Expression regulation of MALATE SYNTHASE involved in glyoxylate cycle during protocorm development in Phalaenopsis aphrodite (Orchidaceae).

Orchid (Orchidaceae) is one of the largest families in angiosperms and presents exceptional diversity in lifestyle. Their unique reproductive characteristics of orchid are attracted by scientist for centuries. One of the synapomorphies of orchid plants is that their seeds do not contain endosperm. Lipids are used as major energy storage in orchid seeds. However, regulation and mobilization of lipid usage during early seedling (protocorm) stage of orchid is not understood. In this study, we compared transcriptomes from developing Phalaenopsis aphrodite protocorms grown on 1/2-strength MS medium with sucrose. The expression of P. aphrodite MALATE SYNTHASE (PaMLS), involved in the glyoxylate cycle, was significantly decreased from 4 days after incubation (DAI) to 7 DAI. On real-time RT-PCR, both P. aphrodite ISOCITRATE LYASE (PaICL) and PaMLS were down-regulated during protocorm development and suppressed by sucrose treatment. In addition, several genes encoding transcription factors regulating PaMLS expression were identified. A gene encoding homeobox transcription factor (named PaHB5) was involved in positive regulation of PaMLS. This study showed that sucrose regulates the glyoxylate cycle during orchid protocorm development in asymbiotic germination and provides new insights into the transcription factors involved in the regulation of malate synthase expression.

Profiling of translatomes of in vivo-grown pollen tubes reveals genes with roles in micropylar guidance during pollination in Arabidopsis.

Transcriptome profiling has been used to identify genes expressed in pollen tubes elongating in vitro; however, little is known of the transcriptome of in vivo-grown pollen tubes due to the difficulty of collecting pollen that is elongating within the solid maternal gynoecium. Using a pollen-specific promoter (ProLAT52) to generate epitope-tagged polysomal-RNA complexes that could be affinity purified, we obtained mRNAs undergoing translation (the translatome) of in vivo-grown pollen tubes from self-pollinated gynoecia of Arabidopsis thaliana. Translatomes of pollen grains as well as in vivo- and in vitro-cultured pollen tubes were assayed by microarray analyses, revealing over 500 transcripts specifically enriched in in vivo-elongating pollen tubes. Functional analyses of several in vivo mutants (iv) of these pollination-enhanced transcripts revealed partial pollination/fertilization and seed formation defects in siliques (iv2, iv4, and iv6). Cytological observation confirmed the involvement of these genes in specialized processes including micropylar guidance (IV6 and IV4), pollen tube burst (IV2), and repulsion of multiple pollen tubes in embryo sac (IV2). In summary, the selective immunopurification of transcripts engaged with polysomes in pollen tubes within self-fertilized florets has identified a cohort of pollination-enriched transcripts that facilitated the identification of genes important in in vivo pollen tube biology.

Rice SIZ1, a SUMO E3 ligase, controls spikelet fertility through regulation of anther dehiscence.

• Sumoylation, a post-translational modification, has important functions in both animals and plants. However, the biological function of the SUMO E3 ligase, SIZ1, in rice (Oryza sativa) is still under investigation. • In this study, we employed two different genetic approaches, the use of siz1 T-DNA mutant and SIZ1-RNAi transgenic plants, to characterize the function of rice SIZ1. • Genetic results revealed the co-segregation of single T-DNA insertional recessive mutation with the observed phenotypes in siz1. In addition to showing reduced plant height, tiller number and seed set percentage, both the siz1 mutant and SIZ1-RNAi transgenic plants showed obvious defects in anther dehiscence, but not pollen viability. The anther indehiscence in siz1 was probably a result of defects in endothecium development before anthesis. Interestingly, rice orthologs of AtIRX and ZmMADS2, which are essential for endothecium development during anther dehiscence, were significantly down-regulated in siz1. Compared with the wild-type, the sumoylation profile of high-molecular-weight proteins in mature spikelets was reduced significantly in siz1 and the SIZ1-RNAi line with notably reduced SIZ1 expression. The nuclear localization signal located in the SIZ1 C-terminus was sufficient for its nuclear targeting in bombarded onion epidermis. • The results suggest the functional role of SIZ1, a SUMO E3 ligase, in regulating rice anther dehiscence.

PeMADS6, a GLOBOSA/PISTILLATA-like gene in Phalaenopsis equestris involved in petaloid formation, and correlated with flower longevity and ovary development.

In this study, we isolated and characterized the function of a GLOBOSA/PISTILLATA-like gene, PeMADS6, from a native Phalaenopsis species, P. equestris. Southern blot analysis showed PeMADS6 as a single copy in the Phalaenopsis genome. Results of the determination of temporal and spatial expression showed that PeMADS6 was expressed and thus participated in the development of the sepals, petals, labellum and column in Phalaenopsis. Further confirmation of the expression pattern of PeMADS6 was carried out with in situ hybridization. Repressed expression of PeMADS6 in the orchid ovary was found to be pollination regulated, which suggests that the gene may have an inhibitory effect on the development of the ovary or ovule. In addition, auxin acted as the candidate signal to regulate the repression of PeMADS6 expression in the ovary. Furthermore, the flowers of transgenic Arabidopsis plants ectopically overexpressing PeMADS6 showed the morphology of petaloid sepals, with a 3- to 4-fold increase in flower longevity. Concomitantly, delayed fruit maturation was also observed in the transgenic Arabidopsis, which is consistent with the inhibitory effect of PeMADS6 on the development of the ovary. Thus, as a B-function gene, PeMADS6, not only specifies floral organ identity but has functions in flower longevity and ovary development in orchids.

Four DEF-like MADS box genes displayed distinct floral morphogenetic roles in Phalaenopsis orchid.

The complex flower organization of orchids offers an opportunity to discover new variant genes and different levels of complexity in the morphogenesis of flowers. In this study, four B-class Phalaenopsis DEF-like MADS-box genes were identified and characterized, including PeMADS2, PeMADS3, PeMADS4 and PeMADS5. Differential expression profiles of these genes were detected in the floral organs of P. equestris, suggesting distinctive roles in the floral morphogenesis of orchids. Furthermore, expressions of these genes were varied to different extents in the peloric mutants with lip-like petals. Expression of PeMADS4 was in lips and columns of wild type, and it extended to the lip-like petals in the peloric mutant. Expression of PeMADS5 was mainly in petals and to a lesser extent in columns in the wild type, whereas it was completely eliminated in the peloric mutant. Disruption of the PeMADS5 promoter region of the peloric mutant was detected at nucleotide +312 relative to the upstream of translational start codon, suggesting that a DNA rearrangement has occurred in the peloric mutant. Genomic structure analysis of the PeMADS5 showed that the exon length was conserved in exons 1-6, similar to DEF-like genes of other plants. Collectively, this is the first report that four DEF-like MADS genes were identified in a single monocotyledonous species and that they may play distinctive morphogenetic roles in the floral development of an orchid.