Progress 09/01/04 to 08/31/08
Outputs
OUTPUTS: During the final phase of the project, we cloned and sequenced RNAs of 15-30 nt from tomato mature leaves as well as fruit at three successive stages of development: flower bud, young fruit at 10 days post anthesis, and mature ripe fruit. We obtained a total of 1,210 nonredundant sequences, among which 761 are expressed in leaf, 105 in flower bud, 123 in young fruit, and 120 in mature fruit. Overall, 80-85% of fruit and leaf small RNAs ranged between 21 and 24 nt in length. The tomato small RNAs were designated as "SlsmR-#," where SlsmR stands for Solanum lycopersicum small RNA and the "#" is the unique number for each small RNA. Letters further differentiate members of a putative family (Itaya et al., 2008). Our further analyses of SlsmR-31a have yielded novel and unexpected findings about the regulatory function of this small RNA and its evolutionary origin. These new findings, summarized below, are being written up for publication. SlsmR-31a guides cleavage of a Ca2+-ATPase (ACA) mRNA, SlACA10 in tomato, by recognizing a target sequence in the 5' untranslated region (UTR) of this mRNA. Over-expression of SlsmR-31a or a cleavage-resistant form of SlACA10 (SlACA10R) in transgenic tomato plants resulted in significant stamen lengthening and abolished or reduced fruit production. We cloned three structurally distinct versions of 31a precursors from several species near the root of Solanaceae phylogeny tree. Two longer versions are transcribed but the transcripts appear to be incompetent for post-transcriptional processing to yield the mature 31a miRNA. They were lost in more evolutionarily advanced Solanaceous species. The third, also shorter, processing-competent version has been retained in these advanced species during evolution. We found that the open reading frame (ORF) of ACA10 is highly conserved in plants. However, the 31a-regulated expression of ACA10 has evolved in only some species of Solanaceae. For instance, the mature 31a miRNA is produced in tomato and potato, but hardly detectable in closely related eggplant and more distantly related tobacco despite presence of the perfect 31a target sequence in the 5'UTR of ACA10 mRNA these latter species. On the other hand, some plants near the root of the Solanaceae phylogeny tree lack the 31a target sequence in their ACA10 even though they produce mature 31a. These and other observations suggest independent evolution of a nonconserved miRNA and its target sequence. PARTICIPANTS: Biao Ding Asuka Itaya Ying Wang Xuehua Zhong TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our new data suggest a potential role of SlsmR-31a in fine-tuning SlACA10 expression critical for the proper development of flower organs and fruit production. They also suggest that post-transcriptional processing of miRNA precursors is a novel limiting factor during the evolution of functional miRNAs. These results not only shed new light on the biological function 31a-regulated ACA10 expression, but also contributed to the development of a powerful Solanaceae experimental system to investigate the broad question of how the emergence of various nonconserved miRNA-based gene regulatory networks has contributed to the evolution of distinct plant developmental processes. Ca2+-ATPases are ubiquitously encoded in all organisms and their functions are essential to many aspects of cellular processes ranging from reproduction to development. Our work provided the first example of miRNA-regulated expression of these universally important genes.
Publications
- Itaya, A., Bundschuh, R., Archual, A.J., Joung, J.G., Fei, Z., Dai, X., Zhao, P.X., Tang, Y., Nelson, R.S., and Ding, B. (2008) Small RNAs in tomato fruit and leaf development. Biochim. Biophys. Acta 1779:99-107.
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: We completed a global identification of small RNAs in tomato leaves and fruits to set a foundation for functional studies of small RNAs in regulating tomato developmental processes. Along this line, we also developed a protocol to perform transient gene silencing assay in tomato, which should enable gene function studies in tomato.
PARTICIPANTS: Individuals who worked on the project at Ohio State University: Itaya, A., Bundschuh, R., Archual, A., Wang, Y., Takeda, R., Zhong, X. and Ding, B. Collaborators and institutions: Joung, J.-G., Fei, Z. (Boyce Thompson Institute, Cornell University) Dai, X., Zhao, P., Harris, A., Tang, Y., Nelson, R.S. (Samuel R. Noble Foundation)
TARGET AUDIENCES: Plant biologists.
Impacts
We developed an Agrobacterium-mediated transient silencing assay in tomato leaves. We have also identified over 350 and 700 small RNAs from tomato fruit and leaf, respectively. Except for conserved microRNAs, more than 90% of the small RNAs are unique to tomato. We confirmed expression of some conserved as well as novel putative microRNAs by Northern hybridization. Our results help form a basis for comparative studies on how small RNA-mediated gene expression have contributed to the evolution of common and distinct developmental pathways of fruits and leaves. We have established the website (http://ted.bti.cornell.edu/digital/sRNA/) for public access to all of our small RNA sequences, their expression patterns in respective tissues, and their matching genes or predicted target genes in a searchable manner. Our work on tomato small RNAs represents a new direction of research in leaf and fruit development in an important crops species. It complements current studies on
the hormonal effects, biochemical processes, and molecular characterization of the protein-coding genes. We expect findings from the project to help accelerate and integrate our understanding of the molecular mechanisms underlying fruit and leaf development in tomato, and also likely in other important crop species. Elucidating the small RNA regulatory network in fruit and leaf formation may enable development of novel RNA-based technologies to produce crops with improved quality, added values, and enhanced resistance to biotic and abiotic stresses.
Publications
- Itaya, A., Bundschuh, R., Archual, A.J., Joung, J.G., Fei, Z., Dai, X., Zhao, P.X., Tang, Y., Nelson, R.S., and Ding, B. (2008) Small RNAs in tomato fruit and leaf development. Biochim. Biophys. Acta 1779:99-107.
- Itaya, A., Zhong, X., Bundschuh, R., Qi, Y., Wang, Y., Takeda, R., Harris, A.R., Molina, C., Nelson, R.S., and Ding, B. (2007). A structured viroid RNA is substrate for dicer-like cleavage to produce biologically active small RNAs but is resistant to RISC-mediated degradation. J. Virol. 81:2980-2994.
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Progress 10/01/05 to 09/30/06
Outputs
The original aims were to investigate the role of miRNA precursors for DCL processing and the role of DCL1 in the specific step of the miRNA biogenesis pathway. During the early course of investigations, other labs published biochemical studies on the biogenesis of Arabidopsis miRNAs and identified the role of DCL1 in the different steps of miRNA biogenesis. Therefore, we made adjustments to our original aims. First, we developed protocols for studying gene silencing in Nicotiana benthamiana protoplasts and tomato leaves. By analyzing the roles of viral silencing suppressors, we demonstrated that the N. benthamiana protoplast offers a useful complementary system to study silencing mechanisms at the cellular level (Qi et al., 2004). The Agrobacterium-mediated transient silencing system in tomato leaves offers a excellent tool for investigating silencing mechanisms in an important crop model (Itaya et al., 2007). Second, we continued mutational analyses to study how the
miRNA precursor structures determine efficiency and specificity of DCL cleavage. We found that the length of the apical loop of the precursor influenced efficiency of processing and the length of the stem region determined specificity (Y. Qi and B. Ding, manuscript in preparation). Third, we decided to initiate a new research direction to investigate the role of small RNAs in tomato development, still within the broad goal of the project to understand how small RNAs function to regulate plant growth and developmental processes. We feel this new direction is of particularly relevant because it addresses one of the most fundamental gene regulatory mechanisms in an important crop species. We sequenced small RNAs from various tomato organs, including leaves, seedlings and fruit at different developmental stages (A. Itaya et al., manuscript in preparation). We obtained sequences of more than 350 small RNAs from tomato fruit and a similar number from tomato leaves. These small RNAs are
classified into several categories. (1) Conserved miRNAs. These include miR168b, miR172h, c, miR159a, miR167a, miR164c and miR424a. Based on the validated miRNA-target pairs reported in Arabidopsis, we have also identified putative target genes for three miRNAs in the tomato genome and EST databases. These putative target genes include AGO1 (SGN-U317175) for miR168b, ARF8 (SGN-U340284) for miR167a, and AP2 (SGN-U314858) for miR172h, c. (2) Sequences that match expressed mRNAs, in either sense or antisense orientation. Eleven of these small RNAs are novel (i.e., they have not been reported in any other species) and have putative target genes. (3) Sequences that match tRNAs and rRNAs. (4) Unknown sequences. These sequences match various plant genome sequences but their nature and function are unclear. (5) Novel sequences. These small RNA sequences do not match any plant DNA sequences available.
Impacts
Short interfering RNA (siRNA)-mediated RNA silencing plays an important role in cellular defense against viral infection and abnormal gene expression in multiple organisms. Many viruses have evolved silencing suppressors for counter-defense. We have developed an RNA silencing system in the protoplasts of Nicotiana benthamiana to investigate the functions of viral suppressors at the cellular level. Our analyses suggest that protoplast-based transient RNA silencing is a useful experimental system to investigate the functions of viral suppressors and further dissect the mechanistic details of the RNA silencing pathway in single cells. Our work on tomato small RNAs represents a new direction of research in leaf and fruit development in an important crops species. It complements current studies on the hormonal effects, biochemical processes, and molecular characterization of the protein-coding genes. We expect findings from the project to help accelerate and integrate our
understanding of the molecular mechanisms underlying fruit and leaf development in tomato, and also likely in other important crop species. Elucidating the small RNA regulatory network in fruit and leaf formation may enable development of novel RNA-based technologies to produce crops with improved quality, added values, and enhanced resistance to biotic and abiotic stresses.
Publications
- Qi, Y. and Ding, B. (2007). Analyses of structural features of microRNA precursors that influence processing efficiency and specificity. (Manuscript in preparation)
- Itaya, A., Zhong, X., Bundschuh, R., Qi, Y., Wang, Y., Takeda, R., Harris, A.R., Molina, C., Nelson, R.S., and Ding, B. (2007). A structured viroid RNA is substrate for dicer-like cleavage to produce biologically active small RNAs but is resistant to RISC-mediated degradation. J. Virol. 81:2980-2994.
- Qi, Y., Zhong, X., Itaya, A., and Ding, B. (2004). Dissecting RNA silencing in protoplasts uncovers novel effects of viral suppressors on the silencing pathway at the cellular level. Nucl Acids Res. 32 (22): e179.
- Itaya, A., Bundschuh, R., Archual, A., Ding, B. (2007) Small RNAs in tomato fruit and leaf development.(Manuscript in preparation)
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