Progress 07/01/02 to 06/30/07
Outputs
OUTPUTS: The report covers the work conducted from January 1, 2007 to June 30, 2007 since this project was terminated on June 30, 2007. Since mutation of individual genes (single mutant) in the laccase family in Arabidopsis did not affect performance of plants under salinity and drought conditions, we created 33 double mutants by crossing single mutants of laccase genes that are expressed in roots. Our short-term and long-term stress experiments did not reveal significant effects of double mutations on plant growth under normal and stress conditions. To examine whether these mutations have any effect on lignin and tannin deposition in tissues, we have collected and fixed various tissues at different growth stages and are currently staining and analyzing the samples. Interestingly, we observed a double mutant showed defect in seed production, a phenotype that was not observed in either single mutant parent, suggesting these two laccase genes may be functional redundant. We are
currently determining which step in the reproductive growth was affected in the double mutant. A manuscript describing a method for identification of new cell wall protein genes or studying subcellullar localization of proteins was submitted to Planta. In addition, a large set of drought-responsive cell wall proteins were identified from maize roots and published in Plant Physiology, as a result of collaborative effort of many researchers from multiple institutes. This large scale proteomic analysis provides novel insights into the complexity of mechanisms that regulate root growth under water deficit conditions.
PARTICIPANTS: Yajun Wu, Project Leader
Impacts
Our results have demonstrated that cell wall proteins have versatile functions in regulating plant growth and responding to environmental stresses. Manipulation of some of the cell wall proteins will eventually lead to an improvement of stress tolerance in crops.
Publications
- Zhu J, Alvarez S, Marsh E, LeNoble ME, Cho I-J, Chen S, Nguyen HT, Sivagurud M, Wu Y, Schachtman DP, Sharp RE 2007. Cell wall proteome in the maize primary root elongation zone. II. Region-specific changes in water soluble and lightly ionically-bound proteins under water deficit. Plant Physiol 145:1533-1548.
- Chen D, Liang M, DeWald DB, Weimer B, Davis EJ, Peel M, Bugbee B, Wu Y* 2007. Identification of drought response genes from two alfalfa cultivars using Medicago truncatula microarrays. Acta Physiologiae Plantarum (in press).
- Xiaojun Qi, Ji Qi, Yajun Wu* 2007. RootLM: A simple color image analysis program for length measurement of primary roots in Arabidopsis. Plant Root 1:10-16.
- Valdivia ER, Wu Y, Li L-C, Cosgrove DJ, Stephenson AG 2007. A group-1 grass pollen allergen (B-expansin) influences the outcome of pollen competition in maize. PLoS ONE 2:e154. doi:10.13.
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Progress 01/01/06 to 12/31/06
Outputs
Our gene expression studies suggested that laccases in Arabidopsis may play important roles in response to salinity and dehydration stress. To provide genetic evidence for their roles in stress response, we identified T-DNA insertional mutants for 12 laccase genes. However, characterization of these mutants did not reveal drastic defect in stress response relative to their wild-type controls. We reasoned that the genetic redundancy may have masked the effect from a single gene mutation, and double or triple laccase mutants are needed to display phenotype in stress response. In the past year, we made crosses among single laccase mutants to create double mutants. Ten mutants, representing 10 different laccase genes whose expression was upregulated in the roots under stress, were chosen for crossing. We so far have identified 36 different double mutants using PCR method. A great effort was made to characterize the homozygous mutants in comparison with controls (single
mutants used for crossing and wild-type plants). No visible difference was observed between any double mutant and their controls under normal growth conditions. All mutants were then subjected to salinity and dehydration stress on MS-Phytagel plates. Based on a root elongation assay, we did not observe significant defect in stress response in the double mutants in comparison with their single mutation controls. The disappointing results from the double mutants testing may again be due to the great genetic redundancy of the laccase gene family. Alternatively, the methods we used may not be effective to detect the phenotype. We are currently taking different approaches to examine these mutants. Further characterization of single laccase mutants, we found that one of the laccase mutants showed earlier flowering than wild-type plants. Limited information in literatures indicates that some secondary metabolites can affect flowering timing in various plants. Due to their potential roles in
lignin and tannin synthesis, a mutation in laccase may cause an accumulation of certain secondary metabolites which could trigger earlier flowering. Our results of the laccase mutant screening and characterization are now reported in three refereed-journal articles. While assaying root elongation of the double mutants on Phytagel plates under different growth conditions, we realized the need of a simple method for root length measurement. In collaboration with the researchers in computer sciences on campus, we developed a simple computer program, RootLM, to measure the length of primary roots in Arabidopsis. The program is free for public download (http://www.cs.usu.edu/xqi/RootLM/).
Impacts
Our research has provided the first genetic evidence for the role of laccase in lignin synthesis. Controlling lignin content through laccase can potentially improve the quality of forage in Utah and the quality of biomass for bioenergy production. Our studies also provide valuable insights into the role of laccase in plant stress response. We also created a valuable collection of laccase mutants which is available to the plant research community. Further studies of these mutants may lead to an understanding of the role of laccase in stress response and an application of using laccase genes to improve drought and salinity tolerance of major crops in Utah.
Publications
- Liang M, Davis E, Gardner D, Cai X, Wu Y* (2006) Involvement of AtLAC15 in lignin synthesis in seeds and in root elongation of Arabidopsis. Planta 224: 1185-1196.
- Cai Xi, Davis EJ, Ballif J, Liang M, Bushman E, Haroldsen V, Torabinejad J, Wu Y* (2006) Mutant identification and characterization of laccase gene family in Arabidopsis. J Exp Bot 57: 2563-2569.
- Liang M, Haroldsen V, Cai X, Wu Y* (2006) Expression of a putative laccase gene, zmLAC1, in maize primary roots under stress. Plant Cell & Environ 29: 746-753.
- Q X-J, Q Ji, Wu Y* (2007) RootLM: A Simple Color Image Analysis Program for Length Measurement of Primary Roots in Arabidopsis. Plant Root (in press)
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Progress 01/01/05 to 12/31/05
Outputs
Laccase Gene Family: Our previous gene expression studies suggested that laccases in Arabidopsis may play important roles in response to salinity and dehydration stress. To provide genetic evidence for their roles in stress response, we focused on mutant screening and characterization this year. We identified 21 mutants for 12 laccase genes (average two independent lines for each gene) and confirmed them using RT-PCR. All mutants were subjected to salinity and dehydration stress. Based on a root elongation assay, we identified a laccase mutant that showed a reduction in growth only under PEG-induced dehydration stress. The reduction was small but statically different. We also identified two mutants for a single laccase gene with altered seed color. Biochemical and molecular analysis revealed that the mutants accumulated proanthocyanidin or condensed tannins but showed a 30% reduction in lignin. We further demonstrated that mutant seeds showed a great reduction in
lignin polymerization activity. In a recent study from another group it was shown that the same laccase mutant showed a defect in tannin synthesis, resulting in an accumulation of tannin with reduced degree of polymerization (i.e. smaller polymers). Together with our results, these studies provide evidence that this particular laccase is involved in both lignin and tannin synthesis. The findings greatly enhance our fundamental knowledge for the role of laccase in plants and also have potential applications in improving the quality of forage plants and biomass production for bioenergy and the paper industry where lignin content needs to be reduced. New Cell Wall Protein Project: We cloned an additional 7 putative new cell wall protein genes which were fused with green fluorescence protein (GFP) in frame and transformed into Arabidopsis. Expression of the fusion proteins was driven by a 35SCaMV promoter. More than 10 independent transformant lines for each gene were obtained. Under a
confocal microscope, most of the transformants showed reasonable or strong fluorescence in the roots. Unfortunately, transformants from 8 genes (including 2 genes from last year) showed cytosolic or membrane localization of GFP protein, indicating that the putative new cell wall protein genes did not encode cell wall proteins. Only one gene appeared to have GFP localization in cell walls. Over-expression of these genes did not seem to have any readily observable phenotype. In summary, our approach appears to be very effective in determining subcellular localization of new proteins. However, our results also have suggested that the number of cell wall proteins may be significantly lower than was predicted in the Arabidopsis genome.
Impacts
Drought and salinity are two major abiotic stresses limiting crop production in Utah and other western states. Manipulation of laccase gene expression can potentially lead to drought and salinity tolerant crops and improve crop production in Utah. In addition, controlling lignin content through laccase can potentially improve the quality of forage in Utah and the quality of biomass for bioenergy production.
Publications
- Zhu J, Alvarez S, Chen S, Schachtman DP, Wu Y, Sharp RE. 2005. Cell wall proteome in the maize primary root growth zone: region-specific responses to water deficit.. Annual ASPB meeting.
- Zhu J, Chen S, Alveraze S, Asirvatham VS, Schachtman DP, Wu Y*, Sharp RE. 2005. Cell wall proteome in the maize primary root elongation zone. I. Extraction and identification of water soluble and lightly ionically-bound cell wall proteins.. Plant Physiol, in press.
- Endo S, Wu Y*. 2005. Identification of key genes in abiotic stress responses in plants . (URCO-undergraduate research, USU).
- Cai X-N, Liang M-X, Davis E, Haroldsen V, MacAdam J, Wu Y*. 2005. Laccase gene expression in maize and Arabidopsis roots under stress. . Annual ASPB meeting.
- Wu Y*, Jeong B-R, Fry SC, Boyer JS. 2005. Change in XET activities, cell wall extensibility and hypocotyl elongation of soybean seedlings at low water potential. . Planta 220:593-601.
- Liang M, Haroldsen V, Cai X, Wu Y*. 2005. Expression of a putative laccase gene, zmLAC1, in maize primary roots under stress.. Plant Cell & Environ, in press.
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Progress 01/01/04 to 12/31/04
Outputs
Laccase Gene Expression under Stresses: Laccases comprise a multigene family in Arabidopsis. They have been proposed to be involved in the lignification process in plants. However, genetic evidence for their physiological function is still missing. As a step toward understanding their in vivo functions, we studied the expression pattern of the laccase genes in Arabidopsis. The transcripts were detected for all 17 annotated laccase genes, indicating they are expressed genes. Different laccase genes have different expression patterns. One laccase is specifically expressed in roots; another is only expressed in silique. The rest of the laccase genes are expressed in more than one tissue. All genes but one are expressed in the roots. Based on quantitative gene expression analysis, 9 laccase genes showed a great increase in transcript level after salinity (150 mM NaCl) and dehydration (40% PEG) treatment. Low concentrations of ABA could enhance laccase gene expression
directly. It is possible that the regulation of these laccase transcripts under stresses may be mediated by ABA. In contrast to Arabidopsis, a maize laccase gene was only affected by salinity treatment and not by PEG. Thus ABA may not be involved in regulation of this maize laccase gene expression. We have identified putative mutants for 12 Arabidopsis laccase genes. Once these mutants are confirmed, we will examine whether the mutants compromise tolerance to salinity and dehydration stress. New Cell Wall Protein Gene Project: We have successfully obtained transformants that are over-expressing two putative cell wall proteins fused with green fluorescence protein (GFP). We observed strong green fluorescence under a confocal microscope from the transformants that are over-expressing one of the putative cell wall protein genes. Very weak or no green fluorescence was detected from the transformants of the other gene. Thus, the vector we have designed is functioning and will facilitate
cloning of other genes. We also observed that the GFP was mainly localized around cells, suggesting an association with cell walls. The GFP localization will be examined more closely after cells are plasmolyzed. The line that exhibited the strongest green fluorescence had much shorter and hairier roots compared with untransformed plants. Thus, this gene may be involved in controlling root growth and development. Meanwhile, we have also done some preliminary phenotyping on the insertional mutants that we have confirmed. Mutants are only available for some of the genes on the 25-gene list. No obvious growth defect has been observed from all the mutants examined under normal growth conditions. Currently we are doing more detailed testing on these mutants, including abiotic stress treatments. We have ordered 18 more putative mutant lines for 11 additional genes. If they are true mutants, we should have mutants for 20 genes on the 25-gene list.
Impacts
Drought and salinity are two major abiotic stresses limiting crop production in Utah and other western states. Manipulation of laccase gene expression can potentially lead to drought and salinity tolerant crops and improve crop production in Utah. In addition, controlling lignin content and other cell wall components through laccase and other cell wall protein genes can greatly improve the quality of forage in Utah.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
We have completed bioinformatic analysis on the top 25 genes that were identified as potential novel cell wall protein genes from our previous work. Based on their limited similarity to other known proteins, some of the protein activity assays will be targeted in the future. A vector that was designed to facilitate cloning and expression of these putative novel cell protein genes has been completed. To test the construct, two genes from the 25-gene list have been cloned, sequenced and transformed into Arabidopsis plants with this vector. If the construct is working as expected, the transformants will over-express these genes and hopefully exhibit visible phenotype which will provide the first genetic evidence for the function of these genes. Since these proteins are fused with green fluorescence protein (GFP), we will be able to determine cellular localization of the proteins. Another reverse genetics approach that we took was to use T-DNA insertional mutants. After
searching the T-DNA insertional mutant database, we found potential mutants for 12 genes on the 25-gene list. By using PCR-based screening method, we have confirmed insertion and identified homozygous mutants for 9 genes. In the coming year, we will identify true transformants from the seeds that we have obtained from transformation and examine cellular localization of GFP-tagged proteins. If the construct works as expected, i.e. overexpressing GFP-fused proteins, 10 more genes from the 25-gene list will be selected for cloning and over-expression in Arabidopsis. However, if the construct is not working properly, modification will be made to the construct. The new construct will be tested again in Arabidopsis plants. We will start to characterize the transformants or/and mutants that are available for the changes in morphology and physiology. The proteins showing localization in cell walls in over-expression transformants or the plants showing obvious phenotype in T-DNA insertional
mutants or in transformants will be selected for more detailed analysis, such as cell wall properties and regulation of gene expression.
Impacts
The approach that we are developing can become a powerful tool for studying other genes/proteins with unknown functions.
Publications
- Wu Y, DeWald D, Chen D, Qian Y (2003) Identification of novel cell wall protein genes in Arabidopsis. August Annual Plant Biologist Society Meeting. (Poster Abstract number: 1466)
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Progress 01/01/02 to 12/31/02
Outputs
The first step of this project is to identify potential novel cell wall protein genes in Arabidopsis genome using bioinformatics tools. As a joined effort with a bioinformatician in the Biotechnology and Genomics Research Center at Utah State University, we have found that 5,779 families or single genes are annotated as unknown or hypothetical proteins or similar to unknown proteins in the Arabidopsis genome. Among them, total 583 families or single genes were found containing signal peptides using SignalP prediction. We have identified 25 genes or representative genes from families with the highest signal peptide prediction score (i.e. mostly possibly to be secreted) and absence of transmembrane domains or known ER retention or vacuolar delivery signature. These genes have cDNAs in database, indicating they are truly expressed proteins. In addition, the representative genes from families were selected from the ones that every member in the family has a signal
peptide.
Impacts
This project will greatly improve our understanding on how cell wall proteins are involved in regulating plant growth and development. Manipulation of some of the cell wall proteins in plants can potentially lead to an improvement of plant growth and production.
Publications
- No publications reported this period
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