| Plant microRNAs (miRNAs) are about 21 nucleotides, endogenously expressed, noncoding RNAs processed by Dicer-like proteins from stem-loop regions of long RNA precursors. Among these miRNAs, miR396 was first identified by Jones-Rhoades in Arabidopsis in 2004, and was predicted to target AtGRF [Arabidopsis thaliana growth regulating factor (AtGRF)] family members (GRF1, GRF2, GRF3, GRF7, GRF8, GRF9) whose cleavage products were discovered by 5'RACE. Further analysis showed miR396 was highly conserved in distantly related plant species, both in terms of primary and mature miRNAs, but especially for mature sequences and their complementary miRNA* sequence. It implicated that miR396 played important and conserved regulatory roles in plant development, such as flower and leaf development. This study was focused on function analysis of miR396 in tobacco by generating transgenic tobacco overexpressing Arabidopsis miR396a.By means of northern blotting, we investigated the expression patterns of miR396 in tobacco (Nicotiana tabacum L.) using miR396a antisence probe, the result showed that miR396 expressed in all tissues suggesting that a tobacco ortholog of miR396 is present with the possibility that the sequences are almost identical. To demonstrate the function of miR396 in tobacco, we inserted the synthetic precursor Ath-MIR396a into the over-expression -vector (pocA30) and transformed into tobacco by the leaf-disk method mediated by Agrobacterium tumefaciens. The inducer increased miR396 levels significantly, indicating appropriate maturation of the synthetic precursor Ath-MIR396a in transgenic tobacco. Moreover, a narrow-leaf phenotype and abnormal development of flower organs were observed in transgenic tobacco, which resembled those in transgenic Arabidopsis overexpressing miR396. Our previous study has shown that miR396 modulated leaf shape by regulating AtGRF1-AtGRF3 in Arabidopsis. We searched the tobacco EST sequence database with the conserved sequences of Arabidopsis thaliana GRF (AtGRF) as query. Consequently, we obtained four putative partial cDNA sequences (GenBank Ace:FG137771, FG165999, FG167390 and FG194560), which contained the match-sites with Ath-miR396 and were highly homologous in both nucleotide and amino sequences. Additionally, the four deduced amino sequences contained a specific GRF DNA-binding sequence defined as the WRC domain.Upon these results, it seemed that these four partial cDNA were from four different NtGRF-like genes. To determine whether endogenous NtGRF-like mRNA levels were significantly reduced in inducer-treated leaves compared with wide-type plant in tobacco. Both of the full length sequence and the cleaved fragment in wild-type and transgenic tobacco were detected by semi-quantitative RT-PCR. The results revealed that the levels of cleavage-site-spanning fragment of three NtGRF-like genes were significantly decreased in transgenic plants but not of the cleaved fragment, indicatng that NtGRF-like genes were likely cleaved by miR396 in tobacco.Then, we analyzed the effect of drought stress on miR396 during 8-week-old plants grown on soil. We discovered miR396 was induced with the increasing time of drought treatment. Furthermore, transgenic miR396 plants were significantly more drought tolerance than wide-type.8-week-old seedlings of transgenic and wild-type plants were withheld from water for 3 weeks, wild-type plants leaves clearly wilted compared to those of transgenic plants, similar result was obtained when 3-week-old-seedlings of wide-type and transgenic plants were treated with PEG6000 (10%) for 4h. To seek the mechanism that decreasing wilting of transgenic plants with miR396 over-expression under drought stress, we detected water loss rate and relative water content of wide-type and transgenic plants respectively. The results revealed that the transgenic tobacco overexpressing miR396 had a lower water loss rate and higher relative water content in contrast to wide-type plants, indicating that the leaves of inducer treated plants had an enhanced ability of water conservation in drought stress condition. Meanwhile we found that the stomata numbers were reduced about 30% to 40% in 35S::MIR396a lines compared with controls. Additionally, both transgenic plants were so shorter and there leaves were so narrower than control that they reduced water vapor exchanged between plants and environment. To sum up, the reduction of stomata number of transgenic plants, narrow leaves, and short plants caused decreasing wilting of transgenic plants with miR396 over-expression under drought stress.We also detected molecular Trafficking of miR396 in phloem by performing micro-grafting experiments using tobacco. The result showed that chimeric plants constitutively over-expressing miR396 in the shoot accumulated mature miR396 species to higher levels in their upper wild-type leaves, suggesting potential signaling roles of miR396 by long-distance regulation of gene expression.In conclusion, our results demonstrated miR396 regulated leaf shape development by down-regulating NtGRF-like in tobacco and along with the small and narrow-leaf phenotype in 35S:miR396, the number of stomata was decreased. These physiological varieties made a contribution to improving the drought tolerance of transgenic plants. In addition, our results also showed that miR396 was a phloem-mobile microRNA, so we speculated that miR396 might be involved in long-distance regulation of gene expression. |
