Progress 01/01/10 to 12/31/14
Outputs
Target Audience: The main target audience for this work is the community of plant biologists conducting public and private research on mechanisms of plant development. The work is of particular interest to plant biotech companies in search of genes with potential applications in agriculture. Changes/Problems: We initially anticipated applying the majority of this funding to a technician's salary, but found it far more effective to devote a substantial amount of the award into equipment and supplies. This allowed us to purchase the real time PCR hardware and reagents for a critical analysis of gene expression patterns in our WOX transgenics by real time PCR analysis. What emerged from this was solid evidence that the WOX gene LAM1 is a parallel function to that performed by PHANTASTICA and ASYMMETRIC LEAVES2; the LAM1 and PHAN pathways are both required for establishment and maintenance of polarity in expanding leaf blades. The no cost extension allowed us to go forward with the analysis of a new set of transgenic plants designed to test the molecular nature of this relationship. What opportunities for training and professional development has the project provided? The project has supported student interns from a number of local colleges including Manchester Community College, Middlesex Community College and the University of New Haven. In addition our project has influenced the direction of research in the labs of two major collaborators: (1)Dr. Million Tadege, Dept. of Plant and Soil Science, Oklahoma State University,(2) Dr. Ross Koning, Dept. of Biology, Eastern CT State University, Willimantic, CT. How have the results been disseminated to communities of interest? The major avenue of dissemination is through publications in scientific journals. In addition, we have made the lam1 bladeless mutant of Nicotiana available to several other research programs as follows: Yuval Eshed, Weizmann Institute, Israel; Kirankumar Mysore, Noble Foundation, Oklahoma; Million Tadege, Oklahoma State Univ., and Ross Koning, Eastern CT State Univ., CT. Our program was the subject of a seminar presentation in the Biology Dept. at Smith College, and was also the subject of this year's work for a student intern from Middlesex Community College. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We identified a WUSCHEL-like homeobox (WOX) gene named LAM1 in Nicotiana, orthologous to STENOFOLIA in Medicago, and MAEWEST in Petunia, and critical to the initiation and lateral growth of the leaf blade. Analysis of a LAM1::GUS reporter gene revealed LAM1 promoter activity in phloem companion cells of young leaf primordia but also in the upper mesophyll and epidermis of the blade. To isolate phloem expression for experimental analysis, we employed the Arabidopsis SUC2 promoter, which is active only in phloem companion cells. The SUC2::LAM1 transgene restored blade formation and mesophyll organization in lam1 mutants, highlighting a pivotal role for vascular expression in the blade pathway, but the leaf margins of complemented mutants (CM) show a localized loss of function. The margin phenotype appeared to be associated with invasion of abaxial (ab) identity into the adaxial (ad) domain, leading to formation of ectopic blades at the aberrant ad/ab boundary. To test this idea, we introduced a GUS-miR166 polarity marker into the SUC2::LAM1 background and confirmed aberrant encroachment of abaxial identity into the adaxial domain at leaf margins. Later in adult leaves, both adaxial and abaxial domains overrun their normal boundaries, creating a dense network of ectopic blades. This failure to maintain adax/abax boundaries indicated that LAM1 is functionally similar to PHAN, a MYB transcription factor critical to leaf polarity. Double mutant analysis confirmed that even partial loss of LAM1 function in SUC2::LAM1 transgenics dramatically enhances phenotypes associated with loss of PHAN in antisense PHAN (AF) transgenics. Because PHAN and LAM1 are coexpressed in the middle mesophyll of the leaf blade, the possibility emerges that they operate together in a multi-protein complex.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Hao Lin, Lifang Niu, Neil A. McHale, Masaru Ohme-Takagi, Kirankumar S. Mysore and Million Tadege. 2013. An Evolutionarily Conserved Repressive Activity of WOX Proteins Mediates Leaf Blade Outgrowth and Development of Floral Organs in Plants. Proc. Natl. Acad. Sciences 110 (1): 366-371.
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Progress 01/01/13 to 12/31/13
Outputs
Target Audience: The main target audience for this work is the community of plant biologists conducting public and private research on mechanisms of plant development. The work is of particular interest to plant biotech companies in search of genes with potential applications in agriculture. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project has supported student interns from a number of local colleges including Manchester Community College, Middlesex Community College and the University of New Haven. In addition our project has influenced the direction of research in the labs of two major collaborators: (1)Dr. Million Tadege, Dept. of Plant and Soil Science, Oklahoma State University,(2) Dr. Ross Koning, Dept. of Biology, Eastern CT State University, Willimantic, CT. How have the results been disseminated to communities of interest? The major avenue of dissemination is through publications in scientific journals. In addition, we have made the lam1 bladeless mutant of Nicotiana available to several other research programs as follows: Yuval Eshed, Weizmann Institute, Israel; Kirankumar Mysore, Noble Foundation, Oklahoma; Million Tadege, Oklahoma State Univ., and Ross Koning, Eastern CT State Univ., CT. Our program was the subject of a seminar presentation in the Biology Dept. at Smith College, and was also the subject of this year's work for a student intern from Middlesex Community College. What do you plan to do during the next reporting period to accomplish the goals? In the next reporting period we will complete the analysis of polarity disruptions in the LAM1 transgenics we have in hand but also generate a new set of transgenics designed to test the nature of the molecular relationship between LAM1 and PHAN. Specifically we want to determine whether the blade initiation and blade expansion functions of LAM1 can be replaced by expression of PHAN from the LAM1 promoter. This addresses the issue of whether all functions of LAM1 are parallel to the ARP-like function of PHAN. Finally with the recent release of a draft genome for N. benthamiana we are moving into an Illumina HiSeq analysis of global changes in gene expression associated with loss of LAM1 function. In particular this is focused on downstream genes in the auxin and cytokinin pathways.
Impacts
What was accomplished under these goals?
We have identified a WUSCHEL-like homeobox (WOX) gene named LAM1 that is critical to the initiation and lateral growth of the leaf blade. Analysis of a LAM1::GUS reporter gene revealed that LAM1 is highly expressed in phloem companion cells of young leaf primordia but also in the upper mesophyll and epidermis of the blade. To mimic phloem expression and isolate this aspect of LAM1 function for analysis, we took advantage of the Arabidopsis SUC2 promoter which is active only in phloem companion cells. The SUC2::LAM1 transgene restores blade formation and mesophyll organization in lam1 mutants, highlighting a pivotal role for vascular expression in the blade pathway, but the leaf margins of complemented mutants (CM) show a localized loss of function. This allows invasion of abaxial (ab) identity into the adaxial (ad) domain and formation of ectopic blades at the aberrant ad/ab boundary, suggesting that LAM1 performs a function that is parallel to that of the MYB transcription factor PHAN. We have employed a double mutant analysis to show that even partial loss of LAM1 function in SUC2::LAM1 transgenics dramatically enhances phenotypes associated with loss of PHAN in antisense PHAN (AF) transgenics. Likewise, we show that loss of LAM1 and loss of PHAN each produces disorganized growth and loss of palisade identity in the upper mesophyll, and according to our polarity analysis with a 35::GUS-miR166 reporter gene, this is not associated with a loss of adaxial identity. We also show that this upper mesophyll phenotype is in both cases associated with overexpression of ASYMMETRIC LEAVES2 (AS2), a lateral organ boundaries (LOB) associated with adaxial leaf identity. Our current hypothesis is that the LAM1 and PHAN proteins each function in physical association with AS2.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Hao Lin, Lifang Niu, Neil A. McHale, Masaru Ohme-Takagi, Kirankumar S. Mysore and Million Tadege. 2013. An Evolutionarily Conserved Repressive Activity of WOX Proteins Mediates Leaf Blade Outgrowth and Development of Floral Organs in Plants. Proc. Natl. Acad. Sciences 110 (1): 366-371.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Assimilation of atmospheric CO2 into carbohydrate by higher plants occurs in the leaf blade, a thin laminar structure with specialized cell layers for light interception and gas exchange. Understanding the genes governing blade initiation and growth will provide valuable tools for eventual engineering of crop plants with higher primary productivity and water use efficiency. We selected Nicotiana sylvestris as a diploid model system for genetic analysis of the blade pathway, which led to the identification of a WUSCHEL-like homeobox (WOX) gene orthologous to Arabidopsis WOX1 which we named LAM1. In mutants lacking LAM1 function, the leaf blade is reduced to a vestigial strip of blade-like tissue that fails to grow outward into an organized lamina. Analysis of a LAM1::GUS reporter gene revealed that LAM1 is highly expressed in phloem companion cells of young leaf primordia but also in the upper mesophyll and epidermis of the blade. To mimic phloem expression experimentally and isolate this aspect of LAM1 function for analysis, we took advantage of the Arabidopsis SUC2 promoter which is active only in phloem companion cells. The SUC2::LAM1 transgene restores blade formation and mesophyll organization in lam1 mutants, highlighting a pivotal role for vascular expression in the blade pathway, but the leaf margins of complemented mutants (CM) show a localized loss of function. This allows invasion of abaxial (ab) identity into the adaxial (ad) domain and formation of ectopic blades at the aberrant ad/ab boundary. Histological analysis of the shoot apex shows an early onset for abaxialization in margins and midveins of young leaf primordia, confirming a role for LAM1 in maintenance of the adaxial domain. Progressive loss of LAM1 function in SUC2::LAM1 transgenics produces an eventual return to the bladeless phenotype in adult leaves, and full loss of LAM1 function in the flowers. Gradual loss of LAM1 during this transition produces leaf blades lacking palisade differentiation, and a highly disorganized pattern of cell division in the upper blade mesophyll, a phenotype identical to that resulting from loss of PHAN in our antisense (AF) transgenics. Though LAM1 operates independently during blade initiation, LAM1 and PHAN appear to function together in the blade to promote palisade development. Expression patterns suggest that both functions are delivered non-autonomously to the upper mesophyll by vascular expression. The genes also operate together in midveins, where abaxialization from partial loss of LAM1 is enhanced severely by coincident loss of PHAN in double mutants. PARTICIPANTS: Dr. Million Tadege, Dept. of Plant and Soil Science, Oklahoma State University Dr. Ross Koning, Dept. of Biology, Eastern CT State University, Willimantic, CT TARGET AUDIENCES: The main target audience for this work is the community of plant biologists conducting public and private research on mechanisms of plant development. The work is of particular interest to plant biotech companies in search of genes with potential applications in agriculture. PROJECT MODIFICATIONS: The project was awarded a one year extension to December 31, 2013 so that the balance of funds could be applied to completion of work.
Impacts
Our project has defined the direction of research in the labs of two major collaborators: (1)Dr. Million Tadege, Dept. of Plant and Soil Science, Oklahoma State University,(2) Dr. Ross Koning, Dept. of Biology, Eastern CT State University, Willimantic, CT
Publications
- Hao Lin, Lifang Niu, Neil A. McHale, Masaru Ohme-Takagi, Kirankumar S. Mysore and Million Tadege. 2012. An Evolutionarily Conserved Repressive Activity of WOX Proteins Mediates Leaf Blade Outgrowth and Development of Floral Organs in Plants. Proc. Natl. Acad. Sciences, in press.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Assimilation of atmospheric CO2 into carbohydrate by higher plants occurs in the leaf blade, a thin laminar structure with specialized cell layers for light interception and gas exchange. Understanding the genes governing blade initiation and growth will provide valuable tools for eventual engineering of crop plants with higher primary productivity and water use efficiency. We selected Nicotiana sylvestris as a diploid model system for genetic analysis of the blade pathway, which led to the identification of a gene named LAM1. In mutants lacking LAM1 function, the leaf blade is reduced to a vestigial strip of blade-like tissue that fails to grow outward into an organized lamina. Parallel mutagenesis work in Medicago truncatula (Noble Foundation, Ardmore, OK) revealed that LAM1 is orthologous to STENOFOLIA (STF), a WUSCHEL-like homeobox (WOX) gene with close homology to WOX1 in Arabidopsis and MAEWEST (MAW) in petunia. To confirm the function of Nicotiana LAM1 we constructed LAM1pro::LAM1 cDNA transgenes for insertion into bladeless lam1 mutants where they fully restored blade formation. Transgenic leaves grow to a normal length, but typically do not reach their full width, and have wavy/dissected margins with a disorganized mesophyll. Because this phenotype occurs only in complemented mutants, it appears that a margin specific aspect of LAM1 function is not provided by the transgene. Analysis of LAM1::GUS reporter transgenics revealed a high level of LAM1 expression in the leaf midvein and all lateral veins of the lamina, which is concentrated primarily in the companion cells of the phloem. But the promoter also shows some activity in the upper mesophyll of the lamina and the upper epidermis. To analyze the function of vascular expression separately, we constructed a transgene where LAM1 cDNA was driven by the Arabidopsis SUC2 promoter, which is active specifically in phloem companion cells of the leaf. The SUC2::LAM1 transgene rescues blade formation in bladeless lam1 mutants and produces leaves with dissected margins, a leaf phenotype very similar to that in the LAM1::LAM1 transgenics. This points to a primary role for vascular tissue in blade morphogenesis, and specifically reveals that vascular expression of LAM1 is critical to this process. Potential insights on the underlying mechanism have come from work on functional parallels between LAM1 and WUSCHEL, the chief organizer of shoot apical meristems operating primarily through regulation of cytokinin signaling. Complementation of the lam1 mutation with a LAM1::AtWUS transgene shows that WUS can fully substitute for LAM1 in the blade pathway. This is consistent with a WUS box in the C terminus of LAM1, which is essential to WUS function and thought to mediate interactions with regulatory partners. The LAM1 3'UTR contains tandem AGAMOUS binding sites nearly identical to those regulating transcriptional repression in the AtWUS 3'UTR. Similar DNA binding domains and interactions with common regulatory partners provide the foundation for our current hypothesis that vascular induction of blade formation in Nicotiana is governed by LAM1 regulation of cytokinin signaling. PARTICIPANTS: Dr. Million Tadege, Dept. of Plant and Soil Science, Oklahoma State University Dr. Ross Koning, Dept. of Biology, Eastern CT State University, Willimantic, CT TARGET AUDIENCES: The main target audiences for this work are biotechnology companies and university professors working on plant developmental biology. Our findings are distributed through seminar presentations and publications in molecular biology journals. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our work is focused on genes controlling the initiation and expansion of the leaf blade, a living solar panel where energy from sunlight drives assimilation of atmospheric CO2 into carbohydrate. These genes will be tools for the eventual engineering of crop plants with higher primary productivity and water use efficiency, two critical issues in the effort to meet growing pressures on the global food supply. The work began with a large scale mutagenesis project where we isolated a range of mutants where blade formation was compromised or eliminated. A series of publications on the LAM1 gene generated particular interest in the scientific community leading to requests for the mutant plant from Dr. Yuval Eshed, Weizmann Institute, Israel; Dr. Kirankumar Mysore, Noble Foundation, Oklahoma, and Dr. Million Tadege, Oklahoma State University. Studies on blade formation in alfalfa at the Noble Foundation led to the isolation of a gene named STENOFOLIA (STF), which was shown to be orthologous to our LAM1 gene. Cloning and sequencing of STF and LAM1 revealed that these genes are direct descendants of WUSCHEL (WUS), the chief organizer of shoot apical meristems which operates primarily through regulation of cytokinin signaling. Connecting blade formation to the cytokinin pathway has refocused the direction of work in both labs. We moved immediately to experiments showing that WUS is functionally so similar to LAM1 that it can restore blade formation in lam1 mutant plants. Armed with this information, we landed a USDA-NIFA competitive grant #2009-03972 to pursue the work, and have our first publication in Plant Cell.
Publications
- Tadege, M., Lin, H., Bedair, M., Berbel, A., Wen, J., Rojas, C.M., Niu, L., Tang, Y., Sumner, L., Ratet, P., McHale, N.A., Maduen F., and Mysore, K.S. 2011. STENOFOLIA regulates blade outgrowth and leaf vascular patterning in Medicago truncatula and Nicotiana sylvestris. Plant Cell 23: 2125-2142.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: One major output has been the distribution of the lam1 bladeless mutant of Nicotiana to several other research programs as follows: Yuval Eshed, Weizmann Institute, Israel; Kirankumar Mysore, Noble Foundation, Oklahoma; Million Tadege, Oklahoma State Univ., and Ross Koning, Eastern CT State Univ., CT. Our program was also the subject of a seminar presentation last year in the Experimental Botany group at Yale University. The program is also the subject of this year's work for a student intern from the Univ. of New Haven. PARTICIPANTS: Our main collaborators are Million Tadege, Dept. Plant and Soil Science, Oklahoma State Univ., and Ross Koning, Dept. of Biology, Eastern CT State University. The project is the subject for traning of student interns from CT universities and community colleges. TARGET AUDIENCES: Our main target audience is university professors and industry researchers in the scientific community. Development of mutant strains and transgenic lines will be available to all. Our materials and publications on the role of WOX genes in morphogenesis will influence the course of investigations in many labs. PROJECT MODIFICATIONS: Our approach to the project has relied heavily on production of transgenic Nicotiana lines designed to reveal the location of gene expression and to determine how the endogenous gene functions. This has proven very effective so there is no major change of approach. As information of gene function emerges from this approach, we have moved into the design of several additional vectors, specifically those aimed at elucidating the vascular function of LAM1 and related WOX genes.
Impacts
Assimilation of atmospheric CO2 into carbohydrate by higher plants occurs in the leaf blade, a thin laminar structure with specialized cell layers for light interception and gas exchange. Understanding the genes governing blade initiation and growth will provide valuable tools for eventual engineering of crop plants with higher primary productivity and water use efficiency. Publications on the role of LAM1 and related WOX genes in morphogenesis will provide a foundation for understanding the molecular mechanisms that regulate formation of all plant organs including leaves and flowers.
Publications
- No publications reported this period
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