Functions Of AP1/SQUA-, REV-, TB1-like Genes And Isolation And Expression Of MicroRNAs In Phyllostachys Praecox

Bamboo (Bambusoideae), as the largest member of the grass family Poaceae, is the long-lived, woody-stemmed perennial grass with important economic value. Unlike common grass species such as rice and maize, bamboos have some features peculiar to themselves in growth and development. These features, including peculiar flowering habits, great vegetative propagation ability and fast growing, have been paid special attention for long time. However, the knowledge about the molecular mechanism of bamboo's growth and development is rather poor. In this study, two AP1/SQUA-like genes were isolated from bamboo (Phyllostachys praecox), theexpression patterns and function of these two genes were analyzed in detail. Meristemrelated REV- and TB1-like genes were cloned from bamboo, the expression of thesetwo genes were analyzed by RT-PCR and in situ hybridization, and the potential useful of TB1-like, genes in phylogenetic analysis of bamboo was also discussed. In addition, through small RNAs library construction, microRNA microarray detection and real-time PCR, the expression of microRNAs during bamboo rhizome and shoot development was profiled. Overall, the results of this study provide useful information for manipulation of bamboo's growth and development. It will be helpful for us to understand the molecular mechanism of bamboo flowering.1. Expression patterns and functional analysis of bamboo (Phyllostachyspraecox) AP1/SQUA-like genes during floral transitionThe switch from vegetative to reproductive growth is a crucial developmental phase in flowering plants and is controlled by multiple pathways that respond to different environmental and endogenous signals. Functional analysis by genetic studies in model eudicots, such as Arabidopsis thaliana, has revealed that plant AP1/SUQA-like MADS-box genes subfamily are critical regulators in floral transition and floral organ identity.In present study, we isolated two novel genes from Phyllostachys praecox and evaluated their functional characteristics. The sequence and phylogenetic analysis indicated that these two genes, named PpMADS1 and PpMADS2, belong to the FUL3 and FUL1 clade of Poaceae AP1/SQUA-like genes, respectively. The PpMADS2 possesses a truncated C terminus lacking the highly conserved paleoAP1 motif. It was further confirmed that the truncated C terminal region was produced by natural sequence deletion in exons, but not by alternative splicing. Ectopic expression of PpMADS1 and PpMADS2 significantly promoted early flowering through up-regulation of AP1 in Arabidopsis. Yeast two-hybrid experiments demonstrated that AP1 protein can interact with PpMADS1 but not PpMADS2, suggesting that these two genes may act differently in signaling early flowering of bamboo plants.Further analysis by RT-qPCR and in situ hybridization revealed distinct expression patterns of these two genes in bamboo. The expression level of PpMADS2 was up-regulated more dramatically than PpMADS1 in flowering bamboos, especially in bamboo shoot apex and leaves. Compared to nonflowering bamboos, PpMADS1 was up-regulated about one fold in bamboo shoot apex of flowering bamboos, while the transcript level of PpMADS2 was increased about 30 fold. Another striking difference was shown in leaves. PpMADS2 was expressed in leaves of flowering branches with 20 fold higher level than in other leaves from non-flowering and flowering bamboos, while no such difference was shown in PpMADS1. The result of in situ hybridization indicated that PpMADS1 and PpMADS2 were expressed throughout floral meristem, but only PpMADS2 was observed in stamen of mature floret. It implied that these two genes were involved in early stage of floral organ development, and PpMADS2 was also related to stamen development in floret.All the results suggested that both PpMADS1 and PpMADS2 were involved in floral transition, and PpMADS2 might play more important roles than PpMADS1 in floral development of Phyllostachys praecox.2. Molecular cloning, expression analyses and primary evolution studies of REV- and TB1-like genes in bambooMost cultured bamboos are perennial woody evergreens that reproduce from rhizomes. It is unclear why some rhizome buds develop into aerial bamboo shoots instead of new rhizomes. REVOLUTA (REV)-like Class III homeodomain leucine-zipper (HD-Zip) proteins and TEOSINTE BRANCHED1 (TB1)-like transcription factors have been shown to play regulatory roles in meristem initiation and outgrowth.We cloned and analyzed the bamboo (Phyllostachys praecox C.D. Chu&C.S. Chao.) REV- (PpHB1) and TB1-like (PpTB1) gene. Their expression was mainly detected by in situ hybridization. PpHB1 expression was detected in the tips of lateral buds, on the adaxial portion of the leaf and within the developing procambium, indicating its close correlation to rhizome bud formation and procambial development. PpTB1 expression was mainly detected on the top of buds at later developmental stages, suggesting that was more likely involved in bud outgrowth. Meristem genes might therefore serve as specific molecular markers of rhizome bud development and could be useful in studies designed to elucidate the mechanisms underlying bamboo shoot development. In addition, meristem genes such as TB1-like sequences may be useful in phylogenetic analyses of bamboo species.3. Isolation and expression profiles of microRNAs (miRNA) during bamboo shoot developmentMiRNAs are involved in multiple developmental processes in plants and animals. In recent years, hundreds of miRNAs of plants and animals have been identified through computational and cloning approaches. However, our understanding on expression and function of miRNAs in bamboo shoot development is largely lacking. To identify miRNAs potentially involved in bamboo shoot development, two complementary approaches were applied: a) establishing a small RNAs library followed by partial sequencing of the library; and b) performing miRNA microarray to establish miRNA expression profiles during various development stages. Six plant homologous and 24 candidate miRNAs were identified through sequence analysis of the library whereas 33 miRNAs were found to be up-regulated in bamboo shoot buds and 12 miRNAs were up-regulated in bamboo rhizome buds with miRNA profiling. Further expression analysis on selected miRNAs, such as osa-miR1561, osa-miR171b, osa-miR319a, Pp-miR168, Pp-miR169 and Pp-miR894, indicated that these miRNAs were highly expressed at early stage of bamboo shoot development, suggesting important roles of miRNAs in fast growing and development of bamboo shoots.