Plant Biotechnology最新文献

Accumulation of anthocyanin provides pigmentation in plant tissues. In petunia, gene expression profiles that lead to anthocyanin production have been extensively characterized in terms of pigmentation in flower petals. Anthers are also pigmented, but the transcriptional control of the genes for anthocyanin biosynthesis in anthers has not been fully characterized. Here we addressed this issue by analyzing the expression of structural genes and genes encoding transcription factors (TFs) of the pathway. Ectopic expression of the PURPLE HAZE (PHZ) gene encoding an R2R3-MYB activator induced pigmentation in anthers. The pigmentation was accompanied by an increase in mRNA levels of AN1, MYB27 and MYBx among the genes encoding TFs. Among the structural genes, mRNA levels of four late biosynthetic genes (LBGs) were higher in the transformants than in the wild type. Analyses of gene expression profile using commercial varieties indicated that mRNA levels of MYB27, MYBx and LBGs and of AN4, responsible for anther pigmentation, were higher in pigmented anthers than in nonpigmented. Differences in the gene expression profile between pigmented anthers induced by ectopic PHZ expression and their nonpigmented control and those between pigmented anthers and nonpigmented anthers of existing varieties were thus remarkably similar. These observations suggest that a high level of expression of the LBGs is characteristic of pigmented anthers and that ectopic PHZ expression in the an4 ^- genetic background induced changes in the transcriptional network toward the state established in pigmented anthers, which is intrinsically brought about by the function of AN4.

Plant cell wall plays important roles in the regulation of plant growth/development and affects the quality of plant-derived food and industrial materials. On the other hand, genetic variability of cell wall structure within a plant species has not been well understood. Here we show that the endosperm cell walls, including both starchy endosperm and aleurone layer, of rice grains with various genetic backgrounds are clearly classified into two groups depending on the presence/absence of β-1,4-linked glucomannan. All-or-none distribution of the glucomannan accumulation among rice varieties is very different from the varietal differences of arabinoxylan content in wheat and barley, which showed continuous distributions. Immunoelectron microscopic observation suggested that the glucomannan was synthesized in the early stage of endosperm development, but the synthesis was down-regulated during the secondary thickening process associated with the differentiation of aleurone layer. Significant amount of glucomannan in the cell walls of the glucomannan-positive varieties, i.e., 10% or more of the starchy endosperm cell walls, and its close association with the cellulose microfibril suggested possible effects on the physicochemical/biochemical properties of these cell walls. Comparative genomic analysis indicated the presence of striking differences between OsCslA12 genes of glucomannan-positive and negative rice varieties, Kitaake and Nipponbare, which seems to explain the all-or-none glucomannan cell wall trait in the rice varieties. Identification of the gene responsible for the glucomannan accumulation could lead the way to clarify the effect of the accumulation of glucomannan on the agronomic traits of rice by using genetic approaches.

In early developing tomato (Solanum lycopersicum L.) fruit, starch accumulates at high levels and is used by various primary metabolites in ripening fruits. ADP-glucose pyrophosphorylase is responsible for the first key step of starch biosynthesis. Although it has been reported that AgpL1 and AgpS1 isoforms are mainly expressed in early developing fruit, their regulatory mechanism has not been elucidated. The present study investigated the transcriptional response of AgpL1 and AgpS1 to various metabolizable sugars, nonmetabolizable sugar analogues, hexokinase inhibitors and proline by an experimental system using half-cut fruits. AgpL1 was upregulated in response to sucrose and constituted hexoses such glucose, whereas the AgpS1 gene almost did not exhibit a prominent sugar response. Further analyses revealed that other disaccharides such maltose and trehalose did not show a remarkable effect on both AgpL1 and AgpS1 expressions. These results indicate that there are two distinct regulatory mechanisms, namely, sugar metabolism-dependent and -independent, for the regulation of AGPase gene expression. Interestingly, the ADP treatment, a hexokinase inhibitors, cancelled the sugar response of AgpL1, indicating that hexokinase-mediated sugar signaling should be involved in the sugar response of AgpL1. These results suggest that sugar-dependent (AgpL1) and sugar-independent (AgpS1) pathways coordinatively regulate starch biosynthesis in immature tomato fruit.

Turfgrasses show a wide range of salinity tolerance. In this study, twenty wild turfgrasses were collected from coastal regions in Japan, and their species; evolutionary lineage; salt tolerance levels; shoot and root K⁺, Na⁺, and proline contents; and amounts of ions secreted from their salt glands were determined. Among them, eighteen turfgrass species were determined based on the internal transcribed spacer 1 sequences. All collected wild turfgrasses were identified as halophytes and were divided into two salt-tolerant levels. They maintained the shoot relative water contents and suppressed excess Na⁺ accumulation in their shoots and roots and K⁺ content homeostasis compared with rice, resulting in the maintenance of a higher K⁺/Na⁺ ratio under salt stress. These characteristics must be part of the salt tolerance mechanisms. Among the four turfgrasses with salt glands, three selectively secreted Na⁺ from their salt glands; however, interestingly, one secreted K⁺ over Na⁺, although it still maintained a K⁺/Na⁺ ratio comparable to that of the other turfgrasses. A significant amount of proline synthesis was observed in most of the turfgrasses in response to salt stress, and the proline content was highly correlated with the salt tolerance, suggesting its key role in the salt tolerance mechanisms. These wild turfgrasses with such diverse ion control mechanisms and proline synthesis profiles are useful materials for investigating the salt tolerant mechanisms and breeding salt tolerant turfgrasses.

The biosynthetic pathway of Catharanthus roseus vinca alkaloids has a long research history, including not only identification of metabolic intermediates but also the mechanisms of inter-cellular transport and accumulation of biosynthesized components. Vinca alkaloids pathway begins with strictosidine, which is biosynthesized by condensing tryptamine from the tryptophan pathway and secologanin from the isoprenoid pathway. Therefore, increasing the supply of precursor tryptophan may enhance vinca alkaloid content or their metabolic intermediates. Many reports on the genetic modification of C. roseus use cultured cells or hairy roots, but few reports cover the production of transgenic plants. In this study, we first investigated a method for stably producing transgenic plants of C. roseus, then, using this technique, we modified the tryptophan metabolism system to produce transgenic plants with increased tryptophan content. Transformed plants were obtained by infecting cotyledons two weeks after sowing with Agrobacterium strain A13 containing a plant expression vector, then selecting with 1/2 B5 medium supplemented with 50 mg l^-1 kanamycin and 20 mg l^-1 meropenem. Sixty-eight regenerated plants were obtained from 4,200 cotyledons infected with Agrobacterium, after which genomic PCR analysis using NPTII-specific primers confirmed gene presence in 24 plants with a transformation rate of 0.6%. Furthermore, we performed transformation into C. roseus using an expression vector to join trpE8 and aroG4 genes, which are feedback-resistant mutant genes derived from Escherichia coli. The resulting transformed plants showed exactly the same morphology as the wild-type, albeit with a marked increase in tryptophan and alkaloids content, especially catharanthine in leaves.

Agrobacterium tumefaciens (Rhizobium radiobacter) is used for the transient expression of foreign genes by the agroinfiltration method, but the introduction of foreign genes often induces transcriptional and/or post-transcriptional gene silencing (TGS and/or PTGS). In this study, we characterized the structural features of T-DNA that induce TGS during agroinfiltration. When A. tumefaciens cells harboring an empty T-DNA plasmid containing the cauliflower mosaic virus (CaMV) 35S promoter were infiltrated into the leaves of Nicotiana benthamiana line 16c with a GFP gene over-expressed under the control of the same promoter, no small interfering RNAs (siRNAs) were derived from the GFP sequence. However, siRNAs derived from the CaMV 35S promoter were detected, indicating that TGS against the GFP gene was induced. When the GFP gene was inserted into the T-DNA plasmid, PTGS against the GFP gene was induced whereas TGS against the CaMV 35S promoter was suppressed. We also showed the importance of terminator sequences in T-DNA for gene silencing. Therefore, depending on the combination of promoter, terminator and coding sequences on T-DNA and the host nuclear genome, either or both TGS and/or PTGS could be induced by agroinfiltration. Furthermore, we showed the possible involvement of three siRNA-producing Dicers (DCL2, DCL3 and DCL4) in the induction of TGS by the co-agroinfiltration method. Especially, DCL2 was probably the most important among them in the initial step of TGS induction. These results are valuable for controlling gene expression by agroinfiltration.

Correct flower organ formation at the right timing is one of the most important strategies for plants to achieve reproductive success. Ectopic overexpression of LATE FLOWERING (LATE) is known to induce late flowering, partly through suppressing expression of the florigen-encoding gene FLOWERING LOCUS T (FT) in Arabidopsis. LATE is one of the C2H2 zinc finger transcription factors, and it has a canonical transcriptional repression domain called the ethylene-responsive element-binding factor-associated amphiphilic repression (EAR) motif at the end of its C terminus. Therefore, LATE is considered a transcriptional repressor, but its molecular function remains unclear. Our genome-edited late mutants exhibited no distinct phenotype, even in flowering, indicating the presence of redundancy from other factors. To reveal the molecular function of LATE and factors working with it, we investigated its transcriptional activity and interactions with other proteins. Transactivation activity assay showed that LATE possesses transcriptional repression ability, which appears to be attributable to both the EAR motif and other sequences. Yeast two-hybrid assay showed the EAR motif-mediated interaction of LATE with TOPLESS, a transcriptional corepressor. Moreover, LATE could also interact with CRABS CLAW (CRC), one of the most important regulators of floral meristem determinacy, through sequences in LATE other than the EAR motif. Our findings demonstrated the possibility that LATE can form a transcriptional repression complex with CRC for floral meristem determinacy.