Circadian Rhythm Clock Genes Mediated Exogenous γ-aminobutyric Acid To Integrate Photoperiodic Flowering Pathway In Promoting Arabidopsis Early Flowering Time

Multiple environmental factors, including day length, light intensity, temperatureand so on, affect the timing of the floral transition in Arabidopsis (Arabidopsisthaliana). γ-aminobutyric acid (GABA), the non-protein amino acid, plays animportant role in plant development. Recent finding indicates that exogenous GABAcould stimulate Arabidopsis early-flowering. Under long day (LD) condition, theplants seedlings sprayed with GABA（1mmol/L）flowered earlier1week than thecontrol (treated with water). However, the mechanism underlying is far more fromunderstood. In this contribution, key players of multiple flowering pathways wereselected as the staring research point, the expression pattern of photoperiodicflowering pathway genes, circadian clock genes were quantitatively assayed byreal-time RT-PCR，exploring the possible relationship of GABA and their expression.The results demonstrated that the promotion of GABA on pay much more attention toelucidate the influence of GABA on Arabidopsis early-flowering phenotype has muchrelation with its redialing the rhythm of innate biological clock in plants, resulting inthe output of photoperiodic flowering pathway which leads to the final early flowerphenotype. The main results are summarized as follows:1. Under Long Day (LD)(16h light/8h dark), compared to plants with waterspaying, the plants sprayed with GABA (1mmol/L) could stimulate Arabidopsisnearly one-week early flowering. To understand the mechanism underling, keyregulatory genes involving flowering pathways was analysed at transcriptional levelin the time sequence (2-4week). Real-Time PCR showed that floral integration gene,FT, a key component photoperiodic pathway gene, has significant up-regulation at theend of4-week, reaching approximately7-fold of the control. Meanwhile, theexpression of another floral integration gene, LFY, floral meristem identity genes,AP1and FUL has significant up-regulation upon GABA treatment. However, theexpression of negative floral repressor gene, FLC, was markedly curbed by GABAtreatment. Furthermore, GABA treatment also obviously up-regulated thetranscription level of blue light receptors gene, CRY1and CRY2. The result indicatedthat the role of GABA in Arabidopsis early flowering is achieved by its up regulationon photoperiodic pathway gene FT to affect flowering output gene expression,including LFY, AP1, and FUL, and at last function in early flowering in Arabidopsis.2. GABA could entrain the pattern of circadian rhythm. Previous investigation indicated that photoperiodic flowering pathway is controlled by circadian rhythm. Tofurther explore whether this GABA treatment is done with the integration of circadianrhythm, clock oscillator gene are studied in the diurnal rhythm period. The treatedplants of3week seedlings with/without GABA treatment were sampled at the sametime at4h intervals for continuous3whole days for18times sampling. The firstsampling time point (at11:00AM) was set as ZT0（Zeitgeber time）. The resultsshowed that the central oscillator gene, TOC1, LHY, CCA1demonstrated diurnalfluctuation. However, the amplitude of gene expression pattern in TOC1wasobviously higher in the GABA treatment than that in the control. This redialed clockrhythm by GABA implies the significant role of GABA in the course of earlyflowering phenotype.3. Notably， diurnal expression pattern of FT was synchronized with theexpression of CRY2，a kind of blue light receptor gene. However, the expression of FTand CRY2was not consistent with the expression of CO, implying another floweringpathway participating in the course. Furthermore, the expression of CRY1wassynchronized with CRY2; the expression of PHYA was consistent with PHYB.Interestingly, the up-regulated expression of CRY1, CRY2and PHYA by GABA wasoccurred in the same diurnal course. To consistent with this, the expression of COup-regulated by GABA was almost occurred at the same diurnal time point, implyingthat the expression was complexly controlled by the expression of CRY1，CRY2å'ŒPHYA.In all, our result indicate that the involvement of GABA in the promotion ofArabidopsis early flowering has relation with its alteration of circadian rhythm tofinally control the photoperiodic flowering pathway. The promotion of exogenousspaying GABA on early flowering phenotype provides a novel idea in the agricultureapplication to control crop flowering time.