No abstract

The main physiological roles of phloem and xylem in higher plants involve the transport of water, nutrients and metabolites. They are also involved, however, in whole plant events including stress responses and long-distance signaling. Phloem and xylem saps therefore include a variety of proteins. In this study, we have performed a shotgun analysis of the proteome of phloem and xylem saps from rice, taking advantage of the complete and available genomic information for this plant. Xylem sap was prepared using the root pressure method, whereas phloem sap was prepared with a unique method with the assistance of planthoppers to ensure the robustness of the detected proteins. The technical difficulties caused by the very limited availability of rice samples were overcome by the use of nano-flow liquid chromatography linked to a mass spectrometer. We identified 118 different proteins and eight different peptides in xylem sap, and 107 different proteins and five different peptides in phloem sap. Signal transduction proteins, putative transcription factors and stress response factors as well as metabolic enzymes were identified in these saps. Interestingly, we found the presence of three TERMINAL FLOWER 1/FLOWERING LOCUS T (FT)-like proteins in phloem sap. The detected FT-like proteins were not rice Hd3a (OsFTL2) itself that acted as a non-cell-autonomous signal for flowering control, but they were members of distinct subfamilies of the FT family with differential expression patterns. These results imply that proteomics on a nano scale is a potent tool for investigation of biological processes in plants.

SUMMARYStem cells are formed at particular times and positions during the development of multicellular organisms. Whereas flowering plants form stem cells only in the sporophyte generation, non-seed plants form stem cells in both the sporophyte and gametophyte generations. Although the molecular mechanisms underlying stem cell formation in the sporophyte generation have been extensively studied, only a few transcription factors involved in the regulation of gametophyte stem cell formation have been reported. The moss Physcomitrella patens forms a hypha-like body (protonema) and a shoot-like body (gametophore) from a protonema apical cell and a gametophore apical cell, respectively. These apical cells have stem cell characteristics and are formed as side branches of differentiated protonema cells. Here, we show that four AP2-type transcription factors orthologous to Arabidopsis thaliana AINTEGUMENTA, PLETHORA and BABY BOOM (APB) are indispensable for the formation of gametophore apical cells from protonema cells. Quadruple disruption of all APB genes blocked gametophore formation, even in the presence of cytokinin, which enhances gametophore apical cell formation in the wild type. All APB genes were expressed in emerging gametophore apical cells, but not in protonema apical cells. Heat-shock induction of an APB4 transgene driven by a heat-shock promoter increased the number of gametophores. Expression of all APB genes was induced by auxin but not by cytokinin. Thus, the APB genes function synergistically with cytokinin signaling to determine the identity of the two types of stem cells.

AINTEGUMENTA (ANT) and APETALA2 (AP2) are transcription factors that are involved in several developmental processes in Arabidopsis thaliana. They are similar in structure, containing two AP2 domains, and have partially redundant functions in reproductive organ development. Expression and functional analyses of ANT and AP2 homologs have been performed previously in almost all angiosperms. In this study, one ANT homolog and two AP2 homologs were isolated from the gymnosperm Pinus thunbergii and were named PtANTL1, PtAP2L1, and PtAP2L2. PtANTL1 is the first reported gymnosperm ANT homologous gene. Based on a gene tree constructed with sequences of all A. thaliana two-AP2-domain-containing genes, it is likely that PtANTL1 and ANT, and likewise PtAP2L1 and AP2, are orthologs. The expression patterns of PtANTL1/PtAP2L1/PtAP2L2 were examined with Southern hybridization of the quantitative RT-PCR products and in situ hybridization. PtANTL1 and PtAP2L1 had almost identical expression patterns in the analyzed organs, and PtANTL1/PtAP2L1/PtAP2L2 were continually expressed in the developing female cone. Our analysis suggests that gymnosperms have orthologs to both ANT and AP2, and that the most recent common ancestor of extant seed plants has two type AP2 subfamily genes, ANT-like and AP2-like, involved in the development of female reproductive organs.

Nitrogen is the most important macronutrient in plants and its supply induces responses in gene expression, metabolism and developmental processes. However, the molecular mechanisms underlying the nitrogen responses remain poorly understood. Here we show that the supply of nitrate but not ammonium immediately induces the expression of a transcriptional repressor gene in rice, designated NIGT1 (Nitrate-Inducible, GARP-type Transcriptional Repressor 1). The results of DNA-binding site selection experiments and electrophoretic mobility shift assays indicated that NIGT1 binds to DNA containing either of two consensus sequences, GAATC or GAATATTC. In transient reporter assays, NIGT1 was found to repress transcription from the promoters containing the identified NIGT1-binding sequences in vivo. Furthermore, NIGT1 repressed the activity of its own promoter, suggesting an autorepression mechanism. Consistently, nitrate-induced NIGT1 expression was found to be down-regulated after a transient peak during nitrate treatment, and the nitrate-induced expression of NIGT1 decreased in transgenic rice plants in which this gene was constitutively overexpressed. Furthermore, the chlorophyll content that could be a marker of nitrogen utilization was found to be decreased in NIGT1 overexpressors of rice grown with nitrate medium but not with ammonium medium. Thus, we propose NIGT1 as a nitrate-inducible and autorepressible transcriptional repressor that may play a role in the nitrogen response in rice. Taken together with the fact that the NIGT1-binding sites are conserved in promoter sequences of Arabidopsis NIGT1 homologs, our findings imply the presence of a time-dependent complex system for nitrate-responsive transcriptional regulation that is conserved in both monocots and dicots.