| Pedicel is the short stem linking and anchoring a single flower to the main axis of inflorescence. Pedicels play a critical role in transporting nutrients to the flower during their development and fruit ripening. In addition, the length and orientation of pedicels directly determine the architecture of the inflorescence or spikes, which may have significant impact on crop yield. Previous works on pedicel development are mostly carried out in the model plant Arabidopsis. A number of genes have shown to be involved in pedicel development including transcription factors BREVIPEDICELLUS(BP), ERECT A, and CRM1/BIG. Despite this, not much is known about pedicel development in other species. We found that a MADS-box gene, NtSVP is required for pedicel development in tobacco. We then studied the functions of NtSVP and its regulatory pathways. The main results are as following:1. Compared with wild-type plants, down regulation of NtSVP by RNAi produced significantly elongated pedicels in the transgenic tobacco plants, leading to a more spread inflorescence architecture. Conversely, overexpression of NtSVP caused quite compact inflorescence with severely shortened pedicels. Paraffin section of the pedicel revealed elongated cells when compared with those of the wild type plant. Thus, the increase length of the pedicel is not caused by increased cell number, but longer pedicel cells.2. Since BP is one of the important genes involved in Arabidopsis pedicel development, we studied the expression patterns of the best homolog of Arabidopsis BP, named NtBPL in the transgenic plants. Real-time PCR showed that down regulation of NtSVP increased the expression of NtBPL.We then knocked down NtBPL expression by RNAi and the transgenic plant showed shortened pedicels and impaired cell elongation, while overexpression of NtBPL caused significant elongation of the pedicels in the transgenic plants. No expression change of NtSVP was found when NtBPL was disturbed, suggesting that NtSVP is positioned at the top of regulatory pathway relative to NtBPL.3. To further identify the relationship between NtSVP and NtBPL, we generated NtBPL-RNAi transgenic plants with those of NtSVP-RNAi. The double down regulation of NtBPL and NtSVP produced shortened pedicels, indicating that genetically NtSVP is indeed located at the upstream of NtBPL in the pedicel regulatory pathway.4. To determine whether NtSVP directly regulates NtBPL or not, we performed yeast one hybrid experiment and electrophoretic mobility shift assay. The results showed that NtSVP can directly bound to the CArG-box [C(A/T)8G] in the NtBPL promoter. Consistent with the reduced transcript levels of NtBPL in NtSVP-OE pedicels, transcription activtiy assay by dual-luciferase reporter system demonstrated that NtSVP acted as a transcriptional repressor of NtBPL.5. Furthermore, we studied the downstream genes of the NtSVP-NtBPL pathway by using tobacco microarray analyses. Comparison of the transcriptome changes between the NtBPL-RNAi line and the wield type plant showed that a large number of genes were regulated by NtBPL. Among them, seven genes are involved in Gibberellin synthesis and metablism including CPS, KS, KAO, GA3ox1, GA3ox2. Down regulation of these genes in the NtBPL-RNAi plant implied that the role of NtSVP-NtBPL pathway on tobacco pedicel development is through GA pathways.Taken together, our work may have revealed a novel regulatory mechanism for plant pedicel development. A working model is proposed where NtSVP suppresses NtBPL expression by directly binding to its promoter, which then affects the GA-biosynthesis for pedicel elongation. This work finds a new regulator upstream BP that is known in Arabidopsis and thus extends the pedicel regulatory pathway by discovering the function of a MADS-box gene NtSVP. Thus, it expands our knowledge in the molecular mechanisms of plant pedicel development, which may be beneficial for crop breeding by modifying inflorescence/spike structure and hence yield improvement. |
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