Functional analyses of an E3 ligase gene AIP2 from wheat in Arabidopsis revealed its roles in seed germination and pre-harvest sprouting.

Pre-harvest sprouting (PHS) seriously affects wheat yield and quality of the grain. ABI3 is a key factor in the activation of seed development and repression of germination in Arabidopsis. An ABI3-interacting protein (AIP2) could polyubiquitinate ABI3, impair seed dormancy and promote seed germination in Arabidopsis. In this study, two wheat AIP2 genes, TaAIP2A and TaAIP2B, were isolated. Subcellular localization assay and yeast two-hybrid analysis revealed that TaAIP2A and TaAIP2B may function through interaction with wheat Viviporous-1 (TaVp1). The transcripts TaAIP2A and TaAIP2B were more abundant in wheat PHS susceptible cultivars than that of resistant ones, and decreased gradually following seed development. Expression of TaAIP2A and TaAIP2B in Arabidopsis aip2-1 mutant lines resulted in earlier flowering, promotion of seed germination, and reduced ABA sensitivity, respectively, somehow mimicking the phenotype of the wild type, with TaAIP2B having a stronger role in these aspects. Furthermore, the expression of upstream genes ABI1 and ABI2 were upregulated, whereas that of downstream genes ABI3 and ABI5 were downregulated in both TaAIP2A and TaAIP2B complemented lines upon ABA treatment. These results suggested that wheat AIP2s could negatively regulate the ABA signaling pathway and play important roles in seed germination, and thus wheat PHS resistance finally.

Identification of a Polygonum cuspidatum three-intron gene encoding a type III polyketide synthase producing both naringenin and p-hydroxybenzalacetone.

Benzalacetone synthase (BAS) is a member of the plant-specific type III PKS superfamily that catalyzes a one-step decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce p-hydroxybenzalacetone. In our recent work (Ma et al. in Planta 229(3):457-469, 2008), a three-intron type III PKS gene (PcPKS2) was isolated from Polygonum cuspidatum Sieb. et Zucc. Phylogenetic and functional analyses revealed this recombinant PcPKS2 to be a BAS. In this study, another three-intron type III PKS gene (PcPKS1) and its corresponding cDNA were isolated from P. cuspidatum. Sequence and phylogenetic analyses demonstrated that PcPKS1 is a chalcone sythase (CHS). However, functional and enzymatic analyses showed that recombinant PcPKS1 is a bifunctional enzyme with both, CHS and BAS activity. DNA gel blot analysis indicated that there are two to four CHS copies in the P. cuspidatum genome. RNA gel blot analysis revealed that PcPKS1 is highly expressed in the rhizomes and in young leaves, but not in the roots of the plant. PcPKS1 transcripts in leaves were inducible by pathogen infection and wounding. BAS is thought to play a crucial role in the construction of the C(6)-C(4) moiety found in a variety of phenylbutanoids, yet so far phenylbutanoids have not been isolated from P. cuspidatum. However, since PcPKS1 and PcPKS2 (Ma et al. in Planta 229(3):457-469, 2008) have been identified in P. cuspidatum, it is possible that such compounds are also produced in that plant, albeit in low concentrations.

Isolation and functional characterization of HvDREB1-a gene encoding a dehydration-responsive element binding protein in Hordeum vulgare.

A gene encoding Hordeum vulgare dehydration-responsive element binding protein 1 (HvDREB1), a member of the A-2 subgroup of the DREB subfamily, was isolated from barley seedlings. A subcellular localization assay revealed accumulation of HvDREB1 protein in the nucleus. As a trans-acting factor, HvDREB1 was able to bind to DRE/CRT elements and transactivate reporter gene expression in yeast cells. A study of various deletion mutants of HvDREB1 proteins indicated that the transactivation activity was localized to the N-terminal region. Expression of the HvDREB1 gene in barley leaves was significantly induced by salt, drought, and low-temperature. In contrast to most A-2 subgroup members in Arabidopsis thaliana, HvDREB1 also responded to exogenous ABA. Overexpression of HvDREB1 activated a downstream gene, RD29A, under normal growth conditions and led to increased tolerance to salt stress in Arabidopsis plants. These results suggest that HvDREB1 produces a DRE-/CRT-binding transcription factor that may have an important role in improving salt tolerance in plants.

Molecular cloning and overexpression of a novel UDP-glucosyltransferase elevating salidroside levels in Rhodiola sachalinensis.

Salidroside is a novel effective adaptogenic drug extracted from the medicinal plant Rhodiola sachalinensis A. Bor. Because this plant is a rare resource and has low yield, there is great interest in enhancing the production of salidroside. In this study, a putative UDP-glucosyltransferase (UGT) cDNA, UGT73B6, was isolated from Rhodiola sachalinensis using a rapid amplification of cDNA ends (RACE) method. The cDNA was 1,598 bp in length encoding 480 deduced amino acid residues with a conserved UDP-glucose-binding domain (PSPG box). Southern blot analysis of genomic DNA indicated that UGT73B6 existed as a single copy gene in the R. sachalinensis genome. Northern blot analysis revealed that transcripts of UGT73B6 were present in roots, calli and stems, but not in leaves. The UGT73B6 under 35S promoter with double-enhancer sequences from CaMV-Omega and TMV-Omega fragments was transferred into R. sachalinensis via Agrobacterium tumefaciens. PCR, PCR-Southern and Southern blot analyses confirmed that the UGT73B6 gene had been integrated into the genome of transgenic calli and plants. Northern blot analysis revealed that the UGT73B6 gene had been expressed at the transcriptional level. High performance liquid chromatography (HPLC) analysis indicated that the overexpression of the UGT73B6 gene resulted in an evident increase of salidroside content. These data suggest that the cloned UGT73B6 can regulate the conversion of tyrosol aglycon to salidroside in R. sachalinensis. This is the first cloned glucosyltransferase gene involved in salidroside biosynthesis.