Progress 09/15/07 to 09/14/11
Outputs
OUTPUTS: Activities included conducting and analyzing experiments, and mentoring three post-docs and one graduate student. PARTICIPANTS: Jason W. Reed (PI) Esther van der Knaap (Co-PI) Miin-Feng Wu (post-doc, UNC) Sara Ploense (post-doc, UNC) Shan Wu (graduate student, OSU) Ning Liu (post-doc, OSU) Karin Ljung, Umea Plant Science Center, Umea Sweden (collaborator) TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Changes in knowledge: Several Auxin Response transcription Factors (ARFs) including ARF6 and ARF8 regulate fruit elongation in Arabidopsis after fertilization. arf6 arf8 double mutants or plants expressing a microRNA (miR167) that targets ARF6 and ARF8, have short fruits even after fertilization. Conversely, partial loss of ARF6 and ARF8 activity (in single mutants) leads to slightly elongated fruit even in the absence of fertilization. Gynoecia of higher-order mutants also deficient in up to three additional ARF genes, NPH4/ARF7, ARF10, and ARF16, had even more elongated fruits in the absence of pollination. These results suggest that five different ARF proteins collectively repress fruit growth before pollination, but promote fruit growth after fertilization. ARF proteins can be switched from gene repressors to gene activators in the presence of auxin, suggesting that fertilization causes an increase in auxin level. Measurements of auxin concentration after pollination confirmed that auxin levels increased in whole gynoecia and in ovules. Analyses of global gene expression in gynoecia before and after pollination revealed a number of genes associated with initiation of fruit growth. Comparisons to similar published data from tomato did not reveal strong overlap of orthologous genes downstream of ARFs in developing fruits of the two species. Direct comparison of fruit growth in unpollinated Arabidopsis ARF-deficient plants and plants heterozygous for mutations affecting the PRC2 polycomb complex revealed that PRC2 deficiency causes a greater degree of parthenocarpic fruit growth than does ARF deficiency. In addition to these studies of Arabidopsis fruit growth, we found that misexpression of repressors of auxin response in developing ovules caused defects in ovule growth and patterning. These defects were suppressed by an arf6 mutation, indicating that ARF6 must be activated for correct ovule development. However, arf6 mutants had normal ovules, so ARF6 must act redundantly with other ARFs in ovules. To explore the functions of the tomato orthologs of ARF6 and ARF8, we overexpressed the Arabidopsis miR167 precursor gene in transgenic tomato plants. We found that transcripts of the four tomato ARF6 and ARF8 genes were decreased in several of these transgenic lines, and that these had defects in vegetative growth and in flower growth. The strongest flower defect was in style elongation, and the transgenic lines were female-sterile. Because of the female sterility, we could not assess whether SlARF6/ARF8 genes are required for fruit growth after fertilization. However, the fruit were not parthenocarpic in the absence of fertilization. Flowers of these plants underexpressed the Style2.1 gene involved in style growth, suggesting a plausible mechanism for part of the phenotype.
Publications
- Wu, S., Xiao, H., Cabrera, A., Meulia, T., and van der Knaap, E. (2011) SUN Regulates Vegetative and Reproductive Organ Shape by Changing Cell Division Patterns. Plant Physiol 157: 1175-1186.
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Progress 09/15/08 to 09/14/09
Outputs
OUTPUTS: Activities included conducting and analyzing experiments, and mentoring one post-doc and one graduate student. PARTICIPANTS: Juan Carlos Serrani Yarce, post-doc at UNC Shan Wu, graduate student at OSU A collaboration was established with Karin Ljung at Umea Plant Science Centre, Umea Sweden. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Arabidopsis fertilization is largely complete within 24 hours of pollination of competent gynoecia. We extracted RNA from duplicate samples of gynoecia at 0, 6, 12, and 24 hours after pollination (with unpollinated controls at the same time points), and analyzed global gene expression using microarrays. Expression of hundreds of genes changed after pollination. We filtered the dataset using available lists of pollen-expressed genes and ovule-expressed genes to identify genes likely to change specifically in gynoecia or ovules after fertilization. In collaboration with Karin Ljung, we are measuring IAA levels in Arabidopsis gynoecia and ovules at 0, 12, and 24 hours after pollination. In the first experiment, auxin levels rose in gynoecia and ovules within 24 hours, and possibly within 12 hours after pollination. We are repeating the experiment in hopes of achieving statistical power for these results. We have surveyed various arf mutant combinations for parthenocarpic fruit development, and the strongest phenotypes are in plants heterozygous for arf6 and homozygous for four other arf mutations. In the absence of pollination, gynoecia of these plants elongate, and they also grow in girth despite lacking developing seeds. This phenotype is much stronger than that of the arf8 single mutant that motivated the project. We have generated duplicate microarray data for three time points of unpollinated fruits of wild-type, arf8 single mutant, and the quadruple mutant gynoecia. We will compare this data to the results from the time course of pollination of wild-type flowers to determine the extent to which parthenocarpy induced by arf mutations resembles normal fruit growth. We are also generating sections of the parthenocarpic fruit to determine the cellular basis of the excess growth. In contrast to these exciting results using arf mutant combinations, we have so far failed to find evidence for involvement of Aux/IAA proteins in similar analyses of Arabidopsis iaa mutants and transgenic plants in which IAA genes are silenced. Multiple ARF proteins may regulate fruit growth in tomato as they do in Arabidopsis. We have generated transgenic tomato plants that overexpress Arabidopsis MIR167a, which targets both ARF6 and ARF8. Of five transgenic plants recovered, two have reduced ARF6 and ARF8 transcripts in flowers compared to control plants. These tomato plants are sterile, suggesting that ARF6 and ARF8 have similar functions in Arabidopsis and tomato. Overexpression of SUN causes elongated fruit shape and changes in vegetative growth reminiscent of auxin overexpressors. For example, the stems and leaf rachises are twisted and the leaf margins are altered. In situ hybridization experiments show that SUN is expressed in developing seed, the placenta, the funiculus and in vascular tissues in tomato fruit collected 7 days post anthesis. This expression pattern overlaps quite closely with those of tomato ARF6 and ARF8. However, different expression patterns of these two auxin signaling genes were not found in the NILs that differ at SUN, suggesting that SUN does not affect fruit shape by altering transcription of ARF6 or ARF8.
Publications
- M.S. thesis, Shan Wu, SUN regulates fruit shape and vegetative growth in tomato, The Ohio State University, 2009.
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Progress 09/15/07 to 09/14/08
Outputs
OUTPUTS: The activities conducted in year one included performing experiments. Results are summarized in the outcomes section. PARTICIPANTS: Miin-Feng Wu, former post-doc in UNC lab, performed some experiments on ARF gene expression in Arabidopsis ovules and fruits. Juan Carlos Serrani Yarce, post-doc in UNC lab, has performed experiments on Arabidopsis fruit formation. Shan Wu, graduate student in U of Ohio lab, has performed experiments on gene expression in tomato fruits. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
To understand the timing of Arabidopsis fertilization, we pollinated male-sterile Arabidopsis plants, using PLAT52:GUS pollen, which we visualized by X-Gluc staining. Three hours after pollination, pollen had germinated but very few fertilization events were visible. After 6 hours, about half of the ovules were fertilized; after 12 hours, about three fourths were fertilized; and after 24 hours fertilization was largely complete. These analyses reveal that the time points chosen span the entire window of fertilization events. We have extracted RNA from gynoecia at 0, 6, 12, and 24 hours after pollination (with unpollinated controls at the same time points), and have submitted the first set of samples for microarray expression analysis. We have generated about 50 transgenic lines carrying a PIAA9:IAA9:GFP/GUS construct, and are assaying these for expression of the GUS transgene and for 3:1 segregation of kanamycin resistance (to check for single locus insertion lines). We also obtained about 20 IAA:GFP lines carrying fusions to different IAA coding regions from Jose Alonso. However, in preliminary analyses we have not been able to detect GFP fluorescence, so these will probably not be useful markers. We have tested parthenocarpic fruit formation in several additional Arabidopsis arf mutants, and find that an nph4-1 arf6-2 double mutant (as well as an arf8 mutant, as previously reported) has partial fruit growth in the absence of pollination. This indicates that at least three ARF proteins, NPH4/ARF7, ARF6, and ARF8, can regulate fruit initiation. Recently published work in tomato also indicates that SlARF7 (orthologous to Arabidopsis NPH4/ARF7) regulates fruit initiation (de Jong et al., Plant J. 57: 160-170). To broaden our efforts to include analyses of multiple ARF genes, we are setting up collaborations with other researchers working on tomato AUX/IAAs and ARFs. We have obtained SlARF7 RNAi tomato plants from Wim Vriezen and are crossing this line to the SUN NILs. We are also currently transforming a P35S:MIR167a construct into tomato to silence both ARF6 and ARF8. Attempts to create transgenic tomato plants that carry a SUN:GFP or auxin responsive PDR5:GUS fusion constructs failed for unknown reasons. We have analyzed SUN expression in tomato ovaries by in situ hybridization, and find that it is expressed in the ee Sun1642 (elongated fruit) genotype and at a very low level or not detectable in the pp LA1589 (round fruit) genotype. In the former, expression was present in the funiculus (arrows) as well as in vascular tissues. In Arabidopsis, ARF6 and ARF8 are expressed in the funiculus. Further experiments will examine how the SUN expression pattern changes after pollination. We have also examined expression of tomato ARF4, ARF9, IAA3, IAA14, IAA16 and PIN7 immediately before and after anthesis. In both ee and pp genotypes, IAA14 expression increased about a day before anthesis and then decreased about two days after anthesis, an expression profile that coincides with that of SUN.
Publications
- No publications reported this period
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