Progress 07/20/05 to 07/19/08
Outputs
OUTPUTS: This project focused on molecular mechanisms governing adaxial/abaxial polarity in leaves and flowers of Nicotiana, with particular attention to NsPHAN and NsPHAV. Previously, we demonstrated that elimination of NsPHAN with an antisense transgene (AF) disrupts the maintenance of adaxial identity. In contrast, we had observed an expansion of adaxial identity in a mutant (phv1) abolishing microRNA (miRNA) regulation of NsPHAV. Our hypothesis, based on work in Nicotiana and Arabidopsis, was that the HD-ZIPIII genes establish adaxial identity, and then NsPHAN maintains this state by suppressing the activity of miR165/166 in the adaxial domain. To test this, we constructed double mutants, where the phv1 mutation was introduced into the antisense NsPHAN (AF) transgenic background. If loss of NsPHAN was disrupting adaxial identity through ectopic expression of miR165/166, we reasoned that this would be suppressed by phv1 mutation, which abolishes miR165/166 regulation of NsPHAV, and this was in fact the case. In leaves of phv1(+/-) AF(+/-) double heterozygotes, we observed suppression of the leaf blade and midvein phenotypes normally associated with loss of NsPHAN. To confirm the role of miR165/166, we constructed a 35S::GUSPLUS reporter gene carrying the miR165/166 recognition site (pRH178) and introduced it into wild-type and antisense NsPHAN backgrounds. Wild-type assays confirmed the expectation miR165/166 activity (loss of GUS staining) is normally confined to the abaxial domain. In AF transgenics, pRH178 revealed that loss of NsPHAN does in fact lead to ectopic miR165/166 activity in the adaxial leaf domain, but this occurs well after the onset of abnormal development in the upper mesophyll. One explanation is that the eventual onset of ectopic adaxial miR165/166 could be a consequence of ectopic adaxial ARF expression. If so, the early stages of random proliferation in the upper leaf mesophyll in AF transgenics may actually result from coexpression HD-ZIPIII and ARF transcription factors. In this case, eventual emergence of ectopic leaf blades would occur only after ectopic miR165/166 produces a new on/off boundary for HD-ZIPIII expression. Analysis of the pRH178 transgene in AF leaves at this stage in fact confirmed strict on/off miR165/166 boundary where ectopic leaf blades emerge from the upper mesophyll. We conclude that NsPHAN normally maintains adaxial development through suppression of ARF, a direct consequence being suppression of miR165/166 and preservation of adaxial HD-ZIPIII expression. Our work on leaf development has been disseminated through seminar presentations and entries into GenBank for genes cloned in Nicotiana. We have also made our transgenics and mutant lines available to a number of investigators, including Dr. Jun-Yi Yang at Rockefeller University, and Dr. Kiran Mysore at the Samuel Roberts Noble Foundation for studies on developmental genetics of legumes. PARTICIPANTS: Dr. Ross E. Koning, Biology Dept., Eastern Connecticut State University was a primary collaborator on the project. Our work was the subject of training in the form of special projects to several senior biology majors at ECSU. In addition, students in the biotechnology program at the Manchester Community and Middlesex Community Colleges completed mentor programs at the CT Agric. Expt. Station working on subjects related to the project. TARGET AUDIENCES: Our main target audience is the scientific community, particularly colleagues studying plant developmental biology. Results of our studies are also included in mini-courses taught for the Federated Garden Clubs of Connecticut as part of their certification for master gardener. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Plant breeders can tailor crop varieties to a broad range of growing conditions by selecting for genes that alter basic patterns of growth and development. Upright stature, bush vs. vine type architecture, and semi-dwarf growth habit are all examples of genetic changes that have allowed farmers worldwide to reduce production costs by growing crops on less acreage. Molecular genetic research is rapidly expanding the list of genes available to plant breeders for this purpose. Genes can now be cloned from any plant, domestic or wild, opening the door to an entirely new repository of genetic resources for crop improvement. This expanded toolbox will allow U.S. farmers to produce our food supply on less acreage, reducing overall production costs, and improving water use efficiency. This is a critical new priority, particularly in Western states where the water demands of a growing population collide with agricultural water use.
Publications
- No publications reported this period
|
Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: This project is aimed at understanding the function of genes regulating formation of leaf and flower primordia in higher plants. Specifically, we are investigating the relationship between PHAN (MYB transcription factor) and the HD-ZIPIII transcription factors PHAB, PHAV and REV, all of which are critical to regulation of lateral organ polarity. We demonstrated through analysis of double mutant plants that the adaxializing effect of the phv1 mutation in the flower's carpels is strictly dependent on the presence of PHAN function. Expression studies confirmed that aberrant abaxial accumulation of phv1 mRNA in the absence of PHAN expression has no effect on carpel development. Our most recent discovery is that this PHAN requirement for PHAV function is confined to lateral organs. In stems and in the shoot apical meristem of Nicotiana, the phv1 mutation produces severe phenotypes in the absence of PHAN function. This is the first indication that the HD-ZIPIII pathway operates in
different ways in different parts of the plant. Particularly for apical meristems, this is not entirely unexpected because PHAN is not expressed in that location. PHAN is expressed in vascular tissue of the stem, but even in its absence, phv1 produces a dramatic phenotype. The results indicate that the HD-ZIPIII pathway functions similarly in apical and vascular meristems, in a manner that is independent of PHAN function. This suggests that PHAN may have evolved exclusively as a polarity mechanism in lateral organs, where it promotes adaxial suppression of auxin response factors, and protects HD-ZIPIII mRNA from microRNA mediated destruction. The results of our work are disseminated in scientific publications and through DNA sequence information posted on the searchable Genbank database at NCBI. In addition, we have shared our mutant strains of Nicotiana with many other investigators. One example is a new project headed by Dr. Jun-Yi Yang in the Plant Molecular Biology program at
Rockefeller University, where ongoing work involves our antisense PHAN transgenic strain. Likewise, our bladeless lam1 mutant is being used by Dr. Kiran Mysore at the Samuel Roberts Noble Foundation in his studies on developmental genetics of legumes.
PARTICIPANTS: Dr. Ross E. Koning, Dept. of Biology, Eastern Connecticut State University is a collaborator on this project.
TARGET AUDIENCES: Additions to our knowledge on the functional interactions between MYB and HD-ZIPIII transcription factors is targeted primarily to other scientists working in plant developmental genetics.
Impacts
Plant breeders can tailor crop varieties to a broad range of growing conditions by selecting for genes that alter basic patterns of growth and development. Upright stature, bush vs. vine type architecture, and semi-dwarf growth habit are all examples of genetic changes that have allowed farmers worldwide to reduce production costs by growing crops on less acreage. Molecular genetic research is rapidly expanding the list of genes available to plant breeders for this purpose. Genes can now be cloned from any plant, domestic or wild, opening the door to an entirely new repository of genetic resources for crop improvement. This expanded toolbox will allow U.S. farmers to produce our food supply on less acreage, reducing overall production costs, and improving water use efficiency. This is a critical new priority, particularly in Western states where the water demands of a growing population collide with agricultural water use.
Publications
- No publications reported this period
|
Progress 01/01/06 to 12/31/06
Outputs
Genes controlling patterns of plant development have been used extensively by plant breeders to produce improved varieties of crop plants. Alterations in growth habit and flowering patterns are examples of developmental changes with applications in agriculture. This project is aimed at identifying the genes that control these morphogenic events, using Nicotiana sylvestris and Nicotiana benthamiana as model systems. The HD-ZIPIII transcription factors PHAB, PHAV and REV are key players in the regulation of leaf and flower development, and all 3 are negatively regulated by the same microRNA molecules (miR165/166). MicroRNA targets HD-ZIPIII transcripts for destruction specifically on one side of young leaf and flower primordia, establishing a critical front/back (adaxial/abaxial) differentiation pattern guiding organ growth and development. Our current studies involve a mutation (phv1) at the miR165/166 recognition site in PHAV, which abolishes miRNA regulation, and
promotes aberrant expression of PHAV in the abaxial domain. This extends adaxial identity throughout the carpels of developing fruit, leading to disorganized growth and formation of exposed ovules on both sides of the carpel wall. It also produces female sterility, because the carpels fail to fuse at the apex, precluding formation of a functional style and stigma. Through analysis of antisense PHAN(AF), phv1 double mutant combinations, we have established that the ability of ectopic PHAV to produce this response is dependent on the PHANTASTICA, a transcription factor in the MYB family. In the absence of PHAN, mutant phv1 carpels revert to wild-type, enclosing the ovules and generating a functional style. This could have meant simply that PHAN is required for PHAV expression, but RTPCR data show that PHAV is expressed at its usual level in double mutant carpels lacking PHAN. Thus, aberrant abaxial expression of PHAV by itself has no consequence; only in the presence of PHAN does this
impose adaxial identity on abaxial tissue. This PHAN requirement pointed to abaxial repression of the auxin response factors (ARF1,2,3) as the critical event disrupting phv1 carpels. We have in fact confirmed that ARF3 is severely downregulated in phv1 carpels, but returns to normal in AF,phv1 double mutants. Together, the data suggest that maintenance of adaxial identity is a self-sustaining cycle: HD-ZIPIII expression promotes PHAN dependent repression of auxin response factors, suppressing miR165/166 activity, which in turn sustains HD-ZIPIII expression.
Impacts
Our work on leaf development has an impact on the direction of investigations in many other research laboratories. The most recent example is a new project headed by Dr. Jun-Yi Yang in the Plant Molecular Biology program at Rockefeller University involving one of our transgenic strains of Nicotiana sylvestris.
Publications
- No publications reported this period
|
Progress 01/01/05 to 12/31/05
Outputs
Genes controlling patterns of plant development have been used by plant breeders to produce improved crop varieties. Alterations in overall plant architecture (bush vs. vine type) and flowering time (early maturity) are examples of developmental changes with direct applications in agriculture. This project is aimed at the identification of gene functions that regulate early events in leaf development, using Nicotiana sylvestris and Nicotiana benthamiana as model systems. One aspect of the project involves PHAVOLUTA, an HD-ZIPIII transcription factor which regulates patterns of proliferation for the indeterminate cells of apical and vascular meristems. PHAV expression is negatively regulated by microRNA (miR165/166). In our phv1 mutant, miRNA regulation of PHAV is abolished by a single nucleotide change at the miRNA recognition site. This produces a severe disruption in growth of the vascular cambium of leaf midveins. In this regard, PHAV is functionally related to
PHANTASTICA, a transcription factor in the MYB family. Loss of PHAN in antisense transgenic leaves of Nicotiana sylvestris alters growth of the midvein cambium in a manner suggesting an aberrant truncation of PHAV expression. To determine whether loss of PHAN was curtailing PHAV expression through regulation of miRNA activity, we introduced our phv1 mutation into the antisense PHAN (AF18B) background. We reasoned that if the vascular phenotype associated with AF18B was mediated through the miRNA pathway, the effect would be reduced or abolished by phv1 which is refractory to miRNA regulation. Our data in fact show that the midvein phenotype of AF18B is fully suppressed by the phv1 mutation. To look for direct in situ evidence of an AF18B effect on miR165/166 activity and to conduct a plant wide assay for domains of miRNA activity, we constructed a 35S::GUSPLUS transgene carrying the miR165/166 recognition site located at the exon 4/exon5 boundary in the Nicotiana ortholog of PHAV.
Analysis of 35S::GUS-miRNA in wild-type plants revealed miR165/166 eliminates GUS activity throughout the abaxial domains of leaves and flowers, consistent with the leaf and flower phenotypes in phv1 mutants. In the AF18B background, the 35S::GUS-miRNA transgene provided direct evidence that loss of PHAN expands the domain of miR165/166 activity. In AF18B leaves, GUS elimination expands out of the abaxial leaf domain, first enveloping the midvein, and then extending outward into the leaf blade, where the GUS on/off boundary coincides with formation of ectopic leaf blades. When AF18B leaves reach the 5cm stage, GUS elimination is observed in the midvein itself and particularly at the leaf tips, where GUS is undetectable in the veins and lamina. Current work is aimed at elucidation of the mechanisms confining miR165/166 to the abaxial domain, and the role of ectopic KNOX in potentiating its expansion into other parts of developing leaves.
Impacts
Our work on leaf development has an impact on the direction of investigations in many other research laboratories. The most direct examples would be Dr. Michael Marcotrigiano at Smith College and Dr. Ross Koning at Eastern Connecticut State University. Mutant strains isolated in our lab have become the main subject of investigation in these labs.
Publications
- No publications reported this period
|
|