Progress 03/01/03 to 09/30/08
Outputs
OUTPUTS: The ubiquitin/26S proteasome pathway of selective protein degradation is essential for proper plant growth and development. It is responsible for the degradation of many short-lived regulatory proteins including those involved in hormone responses, light perception, and cell cycle progression. My efforts have focused on the ubiquitin-specific proteases (UBPs), the enzymes involved in dismantling polyubiquitin chains and detaching ubiquitin from target proteins. We and others have shown that these enzymes are essential for proper ubiquitin pathway function. Whereas, the general functions of the UBPs in the ubiquitin pathway are known, the specific roles performed by each of the 27 UBPs in Arabidopsis remain largely unknown. We have identified mutant plants that do not synthesize specific UBPs and have compared their phenotypes to wild-type plants. We have assembled a collection of UBP mutants in Arabidopsis that we distribute upon request. We concentrated our efforts on the UBP3/4, UBP12/13, and UBP15/16/17/18/19 subfamilies because mutant plants display aberrant phenotypes. Two graduate students are heavily involved in this project. One Ph.D. student investigated the UBP12/13 subfamily. She gained experience in plasmid construction, DNA isolation, DNA analysis, plant growth, and analysis of plant morphology and development especially regarding pollen. She attended the 17th International Conference on Arabidopsis Research in 2006 and the Botany & Plant Biology Joint Congress in 2007 to present her research. These meetings helped her gain a greater appreciation of the breadth of research in plant biology and to meet key scientists in her field. At one of these meetings, she established a collaboration to help get her results published. The other Ph.D. student has learned skills in plasmid construction, DNA isolation/analysis and protein isolation/analysis. She studied the UBP15/16/17/18/19 subfamily and presented her findings during the Davis College of Agriculture, Forestry and Consumer Sciences graduate student research conference at West Virginia University. I concentrated my efforts on UBP3/4 and published my results in Plant Physiology in 2007. Since then, I have assembled UBP3 expression constructs to investigate the role of N-myristoylation and nuclear localization on UBP3 function. I attended the 16th and 17th International Conferences on Arabidopsis Research in 2005 and 2006, and presented my results on UBP functions in Arabidopsis during the poster sessions. During these meetings, I also met with my postdoctoral mentor and several of his post docs/students to discuss findings and future plans. On average, approximately three undergraduate students per year worked in my laboratory to gain experience in laboratory research. Three of them worked on an Honor's Thesis. Three undergraduates each spent a semester or two in my laboratory during 2008 and gained experience in DNA isolation, PCR genotyping, plasmid construction, and conducting genetic crosses. Many of these former undergraduates are currently attending medical, veterinary, or graduate school. PARTICIPANTS: Jed Doelling was the principal investigator and was responsible for overall project design and implementation. I made the decision to concentrate our efforts on the genetic analysis of UBP subfamilies UBP3/4, UBP12/13, and UBP15/16/17/18/19 and the UBC subfamilies UBC4/5/6 and UBC15/16/17/18. I performed most of the bench work associated with UBP3/4 and part of the bench work for the other 4 subfamilies. This included genetic crosses, PCR genotyping of individual plants, construction of complementation cassettes, and phenotypic characterization. I had two Ph.D. students who contributed significantly to this project and about a dozen undergraduates who spent a semester or two working approximately 8 hours per week in my lab. One graduate student, Gulsum Soyler-Ogretim worked extensively on the UBP12/13 subfamily of ubiquitin-specific proteases. She has made significant progress in that we are close to publishing our results and she has become much more independent in her thinking and experimental design. She has characterized the mutant plants at the phenotypic level by comparing their phenotypes to those observed in wild-type plants. In the process, she has gained experience in recombinant DNA technology including plasmid construction, PCR genotyping, and reverse transcriptase PCR. The other graduate student, Crystal Goldyn worked extensively on the UBP15/16/17/18/19 subfamily of ubiquitin-specific proteases. She is still a year or two from graduation, but has obtained experience in the same areas as Gulsum. All of the undergraduate students were able to obtain some experience with recombinant DNA technology, plant growth and preparing lab reports. I had several informal collaborations with scientists at other universities. I worked extensively with Drs. Richard Vierstra (University of Wisconsin), Judy Callis (University of California) and Marisa Otegui (University of Wisconsin) to characterize the UBP3/4 mutants. Marisa Otegui was particularly helpful in conducting electron microscopic analysis of mutant pollen grains. I have also turned to Dr. Mark Johnson (Brown University) for advice regarding the characterization of defective pollen grains. TARGET AUDIENCES: The main target audience is scientists seeking to understand the development of organisms and the role of ubiquitin and ubiquitin-like tags in this process. This has been done via publication, and oral and poster presentations. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Ubiquitin and ubiquitin-like proteins are essential for the growth and development of all eukaryotic organisms including plants. These proteins rely on several enzyme families to control their proper attachment and detachment from target proteins. Ubiquitin-specific proteases, one class of enzymes essential for proper ubiquitin pathway function, can release ubiquitin monomers from ubiquitin chains and some ubiquitin-tagged target proteins. Arabidopsis encodes 27 different UBPs which can be organized into 14 different subfamilies. Whereas the general functions of the UBPs are known, little is known about the specific functions performed by individual UBPs. The large transfer-DNA (T-DNA) insertion collection available for the plant, Arabidopsis thaliana, provided a way to identify plants incapable of producing one or more UBPs. This year, we continued to focus our attention on the ubiquitin-specific protease subfamilies UBP3/4, UBP12/13, and UBP15/16/17/18/19. Single gene and higher order mutant plants were compared to wild-type plants at the phenotypic level. The UBP3/4 subfamily was found to be essential for pollen transmission and important for pollen mitosis II in the male gametophyte. We have assembled several modified UBP3 expression constructs to investigate the role of N-myristoylation and nuclear localization on UBP3 function. Some of these constructs have been introduced into Arabidopsis and analysis is forthcoming. UBP12/13 double mutant pollen appears to be less successful than UBP12 or UBP13 single mutant pollen at fertilizing wild-type ovules. Ovule transmission appears to be normal. UBP12/13 double homozygous mutant plants are dwarfs, never set seed, and produce abnormal flowers. Introduction of a UBP12 cDNA construct expressed under the control of the Cauliflower Mosaic Virus 35S promoter restored seed production in double homozygous mutant plants. A genetic analysis of the five member UBP15/16/17/18/19 subfamily has been complicated because many double homozygous mutant combinations are possible and a UBP19 T-DNA insertion mutant is unavailable. UBP15/16 double homozygous mutants produce perhaps one or two viable seeds per plant, have irregular, elongated leaves, and produce very little pollen. The phenotype of these mutants have been analyzed and documented carefully with regards to seed yield, silique length, leaf shape, and flower morphology. We are in the process of generating a quadruple homozygous mutant in which UBP19 is the only UBP of this subfamily that can be expressed. The ubiquitin conjugating enzymes (UBCs) are also an important class of enzymes in the ubiquitin pathway. Earlier, we attempted to disrupt the expression of UBC subfamilies UBC4/5/6 and UBC15/16/17/18. We had great hope for success because of the availability of T-DNA insertion lines for all seven of these genes. We generated triple homozygous UBC4/5/6 mutants and quadruple homozygous UBC15/16/17/18 mutants. However, we observed no phenotype in either case. We discovered that one or more of the T-DNA insertions did not disrupt the expression of the desired gene.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Ubiquitin and other polypeptide tags are involved in many aspects of plant growth and development. The ubiquitin/26S proteasome pathway is responsible for the degradation of many short-lived, regulatory proteins including those involved in hormone responses, light perception, and cell cycle progression. My efforts have focused on the ubiquitin-specific proteases (UBPs), the enzymes involved in dismantling polyubiquitin chains and detaching ubiquitin from target proteins. Whereas, these general functions of the UBPs are known, the specific roles performed by individual UBPs in Arabidopsis remain largely unknown. The goal of this project is to identify mutant plants that do not synthesize specific UBPs and to compare their phenotypes to wild-type plants. We have assembled a collection of UBP mutants in Arabidopsis that we distribute upon request. We are now concentrating on studying the UBP3/4, UBP12/13, and UBP15/16/17/18/19 subfamilies of UBPs in Arabidopsis because mutant
plants display aberrant phenotypes. Two graduate students are heavily involved in this project. One Ph.D. student is investigating the UBP12/13 subfamily. She has gained experience in plasmid construction, DNA isolation, DNA analysis, plant growth, and analysis of plant morphology and development especially regarding pollen. She attended the Botany & Plant Biology Joint Congress in 2007 and presented some of her research results. These meetings helped her gain a greater appreciation of the breadth of research in plant biology and to meet key scientists in her field. She also established a collaboration to help get her results published. The other Ph.D. student has learned skills in plasmid construction, DNA isolation/analysis and protein isolation/analysis. She is studying UBP15/16/17/18. Both Ph.D. students presented their findings during the Davis College of Agriculture and Consumer Sciences graduate student poster day at West Virginia University. I have had three undergraduate
students spend a semester or two in my laboratory learning experimental techniques (mainly in DNA isolation and analysis) and gathering data. Two have since graduated; one is attending medical school and the other is pursuing graduate studies in biotechnology.
PARTICIPANTS: Jed Doelling (principal investigator) was responsible for overall project design, purchase of experimental reagents, and carry out of the project. I have trained graduate students and undergraduate students in laboratory techniques and in experimental planning. I focused my laboratory work on the UBP3/4 subfamily of ubiquitin-specific proteases. To help with this project, I established collaborations with scientists outside West Virginia University with specialized skills. Drs. Marisa Otegui and Rick Vierstra at the University of Wisconsin-Madison helped with electron microscopy and biochemistry, respectively. Graduate student Gulsum Soyler-Ogretim studied the UBP12/13 subfamily of ubiquitin-specific proteases. She analyzed the progeny of reciprocal crosses between wild-type and mutant plants to investigate transmission rates of wild-type and mutant alleles via pollen and ovules. She also recognized that seed germination is affected by this UBP subfamily and has begun
to investigate this phenomenon. Graduate student Crystal Goldyn is studying the UBP15/16/17/18/19 subfamily of ubiquitin-specific proteases. She has concentrated on the phenotypic characterization of double homozygous ubp15; ubp16 mutants with regards to leaf morphology and extreme reduction in seed production. Three undergraduate students each spent a semester working six to eight hours per week in my lab. This helped them experience what it is like to work in a laboratory and to gain skills in DNA isolation and analysis, plant growth, and preparing lab reports summarizing their findings. These students completed a summary assignment to help them recognize how genetic concepts were used in experimental planning and data analysis.
TARGET AUDIENCES: The main target audience is scientists seeking to understand the development of organisms and the role of ubiquitin and ubiquitin-like tags in this process. This has been done via publication, and oral and poster presentations.
Impacts
Ubiquitin and ubiquitin-like proteins are essential in the growth and development of all eukaryotic organisms including plants. These proteins rely on several enzyme families to control their proper attachment and detachment from target proteins. This year, we focused our attention on the study of the ubiquitin-specific proteases (UBPs), one family of enzymes capable of detaching ubiquitin moieties from target proteins and ubiquitin chains. Whereas the general functions of UBPs are known, little is known regarding the specific functions performed by individual UBPs. The large transfer-DNA (T-DNA) insertion collection available for the plant, Arabidopsis thaliana, provides a way to identify plants incapable of producing one or more UBPs. These mutant plants can then be compared to wild-type plants at the phenotypic and biochemical levels. Arabidopsis encodes 27 different UBPs which can be organized into 14 different subfamilies. At least five of these UBP subfamilies
are important for the proper growth and development of Arabidopsis. The UBP1/2 and UBP14 subfamilies have been characterized earlier. The UBP3/4 subfamily is essential for pollen transmission and appears to be important for the second mitotic cell division in the male gametophyte. However, some double mutant ubp3; ubp4 pollen does appear to undergo pollen mitosis II and to germinate, indicating that UBP3/4 continues to be important during later stages of pollination. In addition, the functions of UBP3/4 do not appear to be pollen-specific because these proteins can be detected in many if not all sporophytic tissues. The UBP12/13 subfamily appears to help pollen transmission (and perhaps ovule transmission) in that double mutant pollen appears less successful than single mutant pollen in fertilizing wild-type ovules. Curiously, some seeds arising from the self fertilization of UBP12/ubp12; ubp13/ubp13 and ubp12/ubp12; UBP13/ubp13 plants germinate much later than their siblings (20 days
until germination rather than the usual 2 days). None of the normal-germinating individuals were determined to be double homozygous mutants, but many of the delayed-germinating individuals were genotyped as double homozygous mutants. The genetic analysis of the five member UBP15/16/17/18/19 subfamily is complicated by the fact that many double homozygous mutant combinations are possible. Our most obvious observations in their analyses are that ubp15; ubp16 double homozygous mutants produce fewer than 5 viable seeds per plant, have irregular, elongated leaves, and produce very little pollen, and that ubp17; ubp18 gametes do not contribute to the next sporophytic generation. Additionally in reciprocal crosses with wild-type plants, ubp15; ubp16 double homozygous mutant individuals function poorly both as pollen donors and as ovule donors.
Publications
- Doelling, J.H., Phillips, A.R., Soyler-Ogretim, G., Wise, J., Chandler, J., Callis, J., Otegui, M.S. and Vierstra, R.D. (2007) The ubiquitin-specific protease subfamily UBP3/UBP4 is essential for pollen development and transmission in Arabidopsis. Plant Physiology 145: 801-813.
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Progress 01/01/06 to 12/31/06
Outputs
The ubiquitin/26S proteasome pathway of selective protein degradation is involved in many aspects of plant growth and development. Much knowledge about how this pathway functions has been reported in journal articles in recent years. I am studying this pathway in the plant, Arabidopsis thaliana by focusing on two classes of enzymes : 1) the ubiquitin-specific proteases (UBPs), and 2) the ubiquitin conjugating enzymes (UBCs). Arabidopsis encodes at least 27 different UBPs and 35 different UBCs. My initial strategy is to eliminate the synthesis of one or more specific UBPs or UBCs and thereafter to characterize the phenotypic consequences. In past years, I have gathered a collection of T-DNA insertion mutants and generated double and triple mutants by genetic crosses. Many of the UBPs and UBCs are products of multigene families, each family member encoding a protein with substantial amino acid sequence similarity. The double or triple mutants are to eliminate from a
single plant multiple proteins of a family in case they perform overlapping functions. I am currently studying the UBP3/4, UBP12/13, UBP15/16/17/18/19, UBC4/5/6, and UBC15/16/17/18 families. The UBP3/4 family is important for the second mitotic division of haploid pollen that generates the two sperm cells from the generative cell and essential for pollen function. UBP12/13 elevates male fertility and is essential for embryo/seed development. Double mutants of the UBP15/16/17/18/19 family show a wide range of phenotypes ranging from the normal (ubp15/17, ubp15/18, ubp16/18) to gametophyte lethal (ubp17/18). The UBP15/16 double mutants appear quite normal until they enter the reproductive stage. They develop weak influorescences and produce very little pollen and therefore, very few seeds per plant (A normal plant typically produces more than a thousand seeds). Currently, it appears that neither UBC4/5/6 nor UBC15/16/17/18 is essential to Arabidopsis growth and development. Additional
tests will soon be conducted to determine if gene expression has been entirely eliminated.
Impacts
The ubiquitin/26S pathway of selective protein degradation is involved in all aspects of plant growth and development. The more than 1000 different proteins that function in this pathway in Arabidopsis thaliana have been functionally classified as ubiquitin activating enzymes, ubiquitin conjugating enzymes (UBCs), ubiquitin protein ligases, ubiquitin-specific proteases (UBPs) and proteasome subunits. Arabidopsis encodes 27 different UBPs and more than 35 different UBCs. The general functions of these proteins in this pathway are known; however, the specific functions of particular UBPs and UBCs are not. By learning the mechanisms by which individual UBPs and UBCs function, various methods to increase the profitability of crops and ornamentals may be discovered.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
The ubiquitin/26S proteasome pathway of selective protein degradation is a key regulatory pathway involved in plant growth and development. The importance of this pathway is manifested by the numerous articles published about this pathway in many different eukaryotic organisms during the last few years. My goal is to investigate the particular roles performed by two families of proteins essential to the ubiquitin/26S proteasome pathway in the model plant species, Arabidopsis thaliana. These two families are the ubiquitin conjugating enzymes (UBCs) and the ubiquitin-specific proteases (UBPs). My main focus during 2005 was to assemble and begin characterization of a collection of Arabidopsis mutants; each member of the collection is unable to synthesize one particular UBC or UBP due to disruption of the corresponding gene by T-DNA insertion. My current collection is composed of one or more mutant seed lines for 23 of the 41 UBC genes and 25 of the 27 UBP genes. Only two
single gene mutants in the collection displayed an obvious phenotype alteration in the homozygous state. Because amino acid sequence comparisons between the 41 different UBCs and the 27 different UBPs in Arabidopsis indicated that many share significant similarity to one another, it is possible that the related proteins serve overlapping and/or similar functions. Based on these sequence comparisons, the family of UBC proteins has been organized into 14 subfamilies of related proteins, and the family of UBP proteins has been organized into 14 subfamilies. Many of the homozygous single gene mutants that do not display an obvious phenotypic consequence involve genes that belong to a multi-gene subfamily. I have chosen to focus my studies on eight specific subfamilies, namely UBP3/4, UBP12/13, UBP15/16/17/18/19, UBP20/21, UBP26, UBC4/5/6, UBC7/13/14, and UBC15/16/17/18. I have begun to assemble double mutants and plan to assemble higher order mutants, if necessary, to determine the
functions of particular protein subfamilies during Arabidopsis development. The UBP3/4 subfamily is essential for pollen transmission. The development of double mutant pollen becomes abnormal at different stages presumably due to the amount of residual protein present in the double mutant cells. Although some double mutant pollen appears to germinate and the resulting pollen tubes do elongate, no example of a successful fertilization by such a pollen grain has been obtained. Most double mutant pollen does not undergo the second mitotic division; hence the pollen contains the expected vegetative nucleus but only one sperm nucleus. Additionally, I have gathered preliminary evidence that the UBP12/13 subfamily is required for embryo development, that the UBP26 subfamily is required for pollen transmission, and that UBP15/16 somehow affects flower development and seed set.
Impacts
Plant growth and development involves the complex interplay of numerous proteins. One very important mechanism by which the levels (and activities) of particular proteins are regulated is ubiquitin-dependent, selective protein degradation. Using genetics as the initial research tool, researchers will gain valuable information regarding the functions of two classes of ubiquitin pathway components, the ubiquitin conjugating enzymes and the ubiquitin-specific proteases, during plant growth and development. The information gained will provide ideas for increasing the profitability of crops and ornamentals using biotechnology.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
The major goals of this research are: 1) to identify all genes encoding ubiquitin-specific proteases (UBPs)and ubiquitin conjugating enzymes (UBCs) in the plant Arabidopsis thaliana, 2) to assemble a collection of T-DNA insertion mutants in which the expression of particular UBPs or UBCs is disrupted, and 3) to characterize the phenotypes of mutant plants. Prior to 2004, Goal 1 was essentially complete; I concentrated on the other two goals during 2004. A search of the genomic DNA sequence of Arabidopsis (http://www.arabidopsis.org) revealed 41 different sequences which may encode UBCs and 27 which may encode UBPs. A search of the Arabidopsis EST databases indicated that full length cDNA sequences have been identified for 37 of the 41 UBC genes and 23 of the 27 UBP genes. The database includes partial cDNA sequences for three additional UBP genes. I have determined that one additional UBC gene is transcribed and that two of the three remaining UBC genes are likely
pseudogenes (sequence analysis suggests that they do not encode complete UBC proteins). I also have evidence that one (UBP17) of the three UBP genes with partial cDNA sequence is in fact a pseudogene with an in frame stop codon. I searched the Salk, Syngenta, Feldman, and Wisconsin Biotech T-DNA collections for lines containing insertions located either within or adjacent to either an UBP gene or an UBC gene. I have obtained 93 insertion lines representing 59 genes and analyzed them by PCR to determine if the insertions are actually present near the expected location. Included are lines for all 27 UBP genes (the insertion is expected outside of the coding region for UBP19 and UBP22) and 32 of the 40 UBC genes (the T-DNA insertion mapped just outside of the coding region for five of these genes). I have confirmed a T-DNA insertion for 49 of the 59 genes (by PCR with appropriate gene-specific and T-DNA-specific primers). For many of these lines, I have conducted backcrosses with
wild-type plants to remove possible second site mutations. A major push in my lab during 2004 was to identify plants where both copies of a specific gene are interrupted by a T-DNA insertion. We have identified 37 homozygous mutant plants that do not display any obvious phenotypic differences from wild-type plants. This was not entirely unexpected since many genes share significant sequence similarity to one or more sequences elsewhere in the genome (these sequences are all included in the 68 genes). Because all related genes might need to be interrupted within the same plant to see a phenotypic change, we have crossed mutant plants together to produce double gene mutants. One of these double mutants (UBP3/4) is defective in pollen transmission. In this mutant, pollen development appears to be defective; however, one particular stage cannot be identified because the developmental abnormalities begin at different stages. Other double and triple mutants are in the process of being
generated. We will begin their analysis and continue to analyze several single gene mutants for which a homozygous plant could not be identified.
Impacts
Plant growth and development involves the complex interplay of numerous proteins. One very important mechanism by which protein levels (and activities) are regulated is selective protein degradation mediated by the polypeptide tag ubiquitin. By studying the specific roles of various ubiquitin conjugating enzymes and ubiquitin-specific proteases using genetics as the primary research tool, researchers will gain valuable information regarding the role of protein degradation in plant growth and development. This information may provide ideas for increasing the profitability of crops and ornamentals using biotechnological approaches.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
My major goals at this time are to 1) identify all genes encoding ubiquitin-specific proteases and ubiquitin conjugating enzymes in the plant Arabidopsis thaliana, 2) assemble a collection of T-DNA insertion mutants in which the expression of particular ubiquitin-specific proteases or ubiquitin conjugating enzymes is disrupted, and 3) phenotypically characterize these homozygous mutants. I have made progress in all three of these goals. A thorough search of the available genomic DNA sequence of Arabidopsis has revealed 27 different sequences which may encode ubiquitin-specific proteases and 40 different sequences which may encode ubiquitin conjugating enzymes. I have attempted to identify mRNAs of the respective genes using PCR amplification of cDNA libraries, RT-PCR, and the Arabidopsis EST databases. Others have identified cDNAs for all but four of the ubiquitin conjugating enzyme genes. I now have preliminary evidence that two of these four genes are transcribed. I
have searched the Salk and Syngenta T-DNA insertion collections for lines having insertions within either an ubiquitin-specific protease gene or an ubiquitin conjugating enzyme gene. I have obtained 63 different insertion lines and have begun to verify that the mutations are actually present. These include lines for all but two of the 27 ubiquitin-specific protease genes and 28 of the 40 ubiquitin conjugating enzyme genes. So far I have confirmed that 32 of these lines actually contain the desired gene disruption and 8 of the lines do not contain the desired gene disruption. This information was obtained using the polymerase chain reaction to amplify the gene/insertion junction using appropriate gene-specific and T-DNA-specific primers. After verifying that the insertion is present, I have backcrossed the mutant lines with wild-type plants to reduce the occurrence of second site mutations in these lines. I have begun the phenotypic characterization of the ubp3/ubp4 double mutant. I
showed earlier that this gene family is essential in Arabidopsis and that pollen must have a functional copy of at least one of the two genes. I recently found that pollen tubes grow from both mutant and wild-type pollen. Hence, the function of the mutant pollen must be impaired after pollen tube growth. It is anticipated that in 2004, several additional mutant lines will be tested for phenotypic abnormalities.
Impacts
Plant growth and development involves the complex interplay of numerous proteins. One very important mechanism by which protein levels (and activities) are regulated is selective protein degradation mediated by the polypeptide tag ubiquitin. By studying the specific roles of various ubiquitin conjugating enzymes and ubiquitin-specific proteases using genetics as the primary research tool, researchers will gain valuable information regarding the role of protein degradation in plant growth and development. This information may provide ideas for increasing the profitability of crops and ornamentals using biotechnological approaches to modify the ubiquitin pathway.
Publications
- No publications reported this period
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