of our work on the postharvest biology of fruits has been an effort to identify the developmental linkage of fruit ripening and increasing fruit susceptibility to pathogens. Work published in this period has identified a strong connection between the cell wall breakdown events that contribute to tomato ripening-related fruit softening and the fruit's increasing susceptibility to the gray mold pathogen, Botrytis cinerea. Tomato fruits whose expression of the cell wall modifying proteins (CWMPs) polygalacturonase (PG) or expansin (Exp) or both PG and Exp was suppressed using transgene approaches were inoculated at the normally most susceptible pink and red-ripe stages. When both PG and Exp had been suppressed in these fruits they were substantially less susceptible to gray mold than controls. In addition, they displayed much less pectin breakdown and were appreciably firmer than unmodified controls. These results indicate that, in spite of the fact that the gray mold pathogen produces many of its own CWMPs when it is attempting to colonize fruit tissues, the pathogen needs help from the fruit's wall modification processes. We are now preparing to submit a manuscript comparing the changes in gene expression patterns in ripening, healthy and infected tomato fruits. Our analysis indicates that inoculation of healthy, mature-green fruit with B. cinerea causes the expression of many of the genes that are also up-regulated during fruit ripening. Since, as described above, fruit ripening contributes to the fruit's increasing pathogen susceptibility, a good pathogen strategy would be to promote ripening when it interacts with fruit tissues and B. cinerea appears to do that. A short visit to our lab by Roberto Gregori, a graduate student from Bologna, resulted in an additional paper dealing with fruit-pathogen interactions. For many years we have examined the role of plant proteins that are inhibitors of pathogen PG (the PG-inhibiting proteins, PGIPs) in plant defense against pathogens. Our report described the ability of a PGIP isolated from apple fruits to inhibit the PG produced by the pathogen Colletotrichum acutatum and set the stage for further work to test whether the apple PGIP contributes to apple fruit defenses against pathogens. During this period we have published short reviews of the (1) roles of cell wall metabolism in plant host-pathogen interactions and (2) the impact of ethylene on cell wall metabolism in fruits. The latter review was published in an issue of Plant Science that memorialized the life and work of the late UC Davis Professor, Shang-fa Yang. In this period we have reported that the flesh reddening that becomes apparent as plum fruits are subjected to prolonged cold storage is exacerbated by exposure to ethylene and that the problem worsens if fruit are allowed to mature prior to storage. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our work linking fruit softening and pathogen susceptibility suggests an excellent strategy for modifying, in a way useful for improved postharvest management, the progression of ripening. The work with apple PGIP adds support to the idea that PGIPs are important plant pathogen-defense proteins.
Publications
- Gregori, R., Mari, M. Bertolini, P., Sanudo-Barrajas, J.A., Tian, J.B. and Labavitch, J.M. 2008. Reduction of Colletotrichum acutatum infection by a polygalacturonase inhibitor protein extracted from apple. Postharvest Biololgy and Technology 48:309-313.
- Manganaris, G.A., Vicente, A.R., Crisosto, C.H. and Labavitch, J.M. 2008. Effect of delayed storage and continuous ethylene exposure on flesh reddening of 'Royal Diamond' plums. Journal of the Science of Food and Agriculture 88:2180-2185.
- Cantu, D.A., Vicente, A.R. Greve, L.C., Dewey, F.M. Bennett, A.B. Labavitch, J.M. and Powell, A.L.T. 2008. The intersection between cell wall disassembly, ripening, and fruit susceptibility to Botrytis cinerea. Proceedings of the National Academy of Sciences USA 105:859-864.
- Cantu, D.A., Vicente, A.R., Labavitch, J.M., Bennett, A.B. and Powell, A.L.T. 2008. Strangers in the matrix: plant cell walls and pathogen susceptibility. Trends in Plant Science 13:610-617.
- Bennett, A.B. and Labavitch, J.M. 2008. Ethylene and ripening-regulated expression and function of fruit cell wall modifying proteins. Plant Science 175:130-136.
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Progress 01/01/07 to 12/31/07
Outputs
We have continued our work on the ripening-related softening of fruits and the interaction of fruit tissues (primarily tomato) with pathogens. Our studies of cell wall changes occurring in ripening boysenberries, raspberries and blueberries (mentioned in last year's report) have now been published. The reports from Vicente et al. demonstrate that the patterns of cell wall disassembly in these small, rapidly softening fruits are quite different, in some cases involving the solubilization of pectins or hemicelluloses, and occasionally, but not always, involving polysaccharide depolymerization. A multi-investigator collaboration led by Prof. J. Rose (Cornell Univ.) has focused on the multiple impacts of the delayed fruit deterioration (DFD) gene on the ripening-associated softening of tomato fruits. The work moves some attention away from cell wall metabolism as the sole explanation of why fruits soften as they ripen. The work shows that the slowly softening mutant fruit
display the same cell wall changes seen in normally ripening tomatoes, but that the fruit cuticle chemistry is altered and that fruit cells maintain their turgor pressure as ripening (based on red color development) occurs. Our work on the interaction of the gray mold pathogen (Botrytis cinerea, Bc) with tomato fruits that have been genetically modified to prevent the expression of polygalacturonase (PG) and expansin (Exp) genes has proven to very informative. Fruits with suppressed PG and Exp soften more slowly than controls and the increase in Bc susceptibility that normally accompanies fruit ripening is substantially reduced. Our work suggests that the normal ripening-associated wall disassembly processes of tomatoes contribute to the success of B. cinerea and, we hypothesize, other pathogens. Dr. George Manganaris, a postdoc working in the lab of our colleague Carlos Crisosto, spent 4 months in our lab and contributed to the on-going interactions of or two groups. Two papers have
been published. These tie together the studies of fruit postharvest management technologies that are a hallmark of Crisosto's program and our expertise in studying cell wall metabolism. The two Manganaris et al. papers describe (1) the testing and use of a 1-MCP liquid formulation on plum fruits and show that the formulation should be useful for managing fruit ripening and (2) compare chilling injury (CI) symptoms in plums with the CI symptoms displayed by other soft fruits. For several years I have teamed with Prof. J. Siriphanich (Kasetsart Univ., Thailand) in the mentoring of Ms. Lampan Khurnpoon, a Ph.D. student. In the past year Lampan has completed her Ph.D. work and a paper describing her work on cell wall metabolism related to durian fruit dehiscence is now in press.
Impacts
Our work with the softening of small fruits makes clear that postharvest strategies aimed at the specific control of fruit ripening must target different enzyme systems in the fruits studied and, by extension, many other fleshy fruits. The team effort led by Rose at Cornell (Saladie et al., 2007) provides additional evidence that turgor pressure plays a big role in fruit texture. When the identity of the DFD gene is clear, this should be useful in development of fruits with longer shelf- and storage-lives. The demonstration of a strong linkage between the wall disassembly processes carried out by fruit enzymes as ripening fruits soften is the first clear explanation of why ripening fruits are increasingly susceptible to pathogens. This is likely to lead to several strategies for management of fruit pathogen susceptibility in the postharvest environment. Dr. Khurnpoon's work is now guiding tests of strategies for managing the postharvest storage and shipping of durian
fruit. This work will occur in Prof. Siriphanich's lab and should prove useful for the important Thai durian industry.
Publications
- Saladie, M. Matas, A.J., Isaacson, T., Jenks, M.A., Goodwin, S.M., Niklas, K.J., Xiaolin, R., Labavitch, J.M., Shackel, K.A., Fernie, A.R., Lytovchenko, A., O'Neill, M.A., Watkins, C. and Rose, J.K.C. 2007. A reevaluation of the key factors that influence tomato fruit softening and integrity. Plant Physiol. 144:1012-1028.
- Vicente, A.R., Powell, A., Greve, L.C. and Labavitch, J.M. 2007. Cell wall disassembly events in boysenberry (Rubus idaeus L. x Rubus ursinus Cham. & Schldl.) fruit development. Functional Plant Biology 34:614-623.
- Vicente, A.R., Ortugno,C., Powell, A.L.T., Greve, L.C. and Labavitch, J.M. 2007. The temporal sequence of cell wall disassembly events in developing fruits: 1. Analysis of raspberry (Rubus idaeus). J. Agric. Food Chem. 55:4119-4124.
- Vicente, A.R., Ortugno, C., Powell, A.L.T., Greve, L.C. and Labavitch, J.M. 2007. The temporal sequence of cell wall disassembly events in developing fruits: 1. Analysis of blueberry (Vaccinium sp.). J. Agric. Food Chem. 55:4125-4130.
- Manganaris, G.A., Vicente, A.R., Crisosto, C.H. and Labavitch, J.M. 2007.Effects of dips in a 1-methylcyclopropene-generating solution on 'Harrow Sun' plums stored under different temperature regimes. J. Agric. Food Chem. 55:7015-7020.
- Khurnpoon, L., Siriphanich, J. and Labavitch, J.M. 2007. Cell wall metabolism during durian fruit dehiscence. Postharvest Biology and Technology (in press)
- Manganaris, G.A., Crisosto, C.H., Vicente, A.R. and Labavitch, J.M. 2007 Cell wall modifications in chilling injured plum fruit Prunus salicina cv. Fortune. Postharvest Biology and Technology (in press)
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Progress 01/01/06 to 12/31/06
Outputs
As an extension of last year's studies of cell wall metabolism in ripening fruits, we have begun a full analysis of the wall changes in normally ripening Ailsa Craig tomato fruits that have been genetically modified to cause the suppression of the ripening-associated of genes encoding polygalacturonase (PG) or Expansin (Exp) or PG and Exp together. We reported last year on studies of non-ripening mutant (rin) lines that were manipulated to express PG, Exp or the combination of these two genes. This year's work with the suppressed lines provides complementary data supporting the idea that PG and Exp act in concert to disrupt cell wall pectin and hemicellulose networks and that this disruption is an important aspect of fruit texture change during ripening. Papers describing this work are almost ready for submission. We also used these transgenic tomato lines in our studies of the interaction of the gray mold pathogen, Botrytis cinerea, with green and ripening fruits.
Suppression of the expression of tomato PG and Exp during ripening dramatically increases the fruit's ability to resist infection. This observation is now being studied at several levels ranging from microarray-based analysis of fruit gene expression during B. cinerea infection, modifications of wall disassembly as the fruit and pathogen interact, and changes in fruit cell integrity during pathogen development. Finally, we have completed a series of studies of ripening-associated changes in cell walls and wall-metabolizing enzymes in small fruits, specifically boysenberries, blueberries and raspberries. A particularly interesting observation is that cell wall changes that are correlated with softening differ considerably between the fruits studied. Papers describing this work have been submitted.
Impacts
Our work with the softening of ripening fruits will eventually identify the proteins that play key roles in fruit cell wall change. Once the genes encoding these proteins are cloned we will be in a position to change ripening-related gene expression and test whether the roles that are apparently played by these proteins in the control of cell wall change are important in fruit softening. Our data for the relationship of fruit ripening to fruit susceptibility to gray mold make clear that one potentially use approach for pathogen control is likely to be management of fruit cell wall metabolism during ripening.
Publications
- Vicente, A.R., Civello, P.M., Martinez, G.A., Powell, A.L.T., Labavitch, J.M., Chaves, A. 2005. Control of postharvest spoilage in soft fruit. Stewart Postharvest Review Journal 1 (4):1-4.
- Vicente, A.R., Greve, L.C., Labavitch, J.M. 2006. Recent findings in plant cell wall structure and metabolism. Stewart Postharvest Review Journal 2(2):1945-1956.
- Vicente, A.R., Saladie, M., Rose, J.K.C., Labavitch, J.M. 2006. The linkage between cell wall metabolism and fruit softening: Looking to the future. Journal of the Science of Food and Agriculture. In Press
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Progress 01/01/05 to 12/31/05
Outputs
Our interest in the relationship of cell wall polysaccharide metabolism to fruit ripening-related softening continues. We have studied several kinds of fruit in the last year. We have used a transgenic tomato line in order to understand the roles of polygalacturonase (PG) and expansin (Exp) in ripening-related wall metabolism. We have used rin mutant lines, transformed to express PG and Exp when the fruits are treated with ethylene. This line was generated by our colleagues A. Powell and A. Bennett. The fruits expressing PG and Exp showed a dramatic shift in the size of pectin and hemicellulose polymers as the time of ethylene exposure was extended. Changes were slightly greater than when the expression of PG or Exp, alone, was promoted in the rin fruits. We have also begun a series of studies of the ripening-related cell wall changes in small fruits. The changes in these fruits should be interesting, because fruits like boysenberries, blueberries and raspberries
continue to grow as they soften. Thus far the changes in boysenberries have been followed. A large down-shift in size of readily-extracted pectins was observed, coincident with later stages of ripening. Samples of raspberries have been collected and cell walls have been prepared. They will be examined soon. In order to make preliminary analysis of cell wall component change in ripening fruits, we have established a collaborative arrangement with Dr. Maureen McCann (Purdue University). Dr. McCann has pioneered the use of near-infrared Fourier transform spectrometry for analysis of polysaccharides in insoluble and solubilized samples. Recently she began an approach by which the system can learn, improving the quality of the data, through use of a neural network system. The system is now learning based on a tomato cell wall ripening series that we have provided. We are also continuing our examination of the interaction of tomato fruits and vegetative tissues with Botrytis cinerea, the
gray mold pathogen. For several years we have been working with a PG-inhibiting protein (PGIP) that had been isolated from tomato fruit and then cloned. A search of data bases revealed that tomato had several gene sequences similar to the one encoding the fruit PGIP. One of these sequences was cloned and the sequence was then expressed in Arabidopsis thaliana. Protein isolated from the transformed A. thaliana had PGIP activity, indicating that the second sequence also encodes PGIP. Affymetrix has recently made available a tomato microarray. We have begun to use this in our studies of the genes expressed when tomato tissues are responding to B. cinerea. Previously we had used PCR-based approaches to follow the expression of several, putative defense-related genes in infected fruits and leaves. The microarray analysis will substantially enhance the amount of gene expression data we can get from inoculation experiments.
Impacts
Our work with the softening of ripening fruits will eventually identify the proteins that play key roles in fruit cell wall change. Once the genes encoding these proteins are cloned we will be in a position to change ripening-related gene expression and test whether the roles that are apparently played by these proteins in the control of cell wall change are important in fruit softening. Our data for fruit PGIPs have already made clear that these proteins play an important role in helping fruit and other tissues defend themselves against pathogen attack. The identification of a new PGIP will add to our ability to study this defense more closely. Our microarray data should eventually help us to understand more fully the scope of the defenses expressed by infected fruits.
Publications
- No publications in the year 2005. Drafts of the work on ripening-related wall change and the defense roles of PGIPs, written by graduate students Liya Yang and Ariel Vicente are now being revised internally.
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Progress 01/01/04 to 12/31/04
Outputs
We have isolated pectin-derived oligosaccharides (PDOs) from tomato pericarp disks that have ripened in culture disks. Structural analysis of these reveals that two pectin polymer digesting enzymes, polygalacturonase(PG) and pectate lyase, are active during ripening. Similar PDOs were isolated from grey mold-infected tomatoes, indicating that the same kinds of pathogen pectolytic enzymes are active during the fungus' colonization of its fruit host. Finally, we have identified similar PDOs in extracts of peaches that have ripened to a juicy texture and fruits ripened to a mealy texture. Mealy fruit also contain larger PDOs, inclding some with remnants of appended arabinan side-chains. Thus suggests that the problem with wall metabolism in mealy ripening is a failure to break down pectin side chains. The PDOs from infected fruits were applied to healthy pericarp disks and this led to the expression of several fruit genes that are also expressed following exposure to the
grey mold fungus, B. cinerea. We have also completed development of homozygous transgenic tomatoes with elevated and suppressed expression of the endogenous PG-inhibiting protein (PGIP) gene. Our transgenic lines with low and high PGIP were inoculated with B. cinerea and the fruits with very low PGIP were much more susceptible to infection than those with elevated PGIP. We have isolated infected tissues from both of the transgenic lines and will soon analyze their PDO content to see if, as proposed by others, PG interaction with PGIP, in vivo, influences the production of elicitor-active PDOs.
Impacts
Our work makes clear that PGIP ids a factor in a plant's defense against pathogens and strongly suggests that that up-regulated expression of plant PGIPs in crop plants, either by conventional breeding or genetic engineering, is likely to result in plants with improved tolerance of pathogens and, perhaps, some insects. Because PDOs may affect the way a plant responds to a pest or pathogen, we also expect to learn more about the specific defense mechanisms of plants.
Publications
- An, H.J., S. Lurie, L.C. Greve, D. Rosenquist, C. Kirmiz, J.M. Labavitch and C.B. Lebrilla. 2004. Determination of pathogen-related enzyme action by MS analysis of pectin breakdown products of plant cell walls. Analytical Biochemistry (in press)
- Shackel, K.A., M.P. Celorio-Mancera, H. Ahmadi, L.C. Greve, L.R. Teuber, E.A. Backus and J.M. Labavitch. 2004. Micro-injection of Lygus salivary proteins to simulate feeding damage in alfalfa and cotton flower. Archives of Insect Biochemistry and Physiology (in press)
- Brummel, David A., Valeriano Dal Cin, Carlos H. Crisosto and John M. Labavitch. 2004. Cell wall metabolism during maturation, ripening and senescence of peach fruit. Journal of Experimental Botany 55:2029-2039.
- Brummell, David A., Valeriano Dal Cin, Susan Lurie, Carlos H. Crisosto and John M. Labavitch. 2004. Cell wall metabolism during the development of chilling injury in cold-stored peach fruit: association of mealiness with arrested disassembly of cell wall pectins. Journal of Experimental Botany 55:2041-2052.
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