MicroRNA393 Regulates Seed Maturation And Germination Through Interaction With ABA In Arabidopsis Thaliana

Seed dormancy and germination are two developmental processes in the life cycle of plants, which are also important for agricultural production. Although the balance of two phytohormones, ABA(abscisic acid) and GA(gibberellin), is known to be a major regulator of dormancy status, recent studies demonstrate that auxin is also critical for dormancy induction and maintenance. miR393 is one of the most conserved miRNA families and its targets encode the TRNSPORT INHIBITOR RESPONSE1(TIR1)/AUXIN SIGNALLING F-BOX(AFB) clade of auxin receptor (TAAR) in Arabidopsis. Our previous data indicated that the expression of miR393 is regulated by ABA and its targets affect ABA sensitivity of seed germination. However, whether miR393 regulates seed maturation and germination and the underlying mechanisms are largely unknown.In this study, we investigated how miR393 regulates seed maturation and germination through TIR1 repression and the interaction of hormone signaling pathways using transgenic report lines and mutants of AtmiR393 and its target. The results are as follows.1 The expression profiles of AtmiR393 and its target genes in seed maturation and germination processes1.1 Expression patterns of miR393 and its targets during seed maturation and germinationSiliques in different stages of maturation and imbibed seeds was GUS stained using pMIR393a/b:GUS, pTIR1:mTIR1-GUS, pTIR1:cTIR1-GUS and DR5:GUS report lines. During seed maturation, the expression of MIR393b reporter increased in metaphase of seed maturation (Stage Ⅲ) and decreased in Stage Ⅳ, while no dramatic change of GUS expression was observed in MIR393a reporter line. The expression of cTIRl was obviously suppressed post-transcriptionally from Stage I to Stage Ⅲ, whereas GUS activity in DR5:GUS report lines was upregulated from Stage Ⅰ to Stage Ⅲ and decreased in Stage Ⅳ.During imbibition, the transcription of miR393 increased. The GUS expression was localized to the cotyledon and radicle in MIR393a reporter line, while was limited to the cotyledon in MIR393b reporter line. Furthermore, the transcription sites of MIR393 were similar to the expression regions of DR5:GUS reporter. cTIR1 was expressed both in dry seeds and imbibed seeds, and its level increased as germination initiated. GUS signal of cTIRlprotein reporter was expressed in cotyledon and elongation zone of radicle, and gradually disappeared in root tip. The expression of mTIR1 (a miR393-resistant form of TIR1) was observed in more extensive regions including root tip. The expression sites of TIR1 and DR5:GUS were overlapped in cotyledon and displayed mutual exclusion pattern in radicle. These data suggested that miR393-mediated posttranscriptional repression plays a regulational role in spatial distribution of TIR1 and auxin response during seed maturation and germination.1.2 Expression levels of AtmiR393 and its targets during seed maturation and germinationThe expression levels of AtmiR393 and its targets were detected by quantitative RT-PCR It showed that the expression of pri-MIR393b was first increased and then down-regulated during seed maturation, and the transcript level of targets, including TIR1, AFB2, AFB3 were decreased, suggesting that miR393 and its targets gradually become inactive when dormancy induces. In imbibed seeds, as germination started, the expression of miR393 and its targets were both significantly up-regulated, which indicated that the accumulation of miR393 is associated with auxin response, and the levels of its targets are likely affected by other factors besides miR393.2 miR393 regulates seed dormancy through interaction with ABA signalling pathway2.1 Exogenous ABA affects the expression of miR393Stratified wild-type seeds were transferred to MS medium supplemented with 5 μM ABA for 1 d under long-day conditions. The results of quantitative RT-PCR revealed that after ABA treatment, the expression of miR393 and its targets was both down-regulated, which indicated that ABA negatively regulates the expression of miR393.2.2 miR393 affects primary dormancyGermination rate and cytoledon green rate of freshly harvested seeds were measured using wild-type, mir393ab,35S:mTIR1 and35S:MIM393 transgenic lines. It showed that, compared with wild-type, the germination rate of 35S:mTIR1 and 35S:MIM393 was significantly decreased, indicating that miR393 may negatively regulates seed dormancy through TIR1.2.3 miR393 affects ABA sensitivity of seed germination and seedling growthTo analyse the ABA sensitivity of seed germination and seedling development, seeds were sown on medium containing different concentrations of ABA. The results showed that, compared to those in wild-type, the germination rate and cytoledon green rate were significantly reduced in mir393ab,35S:mTIR1 and35S:MIM393, and root elongation was only significantly decreased in 35S:mTIRl. It implied that miR393 negatively regulates ABA sensitivity of seed germination and seedling growth.2.4 miR393 affects the expression of genes related to auxin and ABA signalling pathway during seed maturation and germinationThe expression of genes related to auxin and ABA signalling pathway was detected by quantitative RT-PCR. It showed that during seed maturation, the expression of ARF10 increased at first and decreased later. Compared to wild-type, the expression of ARF10 increased significantly at Stage Ⅲ in 35S:mTIR1 transgenic line. During seed germination, the expression of ARF10 increased significantly. Compared to wild-type, the expression levels of ARF10 in dry seeds of 35S:mTIRl and mir393ab were significantly less than that in wild-type, and exhibited more dramatic variation during seed germination.Compared with wild-type, the expression of ABI3 and ABI4 was significantly higher in mir393ab in maturation stage Ⅲ, and in 35S:mTIR1 in DAC1. It suggested that miR393 might regulate the expression levels of ABI3 and ABI4 through TIR1 during seed maturation and germinationIn summary, this study reported that the specific expression of miR393 helps to regulate the spatial distribution of TIR1 during seed maturation and germination, and negatively regulates seed primary dormancy and ABA sensitivity of seed germination and seedling growth. These data provided new evidence for the biological function of miR393 and the interplay of auxin and ABA signals in dormancy induction and release.