Down-regulation of UDP-arabinopyranose mutase reduces the proportion of arabinofuranose present in rice cell walls.

Arabinoxylans may account for up to 25% of the mass of grass cell walls. The interactions of these polysaccharides with themselves and with cellulose and lignin is believed to affect the walls physical properties and increase the walls resistance to biochemical conversion to fermentable sugars. Arabinoxylans have a backbone composed of 1,4-linked ß-D-xylosyl residues, some of which are substituted at O-2 or O-3 with single arabinofuranosyl (Araf) residues. The Araf residues are likely transferred from UDP-Araf to the xylan backbone by arabinofuranosyltransferases. UDP-Araf is itself formed from UDP-arabinopyranose (UDP-Arap) by UDP-arabinopyranose mutase (UAM). In this study, RNA interference (RNAi) was used to suppress UAM expression in rice plants and thereby reduce the amounts of UDP-Araf available for cell wall synthesis. Several of the transgenic plants had reduced proportions of Araf in their walls together with a decrease in the extent of substitution of the xylan backbone, and a reduction of between 25% and 80% in ferulic acid and p-coumaric acid contents of the cell walls. Those transgenic plants with >25% reduction in the amounts of Araf were dwarfed and infertile.

Molecular characterization of FLOWERING LOCUS T-like genes of apple (Malus x domestica Borkh.).

The two FLOWERING LOCUS T (FT)-like genes of apple (Malus x domestica Borkh.), MdFT1 and MdFT2, have been isolated and characterized. MdFT1 and MdFT2 were mapped, respectively, on distinct linkage groups (LGs) with partial homoeology, LG 12 and LG 4. The expression pattern of MdFT1 and MdFT2 differed in that MdFT1 was expressed mainly in apical buds of fruit-bearing shoots in the adult phase, with little expression in the juvenile tissues, whereas MdFT2 was expressed mainly in reproductive organs, including flower buds and young fruit. On the other hand, both genes had the potential to induce early flowering since transgenic Arabidopsis, which ectopically expressed MdFT1 or MdFT2, flowered earlier than wild-type plants. Furthermore, overexpression of MdFT1 conferred precocious flowering in apple, with altered expression of other endogenous genes, such as MdMADS12. These results suggest that MdFT1 could function to promote flowering by altering the expression of those genes and that, at least, other genes may play an important role as well in the regulation of flowering in apple. The long juvenile period of fruit trees prevents early cropping and efficient breeding. Our findings will be useful information to unveil the molecular mechanism of flowering and to develop methods to shorten the juvenile period in various fruit trees, including apple.

Characterization of expressed sequence tags from a full-length enriched cDNA library of Cryptomeria japonica male strobili.

BACKGROUND: Cryptomeria japonica D. Don is one of the most commercially important conifers in Japan. However, the allergic disease caused by its pollen is a severe public health problem in Japan. Since large-scale analysis of expressed sequence tags (ESTs) in the male strobili of C. japonica should help us to clarify the overall expression of genes during the process of pollen development, we constructed a full-length enriched cDNA library that was derived from male strobili at various developmental stages. RESULTS: We obtained 36,011 expressed sequence tags (ESTs) from either one or both ends of 19,437 clones derived from the cDNA library of C. japonica male strobili at various developmental stages. The 19,437 cDNA clones corresponded to 10,463 transcripts. Approximately 80% of the transcripts resembled ESTs from Pinus and Picea, while approximately 75% had homologs in Arabidopsis. An analysis of homologies between ESTs from C. japonica male strobili and known pollen allergens in the Allergome Database revealed that products of 180 transcripts exhibited significant homology. Approximately 2% of the transcripts appeared to encode transcription factors. We identified twelve genes for MADS-box proteins among these transcription factors. The twelve MADS-box genes were classified as DEF/GLO/GGM13-, AG-, AGL6-, TM3- and TM8-like MIKCC genes and type I MADS-box genes. CONCLUSION: Our full-length enriched cDNA library derived from C. japonica male strobili provides information on expression of genes during the development of male reproductive organs. We provided potential allergens in C. japonica. We also provided new information about transcription factors including MADS-box genes expressed in male strobili of C. japonica. Large-scale gene discovery using full-length cDNAs is a valuable tool for studies of gymnosperm species.

The FLOWERING LOCUS T/TERMINAL FLOWER 1 family in Lombardy poplar.

Genes in the FLOWERING LOCUS T (FT) and TERMINAL FLOWER 1 (TFL1) family have been shown to be important in the control of the switch between vegetative and reproductive growth in several plant species. We isolated nine members of the FT/TFL1 family from Lombardy poplar (Populus nigra var. italica Koehne). Sequence analysis of the members of the FT/TFL1 family revealed considerable homology within their coding regions both among family members and to the members of the same family in Arabidopsis, tomato and grapevine. Moreover, members of this family in all four species examined display a common exon-intron organization. Phylogenetic analysis revealed that the genes fall into four different clades: two into the TFL1 clade; five into the FT clade; and one each into the MOTHER OF FT AND TFL1 and BROTHER OF FT AND TFL1 clades. One gene in the TFL1 clade, PnTFL1, is expressed in vegetative meristems, and transgenic Arabidopsis that ectopically expressed PnTFL1 had a late-flowering phenotype. The expression patterns of two genes in the FT clade, PnFT1 and PnFT2, suggested a role for them in the promotion of flowering, and transgenic Arabidopsis that ectopically expressed either PnFT1 or PnFT2 had an early-flowering phenotype.

Functional annotation of 19,841 Populus nigra full-length enriched cDNA clones.

BACKGROUND: Populus is one of favorable model plants because of its small genome. Structural genomics of Populus has reached a breakpoint as nucleotides of the entire genome have been determined. Reaching the post genome era, functional genomics of Populus is getting more important for well-comprehended plant science. Development of bioresorce serving functional genomics is making rapid progress. Huge efforts have achieved deposits of expressed sequence tags (ESTs) in various plant species consequently accelerating functional analysis of genes. ESTs from full-length cDNA clones are especially powerful for accurate molecular annotation. We promoted collection and annotation of the ESTs from Populus full-length enriched cDNA clones as part of functional genomics of tree species. RESULTS: We have been collecting the full-length enriched cDNA of the female poplar (Populus nigra var. italica) for years. By sequencing P. nigra full-length (PnFL) cDNA libraries, we generated about 116,000 5'-end or 3'-end ESTs corresponding to 19,841 nonredundant PnFL clones. Population of PnFL cDNA clones represents 44% of the predicted genes in the Populus genome. CONCLUSION: Our resource of P. nigra full-length enriched clones is expected to provide valuable tools to gain further insight into genome annotation and functional genomics in Populus.

Expression of the autofluorescent protein, DsRed2, in the recombinants of the ectomycorrhizal basidiomycete, Suillus grevillei, generated by Agrobacterium-mediated transformation.

Recombinants were generated from the ectomycorrhizal basidiomycete, Suillus grevillei, through agroinfection using a binary vector carrying the hygromycin B resistance and the autofluorescent protein, DsRed2, markers. DsRed2 was driven by a cis-regulatory region of the glyceraldeyde-3-phosphate dehydrogenase gene (gpd) from the wood-rotting basidiomycete, Coriolus hirsutus, which contains promoters and 5' gpd sequences with first through fourth exons and expressed for the first time in Suillus spp. The transformation system and recombinants expressing an autofluorescent protein may be useful in genetic analysis of the symbiosis.

Experimental determination of proline hydroxylation and hydroxyproline arabinogalactosylation motifs in secretory proteins.

Many secretory and several vacuolar proteins in higher plants contain hydroxylated proline residues. In many cases, hydroxyprolines in proteins are glycosylated with either arabinogalactan or oligoarabinose. We have previously shown that a sporamin precursor is O-glycosylated at the hydroxylated proline 36 residue with an arabinogalactan-type glycan when this protein is expressed in tobacco BY-2 cells (Matsuoka et al., 1995). Taking advantage of the fact that this is the only site of proline hydroxylation and glycosylation in sporamin, we analyzed the amino acid requirement for proline hydroxylation and arabinogalactosylation. We expressed several deletion constructs and many amino acid substitution mutants in tobacco cells and analyzed glycosylation and proline hydroxylation of the expressed sporamins. Hydroxylation of a proline residue requires the five amino acid sequence [AVSTG]-Pro-[AVSTGA]-[GAVPSTC]-[APS or acidic] (where Pro is the modification site) and glycosylation of hydroxyproline (Hyp) requires the seven amino acid sequence [not basic]-[not T]-[neither P, T, nor amide]-Hyp-[neither amide nor P]-[not amide]-[APST], although charged amino acids at the -2 position and basic amide residues at the +1 position relative to the modification site seem to inhibit the elongation of the arabinogalactan side chain. Based on the combination of these two requirements, we concluded that the sequence motif for efficient arabinogalactosylation, including the elongation of the glycan side chain, is [not basic]-[not T]-[AVSG]-Pro-[AVST]-[GAVPSTC]-[APS].

Characterization of full-length enriched expressed sequence tags of stress-treated poplar leaves.

Poplar, whose genome is the first to be sequenced among woody plants, is a favorable model for plant biologists to enable them to understand molecular processes of growth, development and responses to environmental stimuli in trees. The sequence will allow the development of a strategy for improving environmental stress tolerance in forest trees. In this study, we have generated a full-length enriched cDNA library from leaves of axenically grown poplar (Populus nigra var. italica) subjected to environmental stress treatments by dehydration, high salinity, chilling, heat, abscisic acid (ABA) and H2O2. We sequenced >30,000 expressed sequence tags (ESTs) from the cDNA library and consequently collected approximately 4,500 non-redundant clones. We further analyzed cDNAs encoding an ERF/AP2-domain transcription factor which is specific in plants and plays an important role under stress. Thirteen candidates containing the ERF/AP2 domain were found within our EST resource. Some of them showed stress-responsive gene expression. We report here the first collection of full-length enriched stress-related ESTs of poplar and discuss environmental stress responses of forest trees in the light of comparative genomics.

Phytosulfokine stimulates somatic embryogenesis in Cryptomeria japonica.

Phytosulfokine (PSK), which has been identified as a plant growth factor, had a dramatic stimulatory effect on the formation of somatic embryos of sugi (Cryptomeria japonica) in the presence of polyethylene glycol. The resultant somatic embryos germinated with synchronous sprouting of cotyledons, hypocotyls and roots, and most of the seedlings grew normally. A cDNA clone for the precursor to the PSK peptide of C. japonica was identified in an expressed sequence tags database. Our results support the existence of a PSK signaling pathway in C. japonica.