Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: In the flowering plant Arabidopsis thaliana the coordination of patterning and proliferation is necessary within the carpel margin meristem (CMM) to generate ovules that when fertilized will become seeds. The CMM is a vital meristem with regard to reproductive competence of the plant. Additionally, this meristematic structure, when contrasted with other plant meristems, displays a unique pattern of organ initiation and unique molecular regulatory mechanisms. Thus the study of the development of the CMM is of interest to those studying patterns of meristematic development as well as more broadly to those studying the relationships between proliferative and patterning cues. Additionally, I endeavor to integrate my research with my teaching and training responsibilities. I presently mentors two graduate students, and three undergraduates and has previously mentored three postdocs (2 of 3 now assistant profs.), seven additional undergraduates, and a high school teacher. Two undergraduates were from underrepresented ethnic groups. We present data at international meetings and provide scientific materials upon request. Additionally, we lead a faculty/student group that presents hands-on demos in local middle schools. (http://www4.ncsu.edu/~rgfranks/k12outreach.html) PARTICIPANTS: Robert G. Franks - PI Conceived, organized and managed the experimental procedures. Wrote the manuscripts for publication. Disseminated information at scientific meetings. April Wynn - Graduate Student - Carried out experiments, helped analyze data and write publications. received training in aspects of scientific investigation. TARGET AUDIENCES: Other members of the general scientific community. We disseminate our findings through scientific journals and by presenting data at international meetings and at other universities. K-12 students in Wake county elementary schools. We visit schools to lead hands-on science demonstrations. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We have identified a significant number of genes that are preferentially expressed within the medial domain within the context of the gynoecium. We will use this set of genes to identify the DNA control elements that support gynoecial medial domain expression. The identification of these regulatory elements will enable us to better reconstruct the transcriptional hierarchy that supports CMM development and may help us to identify the specific set of transcriptional regulators that drive expression within the CMM. We are using a combination of bioinformatics and wet lab approaches to identify important DNA regulatory elements that support CMM expression of our candidate genes. As important DNA regulatory elements are expected to be conserved across closely-related species, we have used the AVID and CHAOS/DIALIGN algorithms to identify regions of sequence similarity in orthologous gene sequences across five closely-related, fully-sequenced Brassicacea species: A. thaliana, A. lyrata, C. rubella, B. rapa and T. halophila. We have begun to generate promoter fusion constructs, cognizant of position artifacts to determine if these sequences support medial domain expression. In the case of REM15 we have already identified a 1.1Kb fragment required for expression within the stamens and medial portions of the carpel (data not shown). We will identify statistically-overrepresented sequence motifs from our transcriptomics, ChIP and reporter gene data. If there is compelling support for the importance of a given sequence motif/DNA binding site, we will use site-directed mutagenesis to disrupt this site to test the functional significance. Functional motifs will also be multimerized to test if they are sufficient to drive CMM expression in planta. The elucidation of these elements is an important step toward understanding the molecular mechanisms of CMM specification.
Publications
- Larsson, E., Franks, R. and Sundberg. E. (2013) Auxin and the Arabidopsis thaliana gynoecium. Journal of Expermental Biology Special Review Issue (submitted for review)
- Zhang, J., Franks, R.G, Liu, X., Kang, M., Keebler, J.E.M., Schaff, J., Huang, H. and Xiang, Q.-Y.J.. (2012) Comparative 454 transcriptome sequencing in dogwoods (Cornus L.), BMC Genomics - submitted for review
- Liu, X., Xie, D.Y., Feng, C.-M., Franks, R.G., Qu, R., Xiang, Q.-Y.J. (2012) Plant regeneration and genetic transformation of C. canadensis:a non-model plant appropriate for investigation of flower and fruit development in Cornus (Cornaceae) Plant Cell Reports, in press.
- Feng, C.-M., Liu, X., Yu, Y., Xie, D.Y., Franks, R.G., Xiang, Q.-Y.J., (2012). Evolution of Bract Development and B-class MADS Box Gene Expression in Petaloid Bracts of Cornus s. l. (Cornaceae) New Phytologist vol. 196: pp. 631 643. DOI: 10.1111/j.1469-8137.2012.04255.x.
- Scanning Electron Microscopy Analyses of Flower Development in Methods in Molecular Biology - Flower Development. Edited by Frank Wellmer and Jose Luis Riechmann, December, 2012
- Wynn, A.N., Rueschoff, E.E., Franks, R.G. (2011) Transcriptomic characterization of a synergistic genetic interaction during carpel margin meristem development in Arabidopsis thaliana. PloS One 6 (10), e26231
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: The coordination of spatial patterning cues and cellular proliferation underlies diverse biological processes from cancerous growth to reproductive development. A long-term objective of my research program is to understand how proliferative cues are coordinated with spatial information during organogenesis. In the flowering plant Arabidopsis thaliana this coordination of patterning and proliferation is necessary within the carpel margin meristem (CMM) to generate ovules that when fertilized will become seeds. The CMM is a vital meristem with regard to reproductive competence of the plant. Additionally, this meristematic structure, when contrasted with other plant meristems, displays a unique pattern of organ initiation and unique molecular regulatory mechanisms. Thus the study of the development of the CMM is of interest to those studying patterns of meristematic development as well as more broadly to those studying the relationships between proliferative and patterning cues. In order to develop a more complete molecular understanding of CMM development we have initiated a transcriptomic analysis using RNA isolated from staged (floral stage 8 and 9) and hand-dissected gynoecia that had been fixed in ethanol. This allows us to isolate the gynoecia away from the other floral organs and to limit the developmental window of the sample to the period just before and then during ovule primordia initiation; the earliest steps of ovule development. Thus our analyses will contribute to the understanding of the earliest events of ovule development including primordial initiation and the maintenance of meristematic potential in the CMM during gynoecial tube growth. As the seu ant double mutant fails to initiate ovule primordia we reasoned that genes critical for the earliest steps of ovule initiation would display reduced expression in the seu ant double mutant gynoecia, relative to either single mutant or the wild type tissues. We utilized the Arabidopsis ATH1 Array (Affymetrix) to compare transcript levels between four different genotypes (Col-0, seu-3, ant-1, and seu-3 ant-1) with biological quadruplicate sampling. To identify transcripts whose steady state levels were altered in the seu ant double mutant relative to the other genotypes we utilized two statistical approaches. Approach I: PARTICIPANTS: Robert G. Franks - PI Conceived, organized and managed the experimental procedures. Wrote the manuscripts for publication. Disseminated information at scientific meetings. TARGET AUDIENCES: Other members of the general scientific community. We disseminate our findings through scientific journals and by presenting data at international meetings and at other universities. K-12 students in Wake county elementary schools. We visit schools to lead hands-on science demonstrations. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We used a 1-way ANOVA to identify probe sets for which the mean expression level was significantly different in the double mutant relative to the overall expression mean: 210 transcripts displayed reduced accumulation and 128 displayed elevated accumulation in the double mutant. We analyzed these gene sets for GO TERM enrichment using ChipEnrich software (Orlando et al., 2009). The GO terms that were significantly enriched (hypergeometric p value < 0.001) in the gene set with elevated accumulation were response to dehydration stress, response to abscisic acid, and defense response. In the set of genes with reduced accumulation in the seu ant double mutant statistically significant GO categories included transcription factor activity, ad/abaxial polarity specification, flower development, and transmembrane receptor protein kinase activity. We focused our attention on the genes that displayed reduced expression within the seu ant double mutant because: 1) over-represented GO terms suggested a role for this gene set in transcriptional regulation and relevant developmental processes and 2) the reduced accumulation of these transcripts in the seu ant double suggests that they may be preferentially expressed in the CMM in the wild type gynoecium and thus candidates for novel CMM developmental regulators. Surprisingly, seven out of the 31 Approach II candidate genes were members of the B3 superfamily of transcription factors (Swaminathan et al., 2008; Romanel et al., 2009). This enrichment for B3 encoding transcription factors within our sample is statistically highly unlikely to have occurred by chance alone (p = 2.5x10-10 by hypergeometric probability test). The Arabidopsis B3 superfamily consists of 118 genes all of which encode proteins containing one or more B3-type DNA binding domains. The B3 superfamily is comprised of four sub-families: REM (REPRODUCTIVE MERISTEM); LAV(LEAFY COTYLEDON2[LEC2]-ABSCISIC ACID INSENSITIVE3 [ABI3]-VAL); ARF(AUXIN RESPONSE FACTOR); and RAV(RELATED TO ABI3 and VP1). Six of the seven B3 regulators that were identified in our transcriptomics approach are from the REM subfamily (Table 1). Two of these have been previously reported to display CMM-enriched expression (Franco-Zorrilla et al., 2002; Gomez-Mena et al., 2005; Matias-Hernandez et al., 2010). While five of the seven B3 candidate genes are induced by AG activity in the reproductive whorls of the flower (Gomez-Mena et al., 2005). Based upon: 1) the over-representation of B3 genes in our transcriptome profile; the lack of functional data for the REM B3 family members during CMM development; 3) the expression of several family members strongly within the CMM; and 4) the apparent regulation of a number of these B3 genes by AG we have chosen to focus our efforts on testing if these genes function during CMM development . Interestingly ten of the 31 Approach II candidate genes have been previously identified by Gomez-Mena et al. as induced in response to AG (Gomez-Mena et al., 2005) (Table 1). We propose a model that suggests SEU may function in a complex with AG to regulate gene expression in the CMM.
Publications
- Nole-Wilson, S., Rueschhoff, E.E., Bhatti, H. and Franks, R.G., (2010) Synergistic Disruptions in the seuss cyp85A2 Double Mutant Reveal a Role for Brassinolide Synthesis during Gynoecium and Ovule Development. BMC Plant Biology - 2010, 10:198.
- Nole-Wilson, S., Azhakanandam, S., and Franks, R.G., (2010) Polar Auxin Transport together with AINTEGUMENTA and REVOLUTA Coordinate Early Arabidopsis Gynoecium Development. Developmental Biology 346, pp.181-195
- Bao, F., Azhakanandam, S., and Franks, R.G., (2010) SEUSS and SEUSS-LIKE transcriptional adaptors regulate floral and embryonic development in Arabidopsis. Plant Physiology 152, pp.821-836.
- Azhakanandam, S., Nole-Wilson, S., Bao, F. and Franks, R.G., (2008) SEUSS and AINTEGUMENTA Mediate Patterning and Ovule Initiation during Gynoecium Medial Domain Development - Plant Physiology 146, pp. 1165-1181.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: The coordination of spatial patterning cues and cellular proliferation underlies diverse biological processes from cancerous growth to reproductive development. A long-term objective of my research program is to understand how proliferative cues are coordinated with spatial information during organogenesis. In the flowering plant Arabidopsis thaliana this coordination of patterning and proliferation is necessary within the carpel margin meristem (CMM) to generate ovules that when fertilized will become seeds. The CMM is a vital meristem with regard to reproductive competence of the plant. Additionally, this meristematic structure, when contrasted with other plant meristems, displays a unique pattern of organ initiation and unique molecular regulatory mechanisms. Thus the study of the development of the CMM is of interest to those studying patterns of meristematic development as well as more broadly to those studying the relationships between proliferative and patterning cues. Our previously funded proposal focused on the function of three transcription factors, SEUSS (SEU), LEUNIG (LUG) and AINTEGUMENTA (ANT) during CMM development. These three regulators are required for the proper expression of additional transcription factors that pattern the developing gynoecium (i.e. seed pod). Our recently published results indicate that members of the paralogous SEUSS-LIKE (SLK) gene family also function during carpel and ovule development (Bao et al. Plant Physiology, 152:821-836 (2010). Our model, based on our work and the work of others, posits that SEU, LUG, ANT and members of the SLK gene family form multimeric complexes that control key transcriptional events in the CMM required for female fertility. We seek to better define the molecular mechanisms of the SEUSS, SEUSS-LIKE, LEUNIG, and AINTEGUMENTA genes in carpel margin and ovule development. To determine the functions of the SLK genes and investigate the degree of functional redundancy between SEU and SLK genes, we characterized available slk mutant lines in Arabidopsis. Together our data indicates that the SLK genes encode transcriptional adaptors that participate in a diversity of transcriptional regulatory events and share a large degree of functional redundancy with other SLK genes and with SEU. We have also examined the effects of reduced SLK gene function in ant and lug mutant plants. These results indicate that the SLK genes function in a manner similar to that of SEU during carpel margin development. PARTICIPANTS: Robert Franks - PI - has conceived and managed the overall project. Sridevi Azhakanandam (technician) has carried out many of the experiments and was an author on the 2010 Plant Physiology Paper. TARGET AUDIENCES: The target audience is other researchers in the field of Plant Developmental Biology research. We reached this audience through our publications in Plant Physiology and well as our presentations at international meetings for plant biologists. We also maintain a website to disseminate information about our research efforts. http://www4.ncsu.edu/~rgfranks/ PROJECT MODIFICATIONS: Nothing significant to report.
Impacts
The carpel margin meristems give rise to the ovules, the seed precursors. Generating a molecular understanding of the development of the carpel margin meristems and of the molecular mechanisms of ovule development may enable the generation of agricultural varieties with enhanced seed set and thus engender novel strategies to increase agricultural productivity in important crop species. This work has recently been published in Plant Physiology. Plant Physiology 152:821-836 (2010) I briefly summarize the main points here: We have show that mutations in any single SLK gene failed to condition an obvious morphological abnormality. However, by generating double and triple mutant plants we uncovered a degree of redundancy between the SLK genes and between SLK genes and SEU. Our results support a role of the SLK genes in the development of the carpel margin that is similar to that of SEU. Additionally we demonstrate that the SLK genes are also required for optimal auxin responses during development, as well as for aspects of ovule and CMM development. One novel phenotype that has emerged from our genetics experiments suggests a previously unknown role for SEU and SLK2 during the development of the embryonic shoot apical meristem. The seu slk2 double mutant embryos and seedlings show altered development characterized by a loss of the shoot apical meristem and the development of very narrow cotyledons. We are working to better characterize the molecular mechanism that may be responsible for the loss of shoot apical meristem activity in the embryo. Our qRT PCR results indicate that the level of expression of PHB is very reduced in the seu slk2 mutant seedlings relative to either single mutant or the wild type seedlings. Thus it is possible that SEU and SLK2 share an overlapping function in supporting the expression of PHB in the early embryonic shoot apical meristem. Additional analysis will seek to test this model. Our analysis of the effect of slk loss-of-function mutants on ant phenotype indicates that the SLK genes also play an important role in the initiation of ovule primordia from the carpel margin meristem. By decreasing the activity of members of the slk gene family by creating wither homozygous or heterozygous loss-of-function combinations, we show that the ability of the carpel margin meristem to form ovule primordia is very sensitive to the levels of activity within the SLK gene family. These data further support our model suggesting that complexes of SEU, SLK and LUG are required for the meristematic potential of the carpel margin meristem and its ability to generate ovule primordia.
Publications
- Bao F, Azhakanandam S, Franks RG: SEUSS and SEUSS-LIKE Transcriptional Adaptors Regulate Floral and Embryonic Development in Arabidopsis 10.1104/pp.109.146183. Plant Physiol 2010, 152(2):821-836.
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