Progress 01/01/02 to 12/31/06
Outputs
OUTPUTS: The model flowering plant Arabidopsis thaliana is widely used for investigating the growth, development and reproduction of herbaceous plants. Arabidopsis is also a good model for studying the molecular genetics of wood formation, the topic of this project. The most significant project outputs included production and analysis of gene expression profiles of wood-forming tissues (xylem) and sugar-transporting (phloem) tissues, production of cDNA libraries of genes expressed in xylem and phloem, and the discovery and characterization of protein-degrading enzymes and NAC-domain transcription factors involved in wood formation. These outputs were disseminated to multiple audiences: 1) Scientific findings were disseminated to the plant science research community via peer-reviewed publications and professional meetings; 2) cDNA libraries were donated to an undergraduate teaching laboratory at Virginia Tech; 3) Non-technical reports of findings were delivered during Master Gardener
conferences at Virginia Tech.
PARTICIPANTS: Eric Beers, PI, directed project, analyzed data, wrote publications, presented data at scientific meetings and to audiences of Master Gardeners. During the course of the project, six undergraduate students, four graduate students and one postdoctoral were trained in the principles and practices of plant molecular genetics.
TARGET AUDIENCES: The principal target audience consisted of plant scientists studying the differentiation of vascular tissues and in particular the formation of wood (xylem). The secondary audience was undergraduate and graduate students in the classroom where project results were used to illustrate fundamental concepts in plant biology and plant biotechnology.
Impacts
This project pioneered the use of Arabidopsis as a model for the molecular biology of wood formation. As a result, two important changes have been facilitated in the field of wood formation molecular biology. First, scientists interested in modifying wood quality to enhance the suitability of trees and other biomass crops as feedstocks for bioenergy have a greater understanding of how Arabidopsis, while not a true woody plant, can nonetheless contribute to efforts to alter wood properties using breeding and genetic engineering. Second, the first comprehensive list of genes potentially involved in wood-formation was made available as a result of this project, greatly improving the efficiency of selecting genes that are rational targets for strategies aimed at manipulating wood or secondary cell wall production, not only in Arabidopsis but also in biomass crops.
Publications
- Zhao, C., Avci, U., Grant, E.H., Haigler, C.H. and Beers, E.P. (2007) XND1, a member of the NAC domain family in Arabidopsis thaliana, negatively regulates lignocellulose production and programmed cell death in xylem. Plant Journal, doi: 10.1111/j.1365-313X.2007.03350.x. (in press).
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Progress 10/01/05 to 09/30/06
Outputs
Characterizations of the regulation and function of genes expressed in the woody tissue (xylem-specific genes) of Arabidopsis were continued. For three xylem-specific genes, XCP1, XCP2 and XSP1, we are increasing our level of resolution to 10-20 nucleotides of the gene promoters (DNA adjacent to the gene) required for regulation of xylem-specific gene expression. Initial attempts to use this putative regulatory region from the promoter of XCP2 to identify proteins (transcription factors) that interact the promoter to control xylem-specific gene expression were not successful. Follow-up experiments aimed at identifying these transcription factors are underway. We also developed plants that are homozygous for mutations in both XCP1 and XCP2, two redundant cysteine proteases. We have shown by immunoblot analysis that these XCP1/XCP2 double knockouts are indeed lacking XCP1 and XCP2 proteases. Evaluations of knockouts by transmission electron microscopy (TEM) revealed an
interesting ultrastructural defect consisting of persistent cellular debris in tracheary elements of knockouts compared to those in control wild type plants. We are continuing our focus on several members of a family of transcription factors, the NAC-domain family, likely to be important in regulating wood formation. We submitted a manuscript describing our findings for one xylem-specific NAC named XYLEM NAC DOMAIN1 (XND1). The height of homozygous XND1 knockout plants was reduced by as much as one-third compared to wild type Arabidopsis plants. This short-plant phenotype was associated with a corresponding reduction in the average lengths of fibers and vessel members. Leaves and siliques were also significantly shorter in XND1 knockouts. Overexpression of XND1 resulted in extreme dwarfism associated with the absence of xylem vessels. In XND1 overexpressors, vascular bundles that lacked xylem vessels exhibited little or no expression of tracheary element marker genes, while phloem
marker-gene expression and tissue polarity were not altered. TEM observations of the hypocotyl of XND1 overexpressors showed that cells in the incipient xylem zone lacked the secondary wall thickenings that typify functional tracheary elements and contained cytoplasmic contents typical of living parenchyma cells. Overexpression of XND1 fused to the EAR motif transcriptional repressor domain also yielded severe dwarfs with xylem discontinuity, indicating that XND1 normally acts as a transcriptional repressor. The results from overexpression and loss-of-function experiments reported here suggest that XND1 functions as a repressor of secondary cell wall synthesis and programmed cell death in xylem. Our investigations of the flowering-timing genes MYR1 and MYR2, discovered during functional analysis phloem-specific transcription factors, is currently focused on genetic complementation experiments aimed at determining whether specific combinations of MYR1 and MYR2 splice variants
(different forms of the MYR1 and MYR2 proteins expressed from the same gene) are required for complementation of the early flowering phenotype exhibited by the MYR1/MYR2 double knockouts.
Impacts
Xylem and phloem, the two conducting tissues of the plant vascular system, are of tremendous economic importance. Secondary xylem is the wood-forming tissue that provides the raw material for the forest products industry. The transport of amino acids, sucrose and signaling molecules through the phloem determines fruit and seed quality and quantity. Despite the importance of xylem and phloem, remarkably little is known about the genes that regulate vascular tissue development. We are identifying and characterizing genes required for the regulation of wood cell development and for the function of phloem as a mediator of plant reproduction (flowering). Results from these experiments are expected to substantially advance both our understanding of plant vascular tissue development and our ability to manipulate phloem and xylem performance for enhanced food and fiber production.
Publications
- Ko J.H., Beers E.P., Han K.H. (2006) Global comparative transcriptome analysis identifies gene network regulating secondary xylem development in Arabidopsis thaliana. Mol Genet Genomics. DOI 10.1007/s00438-006-0157-1
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Progress 10/01/04 to 09/30/05
Outputs
Characterizations of the regulation and function of genes expressed in the woody tissue (xylem-specific genes) of Arabidopsis were continued. For three xylem-specific genes, XCP1, XCP2 and XSP1, we have identified small (20- to 50-nucleotide) regions of the gene promoters (DNA adjacent to the gene) required for regulation of xylem-specific gene expression. To accomplish this high level of resolution, we analyzed over 40 promoter deletions in transgenic lines of Arabidopsis. The resolution of these small regulatory regions in these promoters has allowed us to develop novel tools for identifying the proteins (transcription factors) that interact with these regulatory regions to control xylem-specific gene expression. Experiments aimed at identifying these transcription factors are underway. We also developed plants that are homozygous for mutations in both XCP1 and XCP2, two redundant cysteine proteases. We have shown by reverse transcriptase-polymerase chain reaction
and immunoblot analyses that these XCP1/XCP2 double knockouts are indeed lacking XCP1 and XCP2 proteases. Differences in growth or development of the knockouts relative to the wild type plants were not evident in preliminary evaluations of these mutants. More detailed characterizations of these mutants at the whole plant, tissue and ultrastructural levels are continuing. We completed a comprehensive gene expression profiling study to identify genes expressed specifically in xylem or phloem. From these sets of genes we have focused on several members of a family of transcription factors, the NAC-domain family, likely to be important in regulating wood formation. To date we have made the most progress on a gene we have named XND1, for XYLEM NAC-DOMAIN1. We have evaluated plants that overexpress XND1 as well as those that lack XND1 expression (XND1 knockouts). XND1 overexpressors are unable to produce xylem, while XND1 knockouts produce xylem fibers and vessel members that are only 70
percent as long as those from wild type plants. Correspondingly, XND1 knockout plants are also shorter, by about 30 percent, than wild type plants. We hypothesize that XND1 is required for preventing differentiation of xylem cells until they have reached their proper size. For our investigations of phloem gene function we chose to characterize two phloem-specific members of a plant specific G2-like family of transcription factors, MYR1 and MYR2. Single knockouts for either MYR1 or MYR2 do not appear to differ from wild type plants in growth or development. However, double knockouts for MYR1 and MYR2 exhibit significantly delayed flowering. Thus we believe we have uncovered new players in phloem-mediated control of flowering timing. Experiments are underway to identify genes that may interact with MYR1/MYR2 in the control of flowering timing.
Impacts
Xylem and phloem, the two conducting tissues of the plant vascular system, are of tremendous economic importance. Secondary xylem is the wood-forming tissue that provides the raw material for the forest products industry. The transport of amino acids, sucrose and signaling molecules through the phloem determines fruit and seed quality and quantity. Despite the importance of xylem and phloem, remarkably little is known about the genes that regulate vascular tissue development. We are identifying and characterizing genes required for the regulation of wood cell development and for the function of phloem as a mediator of plant reproduction (flowering). Results from these experiments are expected to substantially advance both our understanding of plant vascular tissue development and our ability to manipulate phloem and xylem performance for enhanced food and fiber production.
Publications
- Zhao C, Craig JC, Petzold HE, Dickerman AW, Beers EP. 2005. The xylem and phloem transcriptomes from secondary tissues of the Arabidopsis root-hypocotyl. Plant Physiol. 138: 803-818.
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Progress 10/01/03 to 09/30/04
Outputs
Research activities for this project include investigating the regulation of the tracheary element proteases XCP1, XCP2 and XSP1. This is being accomplished by constructing promoter deletions for these three genes in fusion with the reporter gene beta-glucuronidase, for the identification of promoter elements necessary for tracheary element-specific expression. Promoter elements identified from these deletion experiments will be used in yeast one-hybrid experiments to identify transacting factors that activate expression of these tracheary element proteases. In collaboration with Dr. Candace Haigler, North Carolina State University, we have immunolabeled tracheary elements in roots of Arabidopsis seedlings, using anti-XCP2 antibodies, producing what we believe is the first subcellular localization of tracheary element proteins trafficking through the secretory pathway. We are also characterizing the function of XCP2. For functional analysis we have obtained lines of
Arabidopsis plants with T-DNA inserts in XCP2 (XCP2 knockouts) and we have prepared transgenic plants for overexpression of XCP2. These plants are being analyzed for altered phenotypes. We have also performed gene chip analysis of secondary xylem and phloem isolated from Arabidopsis in collaboration with Dr. Allan W. Dickerman, Virginia Bioinformatics Institute. The gene chip experiments have identified several xylem- and phloem-specific transcription factors that may regulate vascular tissue differentiation. We are currently investigating the function of one xylem-specific transcription factor belonging to the NAC domain family and three phloem-specific transcription factors belonging to the G2-like family. Both overexpression and knockout lines for these transcription factors are affected in vascular tissue development or other aspects of radial patterning. In summary, the combination of approaches used for this project--functional genomics, promoter analysis and comprehensive
transcriptional profiling--is leading to new discoveries regarding a variety of processes that impact the development and function of plant vascular tissues.
Impacts
Xylem and phloem, the two conducting tissues of the plant vascular system, are of tremendous fundamental and economic importance. Secondary xylem is the water-conducting, wood-forming tissue and provides the raw material for the forest products industry. The efficiency of amino acid and sucrose transport through the phloem affects yield quality and quantity. The movement of signaling molecules through the phloem is a crucial component of systemic adaptation to the environment and regulation of growth and development. Despite the importance of xylem and phloem, remarkably little is known about the genes that regulate vascular tissue development. We are identifying and characterizing genes required for vascular tissue development through a combination of transcriptional profiling and functional genomics experiments. Results from these experiments are expected to substantially advance both our understanding of plant vascular tissues and our ability to manipulate their
performance for crop improvement.
Publications
- Ismail, I.O. 2004 Function and regulation of xylem cysteine protease 1 and xylem cysteine protease 2 in Arabidopsis. Ph.D. dissertation
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Progress 10/01/02 to 09/30/03
Outputs
Research activities for this project include investigating the regulation and function of the tracheary element proteases XCP1, XCP2 and XSP1. This is being accomplished by constructing promoter deletions for these three genes in fusion with the reporter gene beta-glucuronidase. Additionally, we have obtained several lines of Arabidopsis plants with T-DNA inserts in XCP1 and XCP2 and these plants are being analyzed for altered XCP1 and XCP2 levels and for altered phenotypes in an effort to determine the function of XCP1 and XCP2. We are also performing microarray analysis of secondary xylem and phloem isolated from Arabidopsis in collaboration with Dr. Allan Dickerman, Virginia Bioinformatics Institute. In collaboration with Dr. Candace Haigler, North Carolina State University we are immunolabeling tissues, using anti-XCP1 polyclonal antibodies and monospecific anti-XCP2 polyclonal antibodies, for subcellular localization of these tracheary element proteases. We have
identified the 5' upstream regions necessary for tracheary element gene expression directed by promoters for XCP1 and XSP1. Experiments to determine whether these elements are sufficient to direct tracheary element expression are underway. XCP2 knockout plants appear to develop normally when grown under typical laboratory conditions. Seeds from XCP2 knockouts and wild type plants do not differ in their viability or germination rate or in their response to low phosphate stress. However, preliminary findings using XCP2 knockouts indicate that XCP2 is involved in regulating lateral root formation in response to nitrate availability. Specifically, XCP2 knockouts appear to produce lateral roots earlier and more abundantly than wild type plants when both are grown under low nitrate conditions. We have conducted microarray analysis of xylem gene expression. This work has revealed that of the 30 papain-like cysteine proteases in the Arabidopsis genome, XCP1 and XCP2 are the only
xylem-specific papain-like enzymes. Additionally, xylem microarrays revealed a new xylem-specific subtilisin, At1g20160. Expression of At1g20160 is approximately 10-fold higher than that of XSP1. We are analyzing knockout lines for At1g20160 (in addition to XSP1 knockouts) to determine its function. Our microarray experiments have also revealed the identity of several xylem- and phloem-specific transcription factors and we are investigating the function of one xylem-specific transcription factor, XND1. Overexpression of XND1 leads to the complete suppression of tracheary element formation. In summary, the combination of approaches used for this project--functional genomics, promoter analysis and comprehensive transcriptional profiling--is leading to the discovery of new genes apparently capable of regulating a variety of processes that impact the development and function of plant vascular tissues.
Impacts
Xylem and phloem, the two conducting tissues of the plant vascular system, are of tremendous fundamental and economic importance. Secondary xylem is the water-conducting, wood-forming tissue and provides the raw material for the forest products industry. The efficiency of amino acid and sucrose transport through the phloem affects yield quality and quantity, and the movement of signaling molecules through the phloem is a crucial component of systemic adaptation to the environment and regulation of growth and development. We are identifying the best candidate genes required for vascular tissue development through a combination of microarray transcriptional profiling and functional genomics experiments. We and other investigators interested in modifying wood (and phloem-dependent characteristics) can now concentrate only on those genes identified from this project, as these are the most likely to impact vascular tissues. Consequently, results from our work are expected
to substantially advance our ability to manipulate the characteristics and performance of xylem and phloem.
Publications
- Beers, E.P., A.M. Jones, A.W. Dickerman 2004 The S8 Serine, C1A Cysteine and A1 Aspartic Protease Families in Arabidopsis. Phytochemistry, In Press.
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Progress 10/01/01 to 09/30/02
Outputs
Xylem and phloem, the two tissues that comprise the plant vascular system, are of tremendous economic and fundamental importance. Secondary xylem, the wood-forming tissue, provides the raw material for the forest products industry. The efficiency of sugar transport through the phloem is both a major determinant of yield quality and quantity for fruits, grains and tubers, and an important component of the over wintering success of perennial plant species. Both secondary xylem and phloem are derived from the same population of pluripotent stem cells known as the vascular cambium. Together, xylem and phloem typically consist of seven different cell types, all specified by the vascular cambium. In a cross-sectional view of a woody stem, the vascular cambium forms a continuous two- to four-cell thick ring that produces xylem to the inside and phloem to the outside throughout the life of the stem. Despite the importance of the vascular cambium to plant growth and to human
sustenance, genes that regulate differentiation of xylem and phloem cells produced from cambial initials have not been identified. We are using tracheary element-specific proteases, XCP1, XCP2 and XSP1, as tools to identify genes (trans-acting factors) that regulate gene expression in tracheary elements. We are also determining the function of XCP1, XCP2 and XSP1. Promoter deletion experiments are being performed for these three proteases to identify cis-regulatory elements that are required for tracheary element-specific gene expression. We are also characterizing gene knockout lines for XCP1, XCP2 and XSP1 as part of our functional analysis of these genes. A second component of this project involves near-comprehensive gene expression profiling for xylem and phloem using the Affymetrix Arabidopsis genome chip. To date we have conducted a single experiment for xylem and one for phloem. Replicate experiments are in progress, as are profiling experiment for non-vascular tissue. From our
analyses of vascular tissue-specific gene expression we expect to be able to accomplish two research objectives. First, we will produce a novel, nearly comprehensive catalog of xylem- and phloem-specific genes. Second, by comparing expression profiles from non-vascular tissues of the root with the xylem and phloem profiles, we will make significant progress towards identifying candidate trans-acting factors that control vascular cell fate. These trans-acting factors will then become the subject of additional functional genomics experiments similar to those currently underway for XCP1, XCP2 and XSP1.
Impacts
Secondary xylem, the wood-forming tissue, provides the raw material for the forest products industry. Wood production by plants is totally dependent on the cambium, a thin layer of cells that lies beneath the bark of tree stems. Despite the importance of the cambium, genes that regulate cambial cell activity have not been identified. Using the genetic model plant Arabidopsis, we are identifying features of genes that cause them to be activated in wood. Additionally, we are identifying the genes that regulate the activity of cambium- and wood-specific genes. Discoveries that emerge from these investigations will lead to efficient methods for changing the quality and quantity of wood produced by woody ornamentals, crop plants and economically important tree species.
Publications
- Funk V, Kositsup B, Zhao C, Beers EP (2002) The Arabidopsis xylem peptidase XCP1 is a tracheary element vacuolar protein that may be a papain ortholog. Plant Physiol. 128: 84-94
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