Cytokinin-regulated Flower Development In Arabidopsis

Cytokinins are essential regulators of numerous plant growth and development processes. In Arabidopsis, genes encoding the enzymes responsible for cytokinin metabolic activity such as biosynthesis and degradation have been characterized, and the cytokinin signaling transduction was implicated in control of shoot meristem initiation, stem cell population, apical dominance and fertility. However, little are known about the mechanisms of cytokinin regulation in inflorescence and flower development.The first step of the bulk of cytokinin biosynthesis is catalyzed by seven ATP/ADP isopentenyltransferases (AtIPT1 and AtIPT3-AtIPT8) in Arabidopsis. Here, we tried to increase the cytokinin level in Arabidopsis floral tissues by transgenic expression of AtIPT4 under the control of the promoter of APETALA1 (AP1) that is known to be expressed specifically in flower primordia and perianth in Arabidopsis. Totally, 45 independent AP1::IPT4 lines obtained showed the similar phenotypical alterations with respect to the inflorescence and flower development. In AP1::IPT4 plants, the apical dominance and stem elongation of primary inflorescence were significantly inhibited, which resulted in the formation of sympodial branching architecture. Both the flower and floral organ number, however, were increased obviously in transgenic plants. In addition, many branched trichomes formed on the abaxial surface of sepals. As expected, no obvious morphological changes were observed during the vegetative development. In the longitudinal sections of primary inflorescence apex, the cell number and size of inflorescence meristem were increased in transgenic plants, whereas the meristematic cell size was not altered, indicating elevated cell division rate occurred in the transgenic inflorescence meristem. Scanning electron microscopy analyses indicated that the transgenic primary inflorescence meristem was larger in diameter than wild-type one. There were more flower primordia formed at periphery of inflorescence meristem of AP1::IPT4 plants in comparison with that of wild type plants, however, the floral organ initiation was delayed. Furthermore, GUS staining assay revealed that CLAVATA3 (CLV3) and WUSCHEL (WUS), two genes responsible for stem cell homeostasis maintenance, were implicated in cytokinin-regulated inflorescence and flower development. To uncover the physiological basis for modulating flower development in AP1::IPT4 plants, we analyzed the endogenous cytokinin levels in the young inflorescences from the homozygotic plants of two independent lines with the liquid chromatography--mass spectrometry (LC-MS) method. Compared with wild-type samples, the levels of isopentenyladenine (iP) and its precursor, isopentenyladenosine (iPR), were all elevated significantly, which is consistent with the increase of transcript level of AtIPT4 detected in transgenic lines by RT-PCR analysis. These results support our hypothesis that the phenotypical alterations of AP1::IPT4 plants should be caused by enhanced endogenous cytokinin concentration in inflorescence tissues.To explore the cytokinin-responsive genes in floral tissues, we carried out the analysis for the genome-wide expression profiling in young inflorescences using the Affymetrix Arabidopsis ATH1 GeneChips (Gene Company Limited). The results revealed that 460 gene expression levels were up-regulated and 408 genes down-regulated in floral tissues of transgenic plants. Among them, we are interested in those involved in cytokinin signaling and flower morphogenesis including the type-A ARRs (ARR5, ARR6, ARR7, and ARR15), two CUP-SHAPED COTYLEDON genes (CUC2 and CUC3), three LOB domain protein genes (LBD3, LBD39 and LBD41).Either LBD3 (Naito et al., 2007) or CUC3 overexpression led to stem elongation reduction which is similar to that of AP1::IPT4 plants. In contrast, cuc3 mutation or overexpression of anti-sense LBD3 repressed some aspects of AP1::IPT4 phenotypes including shoot apical dominance and internode length between flowers.These results revealed that both CUC3 and LBD3 contributed to the developmental changes of AP1::IPT4 plants. Genetic and molecular analyses demonstrated that combination of cytokinin receptor mutantion ahk2 and ahk3 blocked almost the phenotypes of AP1::IPT4 plants, and impaired the up-regulation of CUC3 and LBD3 in response to cytokinins. Furthermore, we examined the functions of ARR5, ARR6 and ARR15 in AP1::IPT4 inflorescences. The results suggested that mutation in ARR6 may partially inhibited the developmental alterations of AP1::IPT4 plants, and attenuated LBD3 expression level. Thus, the results indicated that proper development of inflorescence and flower might be partially controlled by cytokinin signaling-mediated CUC3 and LBD3 functions in the inflorescence and flower meristem.