Progress 10/01/08 to 09/30/13
Outputs
Target Audience: Target audiences included: Agricultural seed companies benefitting from new knowledge about cotton fiber and cellulose synthesis. Graduate students in a course taught by Dr. Haigler who benefitted from current research insights. Postdoctoral researchers and undergraduate students participating in research projects in the lab. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Five postdoctoral research associates (two femaies), one PhD graduate student (Hispanic female), and two female (one African American) undergraduate researchers worked on the project. How have the results been disseminated to communities of interest? Four peer-reviewed publications were published in international journals. Six presentations were made at three international research meetings. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
(1) We published a large-scale analysis of the plant cell wall polysaccharide composition in the cotton fibers of the two commercial cotton species, Gossypium hirsutum and Gossypium barbadense. These data revealed differences that may underlie the higher fiber quality parameters of G. barbadense, which can be tested in future experiments. We also worked with a colleague to interpret spectroscopic information from cotton fiber in the biological context, revealing a relatively rapid means to compare key characteristics between different types of cotton fibers. (2) We interpreted the first information about the structure of a plant cellulose synthase, based on a computational model generated in the group of a colleague in NC State College of Engineering (Yara Yingling). This protein is a key player in the synthesis of our most abundant renewable biomaterial, cellulose, and the structural information allows us to make mechanistic predictions about how to alter cellulose synthesis beneficially in future ‘designer’ crop plants. (3) Fluorescence imaging of cotton fiber cross-sections revealed the distribution of particular cell wall polysaccharides across developmental time in two commerically important cotton fiber species. This information was coupled with biochemical analysis to provide clues about key differences that may account for the higher fiber quality in Gossypium barbadense as compared to Gossypium hirsutum. (4) We published information that cotton breeders can use to guide their further work.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Avci U, Pattathil S, Singh B, Brown VL, Hahn MG, Haigler CH (2013) Cotton fiber cell walls of Gossypium hirsutum and Gossypium barbadense have differences related to loosely-bound xyloglucan, PLoS ONE 8(2): e56315. doi:10.1371/journal.pone.0056315
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Sethaphong L, Haigler CH, Kubicki JD, Zimmer J, Bonetta D, DeBolt S, Yingling YG (2013) Tertiary model of a plant cellulose synthase, Proceedings of the National Academy of Sciences USA 110: 7512-7517. doi: 10.1073/pnas.1301027110
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Abidi N, Cabrales L, Haigler CH (2014) Changes in the cell wall and cellulose content of developing cotton fibers analyzed by FTIR spectroscopy. Carbohydrate Polymers 100: 9-16, http://dx.doi.org/10.1016/j.carbpol.2013.01.074
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Slabaugh E, Davis JK, Haigler CH, Yingling YG, Zimmer J (in press) Cellulose synthases: new insights from crystallography and modeling. Trends in Plant Science, http://dx.doi.org/10.1016/j.tplants.2013.09.009
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: Plant-made natural fiber has always been a foundation of human civilization, being used for fuel, building material, textiles, and manufacture of numerous products. All plant fibers, including cotton fiber used for textiles and fibers within plant biomass, have thick secondary cell walls with cellulose as a major component (~45% and ~95% cellulose in wood and cotton fiber, respectively). The abundant cellulose has major effects on the quality characteristics of fiber products (e.g. strength and absorption), as well as being the main target for saccharification to release sugars as part of biofuel production. This project seeks to unify genomic, genetic, molecular, biochemical, microscopic imaging, and computer modeling technologies to obtain a comprehensive understanding of the controls of cellulose synthesis and fiber differentiation and quality. New knowledge generated will be used to design strategies to improve fiber crops, either through biotechnology, marker assisted breeding, or other related techniques. In this reporting period, outputs were: four published peer-reviewed research articles (including two primary research papers and two reviews); one peer-reviewed book chapter; one entry for World Book Encyclopedia; and one PhD dissertation (authored by Lissete Betancur Aberg), seven presentations or posters at four international meetings (five by postdoctoral scholars), and six technical reports to three research sponsors. PARTICIPANTS: Individuals who worked on the project: Dr. Candace Haigler, PI/PD, designed and analyzed research, collected data. Dr. Richard Glick, Research Assistant, collected data and managed the laboratory. Partner Organization: Cotton Inc., Cary, NC Training and Professional Development: This project during this reporting period provided training opportunities to two undergraduate students (David Warren, Heather Hill), three graduate students (L. Betancur, Lora Sigmon Chatham, Leona Tyler), and five postdoctoral fellows (Jon Davis, Mike Stiff, Rich Tuttle, Erin Slabaugh, Katayoun Mansouri). TARGET AUDIENCES: The target audiences of this research is producers of cotton fiber and other fiber crops, as well as international agricultural companies that sell improved seed and fiber products and benefit in their internal research efforts from enhanced knowledge of fundamental aspects of secondary wall cellulose synthesis and fiber development. In addition to guiding this research, which is accomplished through students and advanced researchers, I teach a graduate class that surveys how plants have adapted to the terrestrial environment and control their life processes through genetic, cellular, biochemical, developmental, and signaling mechanisms. This teaching helps to improve the student's ability to plan and perform research as they become broadly knowledgeable about plants. Aspects of my research program are routinely integrated into my teaching. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our peer-reviewed publications and meeting presentations added to knowledge related to objective (a) of the project: Understand how cotton fiber differentiation is regulated, emphasizing cell-wall-based control of cotton fiber quality. We contributed to the analysis of gene sequences encoding cellulose and other cell wall polysaccharides, which was published in Nature under the leadership of Dr. Andy Patterson (Univ. Georgia) as part of describing the first high quality Gossypium (cotton) gene sequence. We also published a paper in Plant Methods describing new tools related to testing the function of cotton genes via down-regulation of their expression through virus-induced gene silencing. Two review articles were published highlighting research progress and potential related to cotton fiber biology and improvement. The PhD dissertation of Lissete Betancur Aberg was finished, which shows a blending of research on arabidopsis and cotton fiber to understand the function of genes expressed in cotton fiber. Related to objectives (b), Determine the cellular organization of the cellulose-synthesizing complex (CSC), and (c) Apply imaging technologies to problems of agricultural importance, a review on moss cell walls and the potential to analyze the cellulose synthesizing complex in moss via freeze fracture electron microscopy was published. Moss provides a model system in which some research questions are more easily addressed than in vascular plant systems. For broad dissemination of knowledge to young students, Haigler wrote the new entry on cellulose for World Book Encyclopedia. Some of the meeting presentations related to objectives (b-d) of the project in other ways, and those outcomes will be itemized in future years once the related publications are available. Changes in knowledge: The first high quality cotton genome sequence is now available, albeit from a diploid ancestor with a smaller genome size but poor fiber. This provides an essential foundation for more effective functional genomics and marker assisted breeding efforts and for future efforts to sequence the larger genome of commercial allotetraploid cotton. Methods are now available for practical and low cost devices for biolistic bombardment of virus-induced gene silencing vectors into cotton, which provide uniquely helpful and novel research and teaching tools. Synthetic review articles on cotton fiber were published, which will stimulate interest and progress in this research area in a broad international arena. A review on a specialized model system, moss, was published, highlighting how it is uniquely suited to address particular questions relevant to plant cellulose synthesis.
Publications
- Paterson AH, Many X, Other Y, Authors Z, and Haigler CH (2012) Repeated polyploidization of Gossypium genomes and the evolution of spinnable cotton fibres. Nature 492: 423-427
- Tuttle JR, Haigler CH, Robertson D (2012) Method: low-cost delivery of the cotton leaf crumple virus-induced gene silencing system. Plant Methods 8:27 (8 pages)
- Roberts AW, Roberts EM, Haigler CH (2012) Moss cell walls: structure and biosynthesis. Frontiers in Plant Science 3: Article 166 (7 pages)
- Haigler CH, Betancur L, Stiff MR, Tuttle JR (2012) Cotton fiber: a powerful single-cell model for cell wall and cellulose research. Frontiers in Plant Science 3: Article 104 (7 pages)
- Stiff MR, Haigler CH (2012) Recent advances in cotton fiber development. In D. Oosterhuis and T. Cothren, eds, Cotton Flowering and Fruiting (http://www.cotton.org/foundation/fandfcontents.cfm), Cotton Physiology Book Series, The Cotton Foundation, Cordova TN, pp. 163-192
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: Plant-made natural fiber has always been a foundation of human civilization, being used for fuel, building material, textiles, and manufacture of numerous products. All plant fibers, including cotton fiber used for textiles and fibers within plant biomass, have thick secondary cell walls with cellulose as a major component (~45% and ~95% cellulose in wood and cotton fiber, respectively). The abundant cellulose has major effects on the quality characteristics of fiber products (e.g. strength and absorption), as well as being the main target for saccharification to release sugars as part of biofuel production. This project seeks to unify genomic, genetic, molecular, biochemical, microscopic imaging, and computer modeling technologies to obtain a comprehensive understanding of the controls of cellulose synthesis and fiber differentiation and quality. New knowledge generated will be used to design strategies to improve fiber crops, either through biotechnology, marker assisted breeding, or other related techniques. In this reporting period, outputs were the publication of three peer-reviewed research articles and five presentations at two international meetings. One publication, which was collaborative with Dr. Earl Taliercio (USDA/ARS-Raleigh), demonstrated an improved tissue culture medium for culture of cotton ovules and fibers in vitro, which will enhance future cotton fiber research. It also pointed to critical intracellular controls of cotton fiber differentiation that can be explored and perhaps exploited for future improvement of cotton fiber length. A second publication, which was collaborative with Dr. Chandrashekhar Joshi (Michigan Tech University) as well as numerous other researchers in the USA and Canada, was featured on the cover of the journal and empirically demonstrated the essential role of cellulose within woody tissue in supporting the tree growth habit. A third publication, which was collaborative with Dr. Brian Sosinski and others at NC State, was a key part of a graduate student's PhD research and demonstrated the feasibility of causing transformed plants to synthesize heat-activatable enzymes while they are alive to facilitate self-processing of their harvested biomass. Similar technologies are expected to be a key part of efficient and economical biomass conversion for future biofuels production. In terms of meeting presentations, we transmitted knowledge on: (a) the controls of cotton fiber development to a workshop focused on cotton breeders; (b) extensions of virus induced silencing (VIGS) in cotton to allow testing of the function of genes that are expressed in cotton fiber (cotton VIGS is a newly emerging key tool for cotton improvement programs in academics, government, and industry); and (c) mechanisms that underpin the synthesis of cellulose, nature's most abundant renewable biomaterial. PARTICIPANTS: Participants were: Dr. Candace Haigler, PI/PD, who designed and analyzed research and collected data; and Dr. Richard Glick, Research Assistant, who collected data. Partner organizations were: Cotton Incorporated, Cary NC; Michigan Tech University; and USDA-ARS, Raleigh, North Carolina. Collaborators and contacts newly added to the project were: Dr. George Allen, Dept. of Horticultural Science, NC State; Dr. Sergei Krasnyanski, Dept. of Horticultural Science, NC State University; Dr. Alex Smirnov, Dept. of Chemistry, NC State University; and Dr. Michael Hahn, Dept. of Plant Biology and CCRC, University of Georgia. Opportunities for training and professional development were provided to: One undergraduate student (Loren Moles), two graduate students (L. Betancur, J.R. Tuttle), and four postdoctoral fellows (R. Alkhatib, J. Davis, M. Stiff, J.R. Tuttle). TARGET AUDIENCES: The target audiences of this research is producers of cotton fiber and other fiber crops, as well as international agricultural companies that sell improved seed and fiber products and benefit in their internal research efforts from enhanced knowledge of fundamental aspects of secondary wall cellulose synthesis and fiber development. In addition to guiding this research, which is accomplished through students and advanced researchers, I teach a graduate class that surveys how plants have adapted to the terrestrial environment and control their life processes through genetic, cellular, biochemical, developmental, and signaling mechanisms. This teaching helps to improve the student's ability to plan and perform research as they become broadly knowledgeable about plants. Aspects of my research program are routinely integrated into my teaching. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our peer-reviewed publications or meeting presentations added to knowledge related to all four objectives of the project: (a) Understand how cotton fiber differentiation is regulated, emphasizing cell-wall-based control of cotton fiber quality. In collaborative work with Dr. Earl Taliercio (USDA/ARS), which was published in the Journal of Cotton Science, we showed that the typical level of calcium in the medium for cotton ovule culture (an in vitro system for studying cotton fiber development) hindered the early elongation of cotton fiber. Lower levels of calcium promoted more rapid elongation. Given that cotton ovules/fiber cultured in vitro are an important research tool in attempts to understand the genetic and cellular controls of cotton fiber development, these results will allow future research to be done with culture conditions that support more normal elongation. The results also suggest that the calcium-mediated signaling helps to control cotton fiber elongation, a finding that can be explored further and potentially exploited as means of improving cotton fiber length. (b/c) Determine the cellular organization of the cellulose-synthesizing complex (CSC)/Apply imaging technologies to problems of agricultural importance. In work reported at the national meeting of the American Chemical Society, we collaborated with Dr. Mark Grimson in using freeze fracture transmission electron microscopy to reveal additional details about the structure of the protein complex that synthesizes cellulose as well as the site of cellulose microfibril formation on the surface of the plasma membrane. This knowledge helps us understand how the cellulose-synthesizing machinery may be able to be manipulated to improve the properties of cellulose in biomass plants. In collaborative work with Dr. CP Joshi (Michigan Tech University) and others that was published in Molecular Plant, we used transmission electron microscopy to reveal the malformed cell walls in woody tissue resulting from down-regulation of a cellulose synthase gene. The transgenic aspen plants were also dwarfed. This work proved the importance of one cellulose synthase enzyme (among many that are present) for normal wood formation and demonstrates the irreplaceable role of cellulose within the xylem in allowing trees and other plants to grow tall. (d) Collaborate with others to link basic biological knowledge to practical strategies for fiber improvement. In collaborative works with Dr. Bryon Sosinski and others at NC State that was published in Biotechnology Progress, we showed that sweet potatoes can be transformed to produce a heat-activatable starch degrading enzyme. Upon heating, this enzyme catalyzed the breakdown of starch in the harvested tubers. This type of technology, when focused on lignin and cellulose-degrading enzymes, may also be extendable in the future to fiber crops targeted for biofuel production.
Publications
- Taliercio E, Haigler CH, The effect of calcium on early fiber elongation in cotton ovule culture. Journal of Cotton Science 15: 154-161 (2011)
- Joshi CP, Thammannagowda S, Fujino T, Gou J-Q, Avci U, Haigler CH, McDonnell LM, Mansfield SD, Menghesa B, Carpita NC, Harris D, DeBolt S, Peter GF, Perturbation of wood cellulose synthesis causes pleiotropic effects in transgenic aspen. Molecular Plant 4: 331-345 (2011)
- Santa-Maria MC, Yencho CG, Haigler CH, Thompson WF, Kelly RM, Sosinski B Starch self-processing in transgenic sweetpotato roots expressing a hyperthermophilic alpha-amylase. Biotechnology Progress 27: 351-359 (2011)
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: Plant-made natural fiber are a foundation of human civilization, being used for fuel, building material, textiles, and manufacture of numerous products. All plant fibers, including cotton fiber used for textiles and fibers within plant biomass, have thick secondary cell walls with cellulose as a major component (~45% and ~95% cellulose in wood and cotton fiber, respectively). The abundant cellulose has major effects on the quality characteristics of fiber products (e.g. strength and absorption), as well as being the main target for saccharification to generate biofuels. This project seeks to unify genomic, genetic, molecular, biochemical, microscopic imaging, and computer modeling technologies to obtain a comprehensive understanding of the controls of cellulose synthesis and fiber differentiation and quality. New knowledge generated will be used to design strategies to improve fiber crops, either through biotechnology, marker assisted breeding, or other related techniques. In this reporting period, major outputs were publications of five peer-reviewed research articles and presentations at two international meetings. One publication, featured on the cover of the journal and collaborative with David Livingston of the USDA-ARS and others, showed novel methods for reconstructing histological sections of plant tissues and highlighting particular features within the 3D volumes (Livingston et al. 2010 in J. Microscopy). This method aids understanding of plant responses to stress and other tissue-level phenomena of agronomic importance. Four other publications focused on cotton fiber. One publication with a graduate student first author showed that wall thickening in leaf trichomes of arabidopsis occurs by different mechanisms compared to cotton fiber, which are seed epidermal trichomes (Betancur et al. 2010 in J. Integrative Plant Biology). This result guides how to make the most effective use of arabidopsis for testing cotton gene function. One publication, which was featured on the cover of the journal, reflected international cotton researchers stressing the importance of sequencing cotton genomes (Paterson et al. in Tropical Plant Biology). One publication, which was cooperative with Univ. of Arizona, revealed cultivar-specific differences in the response of Gossypium hirsutum to virus-induced gene silencing, an important new tool for cotton functional genomics (Idris et al. 2010). One publication (Rapp et al. in BMC Biology), which was collaborative with J. Wendel and coworkers at Iowa State University, showed massive gene changes correlated with domestication of G. hirsutum cotton fiber. This article was highlighted by a commentary and is already designated as "highly accessed"; it contains novel clues about genes that were likely important in generating the improved quality characteristics of modern cotton fiber. In terms of meeting presentations, Haigler's oral presentation at the XII Cell Wall Meeting, Porto, Portugal expanded the interest of others in cotton fiber research. A collaborator, S. Krasnyanski presented a poster on our progress with cotton transformation, research momentum that has attracted outside funding for 2011. PARTICIPANTS: Individuals: Dr. Candace Haigler, PI/PD, designed and analyzed research, collected data. Dr. Richard Glick, Research Assistant, collected data. Partner Organizations: Cotton Incorporated, Cary NC; University of Arizona; University of Iowa; University of Minnesota; University of Rhode Island; USDA-ARS, Raleigh, North Carolina. Collaborators and contacts: Dr. David Livingston, USDA-ARS/Dept. of Crop Science, Raleigh, NC; Dr. Jonathan Wendel, University of Iowa; Dr. Alison Roberts, University of Rhode Island. Training or professional development: The following researchers in training participated in this research project in the Haigler Lab at NC State University: two undergraduate students; two graduate students; one postdoctoral fellow. TARGET AUDIENCES: The target audiences of this research is producers of cotton fiber and other fiber crops, as well as international agricultural companies that sell improved seed and fiber products and benefit in their internal research efforts from enhanced knowledge of fundamental aspects of secondary wall cellulose synthesis and fiber development. A broader target audience includes researchers in all sectors who can benefit from improved imaging techniques to better understand the systems they are working with. In addition to guiding this research, which is accomplished through students and advanced researchers, I teach a graduate class that surveys how plants control their life processes through genetic, cellular, biochemical, developmental, and signaling mechanisms. This teaching impacts students in several plant departments at NCSU, and I anticipate that it helps to improve their own research planning and performance as they become deeply knowledgeable of plants. Aspects of my research program are routinely integrated into my teaching. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Through peer-review publications, we added to knowledge related to two of four objectives of the project: (a) Understand how cotton fiber differentiation is regulated, emphasizing cell wall-based control of cotton fiber quality. In a graduate student research project, we showed that arabidopsis leaf trichomes and cotton fiber (a seed epidermal trichome) use different genetic mechanisms to support secondary wall thickening. This finding tells us that the leaf trichomes of the model plant arabidopsis may not be generally useful as a model for testing the function of cotton genes related to cell wall thickening. Furthermore, the paper showed that cotton fiber wall secondary wall thickening occurs similarly to xylem cells of arabidopsis and other plants. Given that xylem is the main weight component of plant biomass, cotton fiber research and identification of trait-controlling genes in cotton fiber is useful for improvement of biomass plants as well. In cooperation with the University of Arizona, we showed that different cotton cultivars respond somewhat differently to virus-induced gene silencing, an important new tool for cotton functional genomics that was developed at NC State. In this technique, a modified virus vector carries a fragment of an endogenous gene targeted for experimental silencing, and when the plant mounts a defense against the virus the expression of the targeted endogenous gene is also down-regulated. This allows the function of the targeted endogenous gene to be tested. The results showed that under warm growth conditions, the old commercial cultivar Acala SJ-1 has advantages in strong and persistent target gene silencing without extensive symptoms. In cooperation with the University of Iowa, we showed that many genes (over 7,000) showed substantially different expression patterns or magnitude in wild compared to modern cotton fiber. These changes would have occurred under the pressures of ancient domestication followed by breeding for improved cotton fiber quality characteristics. It is remarkable that so many genes could change their expression even within a single cell as trait improvement occurred under human selection pressure. The genes that changed reveal processes that were important in generating the useful properties of modern fiber, and these processes may be manipulated further through modern biotechnological or breeding strategies.(c) Apply imaging technologies to problems of agricultural importance. I assisted the work of Dr. David Livingston, USDA-ARS, on the use of novel image reconstruction technologies to understand mechanisms of freezing sensitivity and tolerance in winter cereals. In addition to revealing the three dimensional aspects of freezing damage and tolerance within an oat seedling over a relatively large spatial scale, the techniques are applicable to understanding many tissue-level phenomena in plants and animals. This knowledge in turn can support the design of strategies to improve agriculturally important organisms.
Publications
- Rapp RA, Haigler CH, Flagel L, Hovav RH, Udall JA, Wendel JF (2010) Gene expression in developing fibers of Upland cotton (Gossypium hirsutum L.) was massively altered by domestication. BMC Biology 2010, 8:139, doi:10.1186/1741-7007-8-139, Open access at http://www.biomedcentral.com/1741-7007/8/139; a Highly Accessed Article with accompanying commentary article.
- Idris AM, Tuttle JR, Robertson D, Haigler CH, Brown JK (2010) Differential cotton leaf crumple virus-VIGS-mediated gene silencing and viral genome localization in different Gossypium hirsutum genetic backgrounds. Physiological and Molecular Plant Pathology 75: 13-22,doi:10.1016/j.pmpp.2010.07.002
- Paterson AH, Rong J-K, Gingle AR, Chee PW, Dennis ES, Llewellyn D, Dure LS III, Haigler C, Myers GO, Peterson DG, ur Rahman M, Zafar Y, Reddy U, Saranga Y, Stewart JM, Udall JA, Waghmare VN, Wendel JF, Wilkins TA, Wright RJ, Elsayed EZ, Hafez EE, Zhu J (2010) Sequencing and utilization of the Gossypium genomes. Tropical Plant Biology 3: 71-74, doi: 10.1007/s12042-010-9051-4. This article was featured on the cover of the journal.
- Livingston DP III, Tuong TD, Gadi SRV, Haigler CH, Gelman RS, Cullen JM (2010) 3D volumes constructed from pixel-based images by digitally clearing plant and animal tissue. Journal of Microscopy 240: 122-129 doi: 10.1111/j.1365-2818.2010.03393.x. This article was featured on the cover of the journal.
- Betancur L, Singh B, Rapp RA, Wendel JF, Marks MD, Roberts AR, Haigler CH (2010) Phylogenetically distinct cellulose synthase genes support secondary wall thickening in arabidopsis shoot trichomes and cotton fiber. Journal of Integrative Plant Biology 52: 205-220, doi: 10.1111/j.1744-7909.2010.00934.x. Invited article for the special issue, THE PLANT CELL SURFACE. Open Access Article: http://www3.interscience.wiley.com/journal/123278612/issue
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Plant-made natural fiber has always been a foundation of human civilization, being used for fuel, building material, textiles, and manufacture of numerous products. All plant fibers, including cotton fiber used for textiles and fibers within plant biomass, have thick secondary cell walls with cellulose as a major component (~45% and ~95% cellulose in wood and cotton fiber, respectively). The abundant cellulose has major effects on the quality characteristics of fiber products (e.g. strength and absorption), as well as being the main target for saccharification to generate biofuels. This project seeks to unify genomic, genetic, molecular, biochemical, microscopic imaging, and computer modeling technologies to obtain a comprehensive understanding of the controls of cellulose synthesis and fiber differentiation and quality. New knowledge generated will be used to design strategies to improve fiber crops, either through biotechnology, marker assisted breeding, or other related techniques. In this reporting period, major outputs were publications of four peer-reviewed research articles and presentations at four international meetings. One publication revealed completely unexpected controlling mechanisms for cotton fiber development. It was the result of an international research effort led by the Haigler lab, and the article was featured on the cover of one of the top three journals in plant science (Singh et al. 2009 in Plant Physiology). One publication reflected the training of an undergraduate researcher (HD Cheek) and showed that the type of auxin is an important variable affecting cellulose synthesis in cultured cotton fiber (Singh et al. 2009 in Plant Cell Reports). One publication reflected our goal to expand the application of imaging technologies to other important problems in agricultural science (Livingston et al. 2009 in Crop Science), and it added to the understanding of mechanisms of freezing damage and tolerance in winter cereal crops. A fourth publication (Santa-Maria et al. 2009 in Biotechnology and Bioengineering) reflected a contribution to the eventual development of biofuels crops containing activatable, self-processing, enzymes. In terms of meeting presentations, Haigler's oral presentation at the Plant Cell Wall Gordon Research Conference (August 2-7, 2009, Bryant University, Smithfield, RI) expanded the interest of others in cotton fiber research. At the same conference, a graduate student's poster (L. Betancur) was given a best poster award because it showed that the dogma about the role of cellulose synthase proteins in different cell wall types is overly simplistic. This information will guide future genetic engineering of biofuels crops. By convention of the meeting, the two Gordon Research Conference abstracts are unpublished: (1) Haigler C, Betancur L, Glick R, Singh B, Avci U Cell wall dynamics mediating cotton fiber differentiation; (2) Betancur L, Craddock ST, Marks DM, Haigler CH, 2009, Cellulose synthase gene family expression in arabidopsis vegetative trichomes. PARTICIPANTS: Individuals working on the project and receiving support from the agency: Dr. Candace Haigler, PI/PD, designed and analyzed research, collected data. Dr. Richard Glick, Research Assistant, collected data. Partner organizations: Cotton Incorporated Cary NC, Michigan State University, Texas Tech University, University of Georgia, University of Minnesota, University of Copenhagen Denmark, USDA-ARS Raleigh NC. Training and professional development: two undergraduate students (H. Cheek and S. Craddock), three graduate students (M. Santa-Maria, L. Betancur, R. Tuttle). TARGET AUDIENCES: The target audiences of this research is producers of cotton fiber and other fiber crops, as well as international agricultural companies that sell improved seed and fiber products and benefit in their internal research efforts from enhanced knowledge of fundamental aspects of secondary wall cellulose synthesis and fiber development. In addition to guiding this research, which is accomplished through students and advanced researchers, I teach a graduate class that surveys how plants control their life processes as well as fundamentals of plant structure and its role in adaptation. This teaching impacts students in several plant departments at NCSU, and I anticipate that it helps to improve their own research planning and performance as they become deeply knowledgeable of plants. Aspects of my research program are routinely integrated into my teaching. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
A Ph.D. research assistant/lab manager paid by NC State in the Haigler lab contributed greatly to our productivity. With his efforts and those of other researchers paid by Cotton Incorporated (Cary NC) and NC State (graduate student teaching assistant support and undergraduate researcher support), we changed the knowledge related to three of four objectives of the project: (a) Understand how cotton fiber differentiation is regulated, emphasizing cell wall-based control of cotton fiber quality. We showed that cotton fiber does not elongate as individual cells. Instead, bundles of fibers form early in elongation through synthesis of an intervening cell wall between adjacent fibers. These bundles become highly organized in the boll, which likely enhances cotton fiber length and yield through allowing thousands of fibers to reach their genetic potential for elongation within the confined boll space. We also showed that a particular form of auxin needs to be added to cotton ovule cultures to promote secondary wall cellulose synthesis, and this will allow better design of future experiments to manipulate cellulose synthesis in culture. (c) Apply imaging technologies to problems of agricultural importance. We assisted the work of Dr. David Livingston, USDA-ARS, on mechanisms of freezing sensitivity and tolerance in winter cereals. Through development of better protocols for sample preparation and reassembly of sections of the oat crown, we showed a three-dimensional view of the tissue's response to freezing injury. Synthesis of an enclosed barrier zone around damaged tissue apparently allows other peripheral tillers to survive and grow after freezing. (d) Collaborate with others to link basic biological knowledge to practical strategies for fiber improvement. We assisted the work of M. Santa-Maria, a graduate student with Dr. Bryon Sosinski, Department of Horticulture, with her research on thermotolerant bacterial enzymes that can be used to produce genetically engineered, self-processing, biofuels crops. This type of technology will reduce the input costs for production of biofuels from plant biomass.
Publications
- Singh B, Avci U, Eichler Inwood SE, Grimson MJ, Landgraf J, Mohnen D, Sorensen I, Wilkerson CG, Willats WGT, Haigler CH (2009) A specialized outer layer of the primary cell wall joins elongating cotton fibers into tissue-like bundles. Plant Physiology 150: 684-699. doi: 10.1104/pp.109.135459. Open Access Article: http://www.plantphysiol.org/cgi/content/full/150/2/684.
- Singh B, Cheek HD, Haigler CH (2009) A synthetic auxin (NAA) suppresses secondary wall cellulose synthesis and enhances elongation in cultured cotton fiber, Plant Cell Reports 28: 1023-1032. doi: 10.1007/s00299-009-0714-2, http://www.springerlink.com/content/w6g86l7867235818/.
- Livingston DP III, Tuong TD, Haigler CH, Avci U, Tallury SP (2009) Rapid microwave processing of winter cereals for histology allows identification of separate zones of freezing injury in the crown. Crop Science 49: 1-6. doi: 10.2135/cropsci2009.02.0077, http://crop.scijournals.org/cgi/content/abstract/49/5/1837.
- Santa-Maria M, Chou C-J, Yencho C, Haigler C, Thompson WF, Kelly RM, Sosinski B (2009) Plant cell calcium-rich environment enhances thermostability of recombinantly produced α-amylase from the hyperthermophilic bacterium Thermotoga maritima. Biotechnology and Bioengineering 104: 947-956, doi: 10.1002/bit.22468, http://www3.interscience.wiley.com/journal/122499737/abstractCRETRY= 1&SRETRY=0.
- Livingston D, Tuong T, Henson C, Duke S, Tallury S, Murphy JP, Haigler C (2009) A three dimensional view of damage in oat crown tissue recovering from freezing, 2009 Annual Meeting-ASA-CSSA-SSSA, Nov. 1-5, AnMtgsAbsts2009.56364.
- Tuttle JR, Roberts AD, Haigler CH, Robertson N (2009) Development of a VIGS system for cotton, Plant Biology 2009: July 18 - 22, Honolulu, Hawaii, Abstract # P15029, http://abstracts.aspb.org/pb2009/public/P15/P15029.html.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Plant cell walls in the form of fuel, building material, forage, extracted chemicals, and fiber are the renewable materials that form the basis of the emerging carbohydrate economy. Meeting the needs of an increasing human population in an ecologically sustainable manner and maintaining the vitality of U.S. agriculture all require optimization of the production and use of fiber crops, including cotton and trees. The cellulose component of these materials is particularly useful to industry, and cellulose is the most abundant renewable biopolymer. Hence, a research focus on cellulose synthesis is needed. An increased understanding of the genetic and cellular regulation of xylem (wood fiber) and cotton fiber differentiation, particularly cellulose synthesis, will lead to development of novel strategies for improvement of crops through genetic engineering or marker assisted breeding. Ultimately the public will benefit from such research through value-added traditional products, novel manufacturing processes and innovative products, and high efficiency use of agricultural land with minimized impact on natural ecosystems. Specifically, through published (or accepted) articles in 2008, we developed and published on a major new tool, virus induced gene silencing, for analyzing gene function in cotton. This technology, which was previously covered by a patent of N. Robertson, has been transferred to several academic labs and industry by the NCSU Office of Technology Transfer. We analyzed Arabidopsis trichomes to demonstrate that there are better models for the wall thickening process in cotton fibers, specifically the Arabidopsis xylem (results on this latter point are in preparation for publication). This established a valid context for using Arabidopsis to test function of orthologs of some cotton genes. Our research on control of xylem autolysis provided the first data on the role of cysteine proteases in this process and demonstrated the essential nature of high quality transmission electron microscopy in uncovering some mutant phenotypes. The timing of xylem autolysis is one control on the extent of secondary wall deposition (and hence biomass productivity), so fully understanding this mechanism may lead to future improvement of biomass crops. In a book chapter on biomass nanotechnology, we reviewed the current state of knowledge about the control of cellulose nanofibril formation by a biological nanomachine, and the article presents knowledge gaps that need to be addressed in further research. In addition to the four full articles mentioned as published or in press above, five related abstracts were published of talks or posters presented at international scientific meetings (Plant and Animal Genome Conference, Beltwide Cotton Conference, Plant Biology 2008, Cell Wall Biosynthesis 3). Another presentation was given at Cotton Incorporated, Cary, NC in a workshop for cotton breeders, highlighting our new fundamental understanding of cotton fiber development and their implications for fiber improvement. PARTICIPANTS: PI/PD, Dr. Candace H. Haigler, North Carolina State University Ph.D. Student then Postdoctoral Associate, Utku Avci, North Carolina State University Ph.D. Student, Lissete Betancur, North Carolina State University Ph.D. Student, J. Rich Tuttle, North Carolina State University Research Associate, Dr. Bir Singh, North Carolina State University Collaborators: Dr. Niki Robertson, North Carolina State University Dr. Eric Beers, Virginia Tech University Dr. Mark Grimson, Texas Tech University Dr. Deb Mohnen, University of Georgia Dr. Judith Brown and Dr. A.M. Idris, University of Arizona Dr. David Marks, University of Minnesota Dr. Alison Roberts, University of Rhode Island Dr. Curtis Wilkerson and Dr. Jeff Landgraf, Michigan State University This project during this reporting period provided training opportunities to three graduate students and two postdoctoral associate at North Carolina State University. TARGET AUDIENCES: The target audiences of this research is producers of cotton fiber and other fiber crops, as well as international agricultural companies that sell improved seed and fiber products and benefit in their internal research efforts from enhanced knowledge of fundamental aspects of secondary wall deposition. In addition to guiding this research, which is accomplished through students and postdoctoral fellows, I teach a graduate class that surveys how plants control their life processes as well as fundamentals of plant structure and its role in adaptation. This teaching impacts students in several plant departments at NCSU, and I anticipate that it helps to improve their own research planning and performance as they become deeply knowledgeable of plants. Aspects of my research program are routinely integrated into my teaching. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Because stable transformation of cotton consumes substantial personnel time and physical space, we worked on other tools for functional gene testing. One paper was published as a Technical Advance article with cover picture (MD Marks first author, see list) to show that Arabidopsis vegetative trichomes, which are often proposed as analogous to cotton fibers, have a distinct type of secondary wall that mixes traditional primary and secondary wall characteristics. The presence of abundant pectin and lignin in the vegetative trichome walls argues against them being a good model for cotton fiber secondary wall thickening. We have other studies in progress, however, which indicate that Arabidopsis xylem is a good model for the process of cellulose deposition that occurs in cotton fiber secondary walls. A second paper was published as a Breakthrough Technologies article (JR Tuttle first author, see list) that established for the first time an effective virus induced gene silencing system for cotton vegetative tissues. (The effectiveness for cotton fiber is still being tested.) A third paper was published including the main Ph.D. project of Utku Avci, who received his Ph.D. under my supervision in August 2007, and Utku Avci was the first author (see list). This paper provided the first functional/genetic data to demonstrate that cysteine proteases have a role in clearing of xylem elements to become functional water conducting units as part of a final programmed cell death process. The data were acquired by technically demanding analyses of genetic mutants by transmission electron microscopy and protein immunolabeling performed in the developmental context. In addition, the data demonstrated a specialized part of autolysis not predicted before that is carried out by two particular cysteine proteases. I co-authored a book chapter (in press, CH Haigler first author, see list), "Biogenesis of cellulose nanofibrils by a biological nanomachine" to highlight the limited current knowledge about the functional operation of the cellulose-synthesizing complex. Evaluation of this project was achieved through the peer-review process resulting in publications in high quality, high impact, international plant science journals, as well as acceptance of a chapter for a book to be published by a major international scientific publisher.
Publications
- Marks MD, Betancur L, Gilding E, Chen F, Bauer S, Wenger J, Dixon RA, Haigler CH. 2008. A new method for isolating large quantities of Arabidopsis trichomes for transcriptome, cell wall, and other types of analyses. Plant Journal 56: 483-492.
- Avci U, Petzold, HE, Ismail IO, Beers EP, Haigler CH. 2008. Cysteine proteases XCP1 and XCP2 aid micro-autolysis within the intact central vacuole during xylogenesis in Arabidopsis roots. Plant Journal 56: 303-313.
- Tuttle JR, Idris AM, Brown JK, Haigler CH, Robertson D. 2008. Geminivirus-mediated gene silencing from cotton leaf crumple virus is enhanced by low temperature in Gossypium hirsutum, Plant Physiology 148: 41-50.
- Haigler CH, Roberts AW. 2009. Biogenesis of cellulose nanofibrils by a biological nanomachine. Biomass Nanotechnology (Lucian Lucia and Orlando Rojas, eds.) Blackwell Publishing: Oxford UK.
- Avci U, Singh B, Grimson MJ, Eichler Inwood SE, Mohnen D, Sorensen I, Willats WGT, Haigler CH. 2008. Cotton fiber develops via a cell-wall-mediated oscillation between individual and multi-cellular development Biosynthesis of Plant Cell Walls, p. 50 (Abstract book of Cell Wall Biosynthesis 3, Asilomar, CA).
- Betancur L, Avci U, Singh B, Marks MD, Haigler CH. 2008. Cell wall synthesis and composition of Arabidopsis vegetative trichomes Biosynthesis of Plant Cell Walls, p. 94 (Abstract book of Cell Wall Biosynthesis 3, Asilomar, CA).
- Haigler CH, Singh B, Wang G, Zhang D. 2008. Genomics of cotton fiber secondary wall deposition and cellulose biogenesis, Abstract W251, Plant and Animal Genome Conference, Intl. Cotton Genome Initiative Conference, San Diego, CA.
- Haigler CH, Avci U, Grimson MJ, Singh B. 2008. Group behavior of cotton fiber during elongation provides new perspectives on possible mechanisms of control of fiber quality, Beltwide Cotton Conference: Cotton Improvement Conference, Nashville, TN.
- Tuttle JR, Haigler CH, Robertson N. 2008. A silencing vector for cotton and its response to temperature. Poster Presentation Number P15017. Annual Meeting of the American Society of Plant Biologists. Merida, Mexico.
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: Objective 1 or a: To understand the cellular organization of the cellulose-synthesizing complex, including development and use of accessible, accurate, specimen preparation methods for light and electron microscopy. The papers mentioned under Objective 3 demonstrate the utility in plants of microwave-assisted chemical fixation (an accessible, accurate, specimen preparation method for transmission electron microscopy) for long-term work to help meet this goal. However, NCSU was lacking the machine required to observe the cellulose-synthesizing complex directly (a Cryo-fracture, Deep-Etch System) until I led the effort to acquire it via grant funding from the NC Biotechnology Center and internal matching funds. Objective 2 or b: To understand metabolic flux related to cellulose synthesis ... A paper was published (C.H. Haigler et al., see list) that demonstrates a transgenic strategy to increase secondary wall thickening is cotton fibers grown under abiotic stress including
cool nights and low light below the canopy in a growth chamber. In addition, a book chapter was published that summarizes my thinking in this area (C.H. Haigler, see list). Objective 3 or c: To understand the control of secondary wall deposition in xylem. Utku Avci received his Ph.D. in Crop Science (August 2007) under my supervision with the dissertation: "Electron microscopy complements genetic manipulation for understanding xylem development". One related paper was published on line (C. Zhao et al., see list) and two more were submitted with decisions pending: (1) U. Avci et al. "Cysteine proteases XCP1 and XCP2 aid micro-autolysis within the intact central vacuole during tracheary element differentiation in Arabidopsis roots"; (2) S. Thammannagowda et al. "Sense cosuppression of a cellulose synthase gene caused severe cellulose deficiency in secondary xylem and altered growth habit of transgenic aspen". Objective 4 or d: To characterize gene expression and protein function related
to cellulose synthesis in secondary wall stage cotton fiber. We cooperated in an effort led by Jeff Chen to highlight the importance and value of sequencing the cotton genome(s) (Chen ZJ et al.; see list). We summarized our research on the genomics of cotton fiber secondary wall deposition and the relationship of cotton fiber gene expression to the Arabidopsis proteome in a book that will appear as the first one on cotton genomics (C.H. Haigler et al. accepted, see list). I gave an invited meeting presentation that explained details of our work to understand the regulation of the transition between primary and secondary wall deposition in cotton fiber (Singh B et al. in press, see list). I cooperated with others working to develop a system for virus-induced-gene-silencing in cotton fiber (paper in preparation with authors: Tuttle JR, Idris AM, Brown JK, Haigler CH, Robertson D).
PARTICIPANTS: PI/PD, Dr. Candace H. Haigler, North Carolina State University; Ph.D. Student, Utku Avci, North Carolina State University; Ph.D. Student, Lissete Betancur, North Carolina State University; Ph.D. Student, J. Rich Tuttle, North Carolina State University; Research Associate, Dr. Bir Singh, North Carolina State University; Collaborators: Dr. Niki Robertson, North Carolina State University; Dr. Eric Beers, Virginia Tech University; Dr. Scott Holaday and Dr. Mark Grimson, Texas Tech University; Dr. Chandrashekhar Joshi, Michigan Tech University; Dr. Deb Mohnen, University of Georgia; Dr. Judith Brown and Dr. A.M. Idris, University of Arizona; Dr. Jodi Scheffler, USDA/ARS, Stoneville, Missippi; Dr. David Marks, University of Minnesota; Dr. Curtis Wilkerson and Dr. Jeff Landgraf, Michigan State University; Dr. Jeff Chen, University of Texas-Austin
TARGET AUDIENCES: The target audiences of this research is producers of cotton fiber and other fiber crops, as well as international agricultural companies that sell improved seed and fiber products and benefit in their internal research efforts from enhanced knowledge of fundamental aspects of fiber development. In addition to guiding this research, which is accomplished through students and postdoctoral fellows, I teach a graduate class that surveys how plants control their life processes as well as fundamentals of plant structure and its role in adaptation. This teaching impacts students in several plant departments at NCSU, and I anticipate that it helps to improve their own research planning and performance as they become deeply knowledgeable of plants. Aspects of my research program are routinely integrated into my teaching.
Impacts
Plant cell walls in the form of fuel, building material, forage, extracted chemicals, and fiber are the renewable materials that form the basis of the emerging carbohydrate economy. Meeting the needs of an increasing human population in an ecologically sustainable manner and maintaining the vitality of U.S. agriculture all require optimization of the production and use of fiber crops, including cotton and trees. The cellulose component of these materials is particularly useful to industry, and cellulose is the most abundant renewable biopolymer. Hence, a research focus on cellulose synthesis is needed. An increased understanding of the genetic and cellular regulation of xylem (wood fiber) and cotton fiber differentiation, particularly cellulose synthesis, will lead to development of novel strategies for improvement of crops through genetic engineering or marker assisted breeding. Ultimately the public will benefit from such research through value-added traditional
products, novel manufacturing processes and innovative products, and high efficiency use of agricultural land with minimized impact on natural ecosystems. Specifically, through published (or accepted) articles in 2007, we demonstrated the usefulness of microwave-assisted chemical fixation in functional genomics related to plant secondary wall deposition. This method will allow others to gain high-resolution knowledge of protein placement and function with more accuracy and efficiency. We provided key data to show that certain transcription factors can be negative regulators of xylem differentiation, which likely relates to plant adaptation under changing environmental conditions. We fostered cotton genomics through promoting whole genome sequencing, and provided evidence that the genetic control of cotton fiber secondary wall deposition is highly similar to xylem. The latter finding implies that research on cotton fiber has added value, beyond leading to cotton fiber improvement
itself, in terms of revealing mechanisms that control cellulose synthesis in fibers of biomass crops. We provided a synthetic summary of the likely biochemical control of secondary wall cellulose synthesis and demonstrated that the extent of fiber filling under stress could be manipulated in transgenic cotton. We demonstrated the need to unify diverse technical approaches (e.g. genomics, microscopy, and enzymology) to generate novel insights on the control of cotton fiber development.
Publications
- JOURNAL ARTICLES Zhao C, Avci U, Grant EH, Haigler CH, and Beers EP. (2007) XND1, a member of the NAC domain family in Arabidopsis thaliana, negatively regulates lignocellulose production and programmed cell death in xylem. Plant Journal Accepted article online: doi: 10.1111/j.1365-313X.2007.03350.x. The electron micrographs of Utku Avci will be featured on the cover of the printed journal, Volume 53:3, in 2008.
- Chen ZJ, Scheffler BE, Dennis E, Triplett B, Zhang T, Chen X, Stelly DM, Rabinowicz PD, Town C,, Arioli T, Brubaker C, Cantrell R, Lacape J-M, Ulloa M, Chee P, Gingle AR, Haigler CH, Percy R, Saha S, Wilkins T, Wright RJ, Deynze AV, Zhu Y, Yu S, Guo W, Abdurakhmonov I, Katageri I, Rahman M, Zafar Y, Yu JZ, Kohel RJ, Wendel J, and Paterson AH (2007) Towards sequencing cotton (Gossypium) genomes. Plant Physiology 145: 1303-1310.
- Haigler CH, Singh B, Zhang D, Hwang S, Wu C, Cai WX, Hozain M, Kang W, Kiedaisch B, Strauss RE, Hequet EF, Wyatt BG, Jividen GM, Holaday AS (2007) Transgenic cotton over-producing spinach sucrose phosphate synthase showed enhanced leaf sucrose synthesis and improved fiber quality under controlled environmental conditions. Plant Molecular Biology 63: 815-832.
- BOOK CHAPTERS Haigler CH (2007) Substrate supply for cellulose synthesis and its stress sensitivity in the cotton fiber. In: Brown RM Jr, Saxena I (eds) Cellulose: Molecular and Structural Biology, Springer: New York, pp. 145-166.
- Haigler, CH, Singh B, Wang G, and Zhang D. (2008) Genomics of cotton fiber secondary wall deposition and cellulose biogenesis. In: Paterson, A.H. (ed.) Genomics of Cotton, Springer: New York, 33 pp (accepted)
- PAPERS IN PROCEEDINGS Singh B, Avci U, Grimson MJ, Eichler Inwood SE, Landgraf J, Mohnen D, Sorensen I, Wilkerson CG, Willats WGT, Haigler CH. (2008) New controls of cotton fiber development and quality illuminated through integration of genomic, cell biological, and biochemical analyses. Proceedings of the World Cotton Research Conference-4, Lubbock, TX (in press)
- ABSTRACTS Tuttle JR, Brown JK, Haigler CH, Robertson D. (2007) Cotton leaf crumple virus as a vector for virus induced gene silencing in Gossypium hirsutum. 5th International Geminivirus Symposium and 3rd International ssDNA Comparative Virology Workshop. May 20 -26, Ouro Preto, Brazil, http://studium.ppg.br/sites/virologia/index.php.
- Inwood SEE, Shield MP, Singh B, Haigler CH, Mohnen D (2007) Cell wall carbohydrate composition changes during development of cotton fibers. XIth Cell Wall Meeting, Copenhagen, Denmark, August 12-17, (in press in Physiologia Plantarum).
- Tuttle JR, Haigler CH, Idris AM, Brown JK, Robertson D (2007) Using virus-induced gene silencing and gene expression as tools to understand virus infections in the cotton plant, Proceedings of the World Cotton Research Conference-4, Lubbock, TX, September 10-14, (in press).
- Scheffler JA, Shields EM, Singh B, Holaday AS, Gannaway J, Haigler CH (2007) SPS over expression in cotton and its effect on lint yield and fiber quality, Proceedings of the World Cotton Research Conference-4, Lubbock, TX, September 10-14, (in press).
- Betancur L, Avci U, Singh B, Marks MD, Haigler CH (2007) Consideration of xylem sclerenchyma and leaf and stem trichomes as potential models in Arabidopsis for cotton fiber differentiation, Proceedings of the World Cotton Research Conference-4, Lubbock, TX, September 10-14, (in press).
- Grimson M, Avci U, Singh B, Haigler CH (2007) New insights into cotton fiber development obtained by imaging technologies, Proceedings of the World Cotton Research Conference-4, Lubbock, TX, September 10-14, (in press).
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Progress 10/01/05 to 09/30/06
Outputs
Progress was made in this reporting period as follows. Objective 2: To understand metabolic flux related to cellulose synthesis under normal and temperature-stressed conditions. A complex, multi-faceted manuscript was prepared and accepted for publication in Plant Molecular Biology in 2007: "Transgenic cotton over-producing spinach sucrose phosphate synthase showed enhanced leaf sucrose synthesis and improved fiber quality under controlled environmental conditions", Haigler CH, Singh B, Zhang D, Hwang S, Wu C, Cai WX, Hozain M, Kang W, Kiedaisch B, Strauss RE, Hequet EF, Wyatt BG, Jividen GM, and Holaday AS. This paper demonstrates a transgenic strategy to increase secondary wall thickening is cotton fibers grown under abiotic stress including cool nights and low light below the canopy in a growth chamber. Objective 3: To understand the control of secondary wall deposition in xylem. Ph.D. student, Utku Avci, continued to investigate the localization of cysteine
proteinases in differentiating xylem cells of Arabidopsis. In 2007, he used electron microscopy to find a delayed autolysis phenotype in mutants with knock-outs in the cysteine proteinase genes. This definitively established the cellular role of the protein products of these genes. We began work on the related publication. We also collaborated with Eric Beers at VPI and Shekhar Joshi at MTU to characterize plants (Arabidopsis and poplar) with altered aspects of secondary wall deposition. One paper was submitted (E. Beers, lead author), and is currently under revision for resubmission. The second set of results should be submitted in 2007 (S. Joshi, lead author). Both sets of results will be included in the Ph.D. dissertation of Utku Avci in 2007. Objective 4: To characterize gene expression and protein function related to cellulose synthesis, especially at the secondary wall stage of cotton fiber development when almost pure cellulose is being synthesized. We did research with
molecular biology, electron microscopy, and cotton tissue culture to follow up the invention disclosure on novel mechanisms regulating cotton fiber development that was filed with NCSU in 2005. The results continue to be very exciting, and we expect to submit a publication in 2007. A paper that was "in press" last year was published: Udall JA, Swanson JM, Haller K, Rapp RA, Sparks ME, Hatfield J, Yu Y, Wu Y, Dowd C, Arpat AB, Sickler BA, Wilkins TA, Guo JY, Chen XY, Scheffler J, Talierco E, Turley R, McFadden H, Payton P, Allen R, Zhang D, Haigler C, Wilkerson C, Suo J, Schulze SR, Pierce ML, Essenberg M, Kim H, Llewellyn DJ, Dennis ES, Kudrna D, Wing R, Paterson AH, Soderlund C, Wendel JF. 2006. A global assembly of cotton ESTs. Genome Research 16: 441-50 A related short review, "in press" last year, was published: Haigler, C.H. 2006. Establishing the cellular and biophysical context of cellulose synthesis. In: T. Hayashi, ed. The Science and Lore of the Plant Cell Wall:
Biosynthesis, Structure and Function, BrownWalker Press: Boca Raton, FL, pp. 97 - 105. Related posters were presented at two meetings, The Plant Cell Wall Gordon Conference and Plant Biology 2006; the latter abstract was published.
Impacts
Plant cell walls in the form of fuel, building material, forage, extracted chemicals, and fiber are the renewable materials that form the basis of the emerging carbohydrate economy. Meeting the needs of an increasing human population in an ecologically sustainable manner and maintaining the vitality of U.S. agriculture all require optimization of the production and use of fiber crops, including cotton and trees. The cellulose component of these materials is particularly useful to industry, and cellulose is the most abundant renewable biopolymer. Hence, a research focus on cellulose synthesis is needed. An increased understanding of the genetic and cellular regulation of xylem (wood fiber) and cotton fiber differentiation, particularly cellulose synthesis, will lead to development of novel strategies for improvement of crops through genetic engineering or marker assisted breeding. Ultimately the public will benefit from such research through value-added traditional
products, novel manufacturing processes and innovative products, and high efficiency use of agricultural land with minimized impact on natural ecosystems.
Publications
- Singh B, Landgraf J, Wilkerson C, Haigler C. 2006. Genes and pathways associated with secondary wall deposition in cotton fibers. Abstract Book: Plant Biology 2006, p. 190, http://abstracts.aspb.org/pb2006/public/P18/P18018.html
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Progress 10/01/04 to 09/30/05
Outputs
Progress during this reporting period is summarized according to the original objectives. Objective 1: To understand the cellular organization of the cellulose-synthesizing complex, including improvement of sample processing methods for microscopy. I continued to interact with Dr. Mark Grimson at Texas Tech University to obtain high-resolution details of the cellulose synthesizing complex with freeze fracture electron microscopy. The appropriate data set and analyses for a publication are still being accumulated. Objective 2: To understand metabolic flux related to cellulose synthesis under normal and temperature-stressed conditions. I communicated with Dr. Scott Holaday and Dr. Bir Singh at Texas Tech University regarding our ongoing research on transgenic cotton plants that over-express sucrose phosphate synthase (SPS). A poster by Hamill et al. on field tests of transgenic cotton was presented at the 2005 Beltwide Cotton Conference. A paper by Singh et al. was
published in Functional Plant Biology. The main conclusions published so far were: (1) SPS transgenic cotton shows potential for positive effects on cotton fiber quality in the field, but testing of additional lines is needed; and (b) these effects were not derived from changes in photosynthetic parameters as determined in greenhouse experiments. Objective 3: To understand the control of secondary wall deposition in xylem. Ph.D. student, Utku Avci, continued to investigate the localization of cysteine proteinases in differentiating xylem cells of Arabidopsis. He revised his first conclusion; we now see evidence that these are retained in the cytoplasm rather than being loaded into the vacuole. The transmission electron microscope images are consistent with their role in autolysis, but their cellular trafficking is different than hypothesized. He presented a poster on these findings at the 16th International Conference on Arabidopsis Research. Objective 4: To characterize gene
expression and protein function related to cellulose synthesis, especially at the secondary wall stage of cotton fiber development. A unique set of expressed sequence tags (ESTs) from cotton fiber was described in Physiologia Plantarum by Haigler et al., and they were incorporated into the first comprehensive assembly of cotton ESTs, as described Genome Research by Udall et al. Ongoing work to derive biological significance from this sequence set using bioinformatics and microarrays (collaborative with C. Wilkerson and J. Langraf at Michigan State University) was discussed by Haigler et al. in an invited presentation at the meeting, Biosynthesis of Plant Cell Walls. It is clear that this sequence set has potential to reveal novel clues about the control of cellulose synthesis and cotton fiber maturity. Haigler also wrote a related invited review for the book, Frontiers in Plant Cell Walls. 2005 Invention disclosures: NCSU, Novel mechanisms regulating cotton fiber development NCSU
#06-052, Mechanism for stimulating plant growth in general or secondary wall deposition in particular
Impacts
Plant cell walls in the form of fuel, building material, forage, extracted chemicals, and fiber are the renewable materials that form the basis of the emerging carbohydrate economy. Meeting the needs of an increasing human population in an ecologically sustainable manner and maintaining the vitality of U.S. agriculture all require optimization of the production and use of fiber crops, including cotton and trees. The cellulose component of these materials is particularly useful to industry, and cellulose is the most abundant renewable biopolymer. Hence, a research focus on cellulose synthesis is needed. An increased understanding of the genetic and cellular regulation of xylem (wood fiber) and cotton fiber differentiation, particularly cellulose synthesis, will lead to development of novel strategies for improvement of crops through genetic engineering or marker assisted breeding. Ultimately the public will benefit from such research through value-added traditional
products, novel manufacturing processes and innovative products, and high efficiency use of agricultural land with minimized impact on natural ecosystems.
Publications
- Haigler, C.H., Zhang, D., Wilkerson, C.G. 2005. Biotechnological improvement of cotton fiber maturity. Physiologia Plantarum 124: 285-294
- Singh B, Haley L, Nightengale J, Kang WH, Haigler CH, Holaday AS. 2005. Long-term night chilling of cotton, Gossypium hirsutum, does not result in reduced CO2 assimilation. Functional Plant Biology 32: 655-666
- Udall JA, 33 others including Zhang D, Haigler C, Wilkerson C, Wendel JF. 2006. A global assembly of cotton ESTs. Genome Research, In press.
- Haigler, C.H. 2006. Establishing the cellular and biophysical context of cellulose synthesis. In: T. Hayashi, ed. Frontiers in Plant Cell Walls, In Press.
- Avci U, Beers E, Haigler C. 2005. Subcellular localization of xylem cysteine proteinase 2 (XCP2) within differentiating tracheary elements of Arabidopsis thaliana determined by use of microwave-assisted sample processing for electron microscopy, Abstract Book: 16th International Conference on Arabidopsis Research, Abtract #78, http://www.union.wisc.edu/meetings/arabidopsis/posters.html
- Haigler CH. 2005. Secondary wall stage cotton fiber provides an optimum platform for analysis of gene expression related to cellulose synthesis. Abstract Book: Biosynthesis of Plant Cell Walls, Pacific Grove, CA, August 4-7, p. 5.
- Hamill M, Haigler CH, Deshui Z, Singh B, Holaday AS and Hwang S. 2005. Evaluation of sucrose phosphate synthase transgenic cotton lines under field conditions in West Texas. Beltwide Cotton Conference, New Orleans, LA, USA, Jan 4-7, Paper # 2763
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Progress 10/01/03 to 09/30/04
Outputs
Progress during this reporting period is summarized according to the original objectives. Objective 1: To understand the cellular organization of the cellulose-synthesizing complex, including development and use of accessible specimen preparation methods for light and electron microscopy that yield accurate results. Utku Avci perfected a microwave-based protocol for immunological and ultrastructural analysis of whole Arabidopsis tissues. These methods are fast and deliver superior results and analysis potential compared to conventional methods. A poster by Avci et al. was presented at the annual meeting of the American Society of Plant Biologists. I continued to interact with Dr. Mark Grimson at Texas Tech University to obtain high-resolution details of the cellulose synthesizing complex with freeze fracture electron microscopy. The appropriate data set and analyses for a publication are still being accumulated. Objective 2: To understand metabolic flux related to
cellulose synthesis under normal and temperature-stressed conditions. I continued to communicate with Dr. Scott Holaday and Dr. Bir Singh at Texas Tech University regarding our ongoing research on transgenic cotton plants that over-express sucrose phosphate synthase. A poster by Singh et al. was presented at the annual meeting of the American Society of Plant Biologists. The publication listed below by Martin et al. also relates to this objective. Objective 3: To understand the control of secondary wall deposition in xylem. Utku Avci was able to meet a main goal of the grant that is funding his Ph.D. work, which was to use electron microscopic immunocytochemistry to localize a cysteine proteinase in differentiating xylem cells of Arabidopsis. He obtained evidence that the proteinase is made in the endoplasmic reticulum and transported to the vacuole where it is stored prior to release during the autolytic event that creates a functional water conducting element. Although dogma states
that such a process happens, these may be the first micrographs that document it. The publication listed below by Kiedaisch et al. also relates to this objective. Objective 4: To characterize gene expression and protein function related to cellulose synthesis, especially at the secondary wall stage of cotton fiber development when almost pure cellulose is being synthesized. In collaboration with Curt Wilkerson at Michigan State University, we collected and released a unique set of expressed sequence tags (ESTs) from secondary wall stage cotton fiber. We released 9, 121 high quality ESTs to GenBank; 15% of these represent previously unknown genes and 28.4% of these represent new genes for the cotton fiber gene expression set. A poster by Zhang et al. was presented at the annual meeting of the American Society of Plant Biologists. I also gave an invited presentation related to this project at the Annual Meeting of the Southern Section of the American Society of Plant Biologists,
Biotechnological Improvement of Cotton Fiber Maturity, and this presentation was converted to an article and accepted for publication. The publication listed below by Zhang et al. also relates to this objective.
Impacts
Plant cell walls in the form of fuel, building material, forage, extracted chemicals, and fiber are the renewable materials that form the basis of the emerging carbohydrate economy. Meeting the needs of an increasing human population in an ecologically sustainable manner and maintaining the vitality of U.S. agriculture all require optimization of the production and use of fiber crops, including cotton and trees. The cellulose component of these materials is particularly useful to industry, and cellulose is the most abundant renewable biopolymer. Hence, a research focus on cellulose synthesis is needed. An increased understanding of the genetic and cellular regulation of xylem (wood fiber) and cotton fiber differentiation, particularly cellulose synthesis, will lead to development of novel strategies for improvement of crops through genetic engineering or marker assisted breeding. Ultimately the public will benefit from such research through value-added traditional
products, novel manufacturing processes and innovative products, and high efficiency use of agricultural land with minimized impact on natural ecosystems.
Publications
- Avci U, Grimson MJ, Haigler CH. 2004. Development of microwave-assisted specimen preparation methods for electron microscopic immunolocalization in whole Arabidopsis tissues. Proc Plant Biology 2004, Abs # 914, http://abstracts.aspb.org/pb2004/public/P72/7642.html
- Kiedaisch BM, Blanton RL, Haigler CH. 2003. Characterization of a novel cellulose synthesis inhibitor. Planta 217: 922-930
- Martin, L.K. and C.H. Haigler. 2004. Cool temperature hinders flux from glucose to sucrose during cellulose synthesis in secondary wall stage cotton fibers. Cellulose 11: 339-349
- Singh B, Haley L, Nightengale J, Haigler, CH and Holaday AS. 2004. The effect of night chilling on photosynthesis for cotton (Gossypium hirsutum). Proc Plant Biology 2004, Abs # 138, http://216.133.76.127/pb2004/public/P34/7717.html
- Zhang D, Hrmova M, Wan C-H, Wu C, Balzen J, Cai W, Wang J, Densmore LD, Fincher GB, Zhang H, Haigler CH. 2004. Members of a new group of chitinase-like genes are expressed preferentially in cotton cells with secondary walls. Plant Molecular Biology 54: 353-372
- Zhang D, Wilkerson C, Haigler CH. 2004. Large scale identification of genes that are preferentially expressed during cotton fiber secondary wall deposition. Proc Plant Biology 2004, Abs # 618, http://abstracts.aspb.org/pb2004/public/P57/7661.html
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