Cadmium (Cd) accumulation in agricultural soils is an increasingly serious problem, as plants absorb Cd, which inhibits their growth and development. Nonetheless, the molecular mechanisms underlying Cd detoxification and accumulation in wheat (Triticum aestivum L.) are unclear. Here, we isolated the U-box E3 ligase TaPUB1 from wheat and reported the functional characterization of TaPUB1 in Cd uptake and tolerance in wheat. Under Cd stress, TaPUB1 overexpression lines displayed higher photosynthetic rates than the wild type; opposite results were observed in the TaPUB1-RNAi lines. In addition, TaPUB1 overexpression lines showed reduced Cd uptake and accumulation, whereas RNAi plants exhibited a significant increase in Cd accumulation after Cd treatment. We further found that TaPUB1 enhanced the resistance of wheat to Cd stress in three ways. First, TaPUB1 interacts with and ubiquitinates TaIRT1, resulting in the inhibition of Cd uptake. Second, TaPUB1 interacts directly with and ubiquitinates TaIAA17, facilitates its degradation, and results in primary root elongation by activating the Aux signaling pathway under Cd stress. Moreover, TaPUB1 decreases ROS accumulation by regulating antioxidant-related gene expression and antioxidant enzyme activity under Cd stress. Thus, a molecular mechanism by which TaPUB1 regulates Cd uptake and tolerance by modulating the stability of TaIRT1 and TaIAA17 proteins was revealed.

Abscisic acid (ABA) is an important regulator of plant growth, development, and biotic and abiotic stress responses. Ubiquitination plays important roles in regulating ABA signaling. E3 ligase, a key member in ubiquitination, actively participates in the regulation of biosynthesis, de-repression, and activation of ABA response and degradation of signaling components. In this study, we found that that overexpression of wheat E3 ligase TaPUB1 decreased the sensitivity of wheat seedlings to ABA, whereas TaPUB1-RNA interference (TaPUB1-RNAi) lines increased wheat sensitivity to ABA during germination, root growth, and stomatal opening. TaPUB1 influenced the expression of several ABA-responsive genes, and also interacted with TaPYL4 and TaABI5, which are involved in ABA signal transduction, and promoted their degradation. Additionally, we observed that TaPUB1-OE lines resulted in lower single-split grain numbers, larger seed size, and higher thousand kernel weight, when compared with the WT lines. Contrasting results were obtained for TaPUB1-RNAi lines. It suggests that TaPUB1 acts as a negative regulator in the ABA signaling pathway by interacting with TaPYL4 and TaABI5, subsequently affecting seed development in wheat. In addition, the enhanced abiotic tolerance of overexpression lines due to enhanced photosynthesis and root development may be related to the degradation of TaABI5 by TaPUB1.

Expansins loosen plant cell walls and are involved in cell enlargement and various abiotic stresses. In previous studies, we cloned the expansin gene TaEXPA2 from the wheat cultivar HF9703. Here, we studied its function and regulation in wheat drought stress tolerance. The results indicated that TaEXPA2-overexpressing wheat plants (OE) exhibited drought tolerant phenotypes, whereas down-regulation of TaEXPA2 by RNA interference (RNAi) resulted in elevated drought sensitivity, as measured by survival rate, photosynthetic rate and water containing ability under drought stress. Overexpression of TaEXPA2 enhanced the antioxidant capacity in wheat plants, via elevation of antioxidant enzyme activity and the increase of the transcripts of some ROS scavenging enzyme-related genes. Further investigation revealed that TaEXPA2 positively influenced lateral root formation under drought conditions. A MYB transcription factor of wheat named TaMPS activates TaEXPA2 expression directly by binding to its promoter. Overexpression of TaMPS in Arabidopsis conferred drought tolerance associated with improved lateral root number, and the close homolog genes of TaEXPA2 were up-regulated in Arabidopsis roots overexpressing TaMPS, which suggest that TaMPS may function as one of the regulator of TaEXPA2 gene expression in the root lateral development under drought stress. These findings suggest that TaEXPA2 positively regulates drought stress tolerance in wheat.

U-box E3 ubiquitin ligases play important roles in the ubiquitin/26S proteasome machinery and in abiotic stress responses. TaPUB1-overexpressing wheat (Triticum aestivum L.) were generated to evaluate its function in salt tolerance. These plants had more salt stress tolerance during seedling and flowering stages, whereas the TaPUB1-RNA interference (RNAi)-mediated knock-down transgenic wheat showed more salt stress sensitivity than the wild type (WT). TaPUB1 overexpression upregulated the expression of genes related to ion channels and increased the net root Na+ efflux, but decreased the net K+ efflux and H+ influx, thereby maintaining a low cytosolic Na+ /K+ ratio, compared with the WT. However, RNAi-mediated knock-down plants showed the opposite response to salt stress. TaPUB1 could induce the expression of some genes that improved the antioxidant capacity of plants under salt stress. TaPUB1 also interacted with TaMP (Triticum aestivum α-mannosidase protein), a regulator playing an important role in salt response in yeast and in plants. Thus, low cytosolic Na+ /K+ ratios and better antioxidant enzyme activities could be maintained in wheat with overexpression of TaPUB1 under salt stress. Therefore, we conclude that the U-box E3 ubiquitin ligase TaPUB1 positively regulates salt stress tolerance in wheat.

The phytohormone abscisic acid (ABA) regulates plant growth and development, as well as responses to various stresses, such as salt and drought. The wheat TaFBA1 gene, which encodes an F-box protein, was previously identified in our laboratory by homologous cloning. We previously found that TaFBA1 expression was induced by ABA and drought stress. In this study, wild-type (WT), TaFBA1 over-expressing (OEs), TaFBA1 homologous gene mutants, and TaFBA1 recovery (Rs) Arabidopsis plants were used. We found that the germination rate, the cotyledon greening rate, the root length, and the photosynthetic performance of TaFBA1 OE plants were better than those of WT under drought and ABA conditions, but mutant plants showed the opposite trend, and overexpression of TaFBA1 in mutants can recover their phenotype. In addition, TaFBA1 was found to be a negative regulator of ABA-induced stoma movement; mRNA transcription of certain ABA signaling-related genes was lower in TaFBA1 OE plants than in WT plants following ABA treatment. Further, we found that TaFBA1 can interact with RCAR1 (an ABA receptor) and ABI5. BiFC assay showed that TaFBA1 may interact with RCAR1 in the plasma membrane. In addition, accumulation of ROS and MDA in TaFBA1 OE plants was lower than that in the WT plants after ABA and drought treatments. Based on these results, we suggest that TaFBA1-regulated ABA insensitivity may be dependent on regulating ABA-mediated gene expression through interacting with RCAR1 and ABI5. Increased antioxidant competence and decreased ROS accumulation may be an important mechanism that underlies improved drought tolerance in TaFBA1 OE plants.

Two wheat near-isogenic lines (Nils; -GS (small grain size) and + GS (large grain size)) were obtained by backcrossing. We found that the grain volume and weight of + GS were significantly higher than those of -GS. There was a corresponding increase in grain yield associated with the + GS line. For analysis of causes, we investigated and compared the anatomic and physiological characteristics of two NILs during the grain filling stage. The bigger grain size in + GS likely resulted from the longer endosperm cells, which is related to the higher IAA content and expansin activity accompanied by the up-regulation of some expansin-related genes. For the higher grain weight of + GS, starch accumulation was higher in + GS than in -GS, which was consistent with relatively higher glucose (Glu), fructose (Fru), and sucrose (Suc) contents. Elevated sugar level may be correlated with sucrose synthase (SuSy) activity and up-regulation of GBSS I, SSS III, and SBE I. The amount of soluble protein in + GS grains also exceeded that in -GS grains, possibly because of increased NR, NiR, and GS activities and up-regulation of nitrogen assimilation and nitrate transporter genes in + GS grains. Furthermore, the photosynthetic rate was higher in + GS and -GS at the early filling stage before 21 DAA. The same trends were observed in sugar content (Suc, Fm, and Glu) and SPS activity. However, the increase in the rate of grain length and width in + GS plants at the late filling stage was significantly higher than that at the early filling periods. These suggest that high accumulation of dry matter (such as starch and protein) at early filling stage was the important factor in achieving the final increased size of wheat grains, besides cell expansion.

Expansins play an important role in plant stress tolerance. In a previous study, we cloned the wheat expansin gene TaEXPA2. Here, we analyze its involvement in oxidative stress tolerance. First, we observed that the expression of TaEXPA2 in wheat seedlings was upregulated during H2O2 stress. Then, we assembled a TaEXPA2 gene expression vector, transformed it to Arabidopsis, and obtained transgenic plants overexpressing TaEXPA2 (labeled OE). When exposed to H2O2, both OE and wild-type (Col) plants were damaged by oxidative stress, as indicated by decolored leaves and increased malondialdehyde (MDA) content. Damage in OE plants was less severe than in Col plants (WT), and this was accompanied by higher activity of cell wall peroxidase (POD) enzymes, including soluble POD, ionically bound POD, and covalently bound POD. The expansin activities of the OE plants were also higher than WT under oxidative stress. We further obtained the Arabidopsis mutant atexpa2 (AtEXPA2 is homologous to TaEXPA2), and found that the antioxidant ability of atexpa2 was lower than that in Col plants, accompanied by depressed activity of POD enzymes and expansins in cell walls. We transformed wheat TaEXPA2 to atexpa2 and obtained plants (labeled Rs) capable of recovering the antioxidant capacity. Oxidative stress tolerance in Rs plants was higher than that of Col plants, and the Rs plants also had higher levels of cell wall POD enzyme and expansin activity. Finally, we identified 13 POD genes in Arabidopsis thaliana and analyzed their expression patterns using quantitative real-time PCR. The expression of 4 of these genes (AtPOD31, AtPOD33, AtPOD34 and AtPOD71) was significantly upregulated during exposure to H2O2. We speculate that the 4 genes upregulated by H2O2 treatment are involved in the increased activity of POD in the cell wall. We suggest that TaEXPA2 may regulate antioxidant capacity in plants by regulating the activity of cell wall peroxidase.

A series of methacrylamide-based C16 monolithic columns were prepared and characterized to determine how their porous structural properties and chromatographic behavior are affected by the percentages of functional monomer, base monomer, and cross-linker in the polymerization solution. Baseline separation of 6 neutral compounds can be readily obtained in an optimized column. Furthermore, the effects of organic additive in the mobile phase, operating voltages, and temperature on retention behaviors and separation efficiencies were also studied. The separation mechanism is also discussed. High column efficiency and good reproducibility indicate that the monolithic columns hold considerable promise.

trans-Resveratrol and cis-resveratrol were analyzed using pressurized-capillary electrochromatography (CEC). The effects of illumination by sunlight to the samples were investigated. The concentration of resveratrol in wines was determined with qualitative and quantitative analysis of resveratrol. As a technique with high efficiency and high resolution, CEC proved to be a viable alternative for the quantitative analysis of wines.

A novel monolithic stationary phase having long alkyl chain ligands was introduced and evaluated in pressurized-capillary electrochromatography of small neutral and charged compounds. The monolithic column was prepared by the in situ copolymerization of ethylene dimethacrylate, 1-hexadecene, allyl alcohol and 2-acrylamido-2-methyl-1-propanesulfonic acid in a quaternary porogenic solvent mixture consisting of 1,4-butanediol, cyclohexanol, dodecanol and water.

A novel monolithic stationary phase with long alkyl chain ligands was introduced and evaluated in pressurized capillary electrochromatography (p-CEC) by using a mixture of neutral and charged compounds. The capillary monolithic-column was prepared by in situ copolymerization of ethylene dimethacrylate, alkyl alcohol and 2-acrylamido-2-methyl-1-propanesulfonic acid in a quadruple porogenic solvent consisting of 1, 4-butanediol/cyclohexanol/docecanol/water. Effects of experimental parameters in p-CEC, such as pressure and electrical field strength, on the separation were investigated. The baseline separation of the components in the mixture was achieved. The monolithic column can be used as an alternative to the conventional capillary column such as the reversed phase ODS.