Dekuan Li

UDP-Glucosyltransferase71C5, a Major Glucosyltransferase, Mediates Abscisic Acid Homeostasis in Arabidopsis

A unique UDP-glucosyltransferase plays an important role in ABA homeostasis by glucosylating ABA to ABA-glucose ester. Abscisic acid (ABA) plays a key role in plant growth and development. The effect of ABA in plants mainly depends on its concentration, which is determined by a balance between biosynthesis and catabolism of ABA. In this study, we characterize a unique UDP-glucosyltransferase (UGT), UGT71C5, which plays an important role in ABA homeostasis by glucosylating ABA to abscisic acid-glucose ester (GE) in Arabidopsis (Arabidopsis thaliana). Biochemical analyses show that UGT71C5 glucosylates ABA in vitro and in vivo. Mutation of UGT71C5 and down-expression of UGT71C5 in Arabidopsis cause delay in seed germination and enhanced drought tolerance. In contrast, overexpression of UGT71C5 accelerates seed germination and reduces drought tolerance. Determination of the content of ABA and ABA-GE in Arabidopsis revealed that mutation in UGT71C5 and down-expression of UGT71C5 resulted in increased level of ABA and reduced level of ABA-GE, whereas overexpression of UGT71C5 resulted in reduced level of ABA and increased level of ABA-GE. Furthermore, altered levels of ABA in plants lead to changes in transcript abundance of ABA-responsive genes, correlating with the concentration of ABA regulated by UGT71C5 in Arabidopsis. Our work shows that UGT71C5 plays a major role in ABA glucosylation for ABA homeostasis.

Optimization of virus-induced gene silencing in pepper (Capsicum annuum L.).

Virus-induced gene silencing is currently a powerful tool for the study of gene function in plants. Here, we optimized the protocol for virus-induced gene silencing, and investigated factors that affect the efficiency of tobacco rattle virus-induced gene silencing in pepper plants. Consequently, an optimal protocol was obtained by the syringe-infiltration method in the leaves of pepper plants. The protocol involves 2-leaf stage plants, preparing the Agrobacterium inoculum at a final OD600 of 1.0 and then growing the inoculated plants at 22°C. Using this protocol, we achieved high efficiency in silencing CaPDS in different cultivars of pepper plants. We further used the CaPOD gene to illustrate the general reliability of this optimized protocol. Viral symptoms were observed on the leaves of inoculated plants of the Early Calwonder cultivar 25 days post-inoculation, indicating that this protocol can also be used to silence other genes in pepper plants. Real-time polymerase chain reaction analyses revealed that the expression levels of CaPDS and CaPOD were dramatically reduced in inoculated leaves compared to control plants. These results demonstrate that the optimized protocol can be applied to functional genomic studies in pepper to investigate genes involved in a wide range of biological processes.

The Arabidopsis F-box E3 ligase RIFP1 plays a negative role in abscisic acid signalling by facilitating ABA receptor RCAR3 degradation.

The phytohormone abscisic acid (ABA) plays a vital role in plant growth and development. The function of ABA is mediated by a group of newly discovered ABA receptors, named PYRABACTIN RESISTANCE 1/PYR-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORs (PYR1/PYLs/RCARs). Here, we report that an Arabidopsis thaliana F-box protein RCAR3 INTERACTING F-BOX PROTEIN 1 (RIFP1) interacts with ABA receptor (RCAR3) and SCF E3 ligase complex subunits Arabidopsis SKP1-LIKE PROTEINs (ASKs) in vitro and in vivo. The rifp1 mutant plants displayed increased ABA-mediated inhibition of seed germination and water loss of detached leaves, while the overexpression of RIFP1 in Arabidopsis led to plants being insensitive to ABA. Meanwhile, the rifp1 mutant plants showed greater tolerance to water deficit. In addition, the RCAR3 protein level was more stable in the rifp1 mutant plants than in the wild-type plants, indicating that RIFP1 facilitates the proteasome degradation of RCAR3. Accordingly, the loss of RIFP1 increased the transcript levels of several ABA-responsive genes. Taken together, these data indicate that RIFP1 plays a negative role in the RCAR3-mediated ABA signalling pathway and likely functions as an adaptor subunit of the SCF ubiquitin ligase complex to regulate ABA receptor RCAR3 stability.

Over-expression of ArathEULS3 confers ABA sensitivity and drought tolerance in Arabidopsis

Abscisic acid (ABA) is an important phytohormone involved in the regulation of plant growth, development and adaption to various environmental challenges. Regulatory component of ABA receptor 1 (RCAR1, also known as PYL9) acts as a newly discovered ABA receptor in Arabidopsis. To identify interacting partners of RCAR1, we have carried out a yeast two-hybrid screen. One protein was identified, ArathEULS3, which belongs to the Euonymus europaeus lectin (EUL) family of plant lectins. The interaction between RCAR1 and ArathEULS3 was confirmed by GST pull-down assay. Transient expression of RCAR1-EGFP and ArathEULS3-EGFP in Arabidopsis protoplasts revealed that both proteins were mainly expressed in cytoplasm and nucleus. Real time qRT-PCR analysis showed that over-expression of RCAR1 increased the expression of ArathEULS3. Furthermore, up-regulating ArathEULS3 in Arabidopsis conferred ABA hypersensitivity during post-germination growth and enhanced drought tolerance, but did not affect the expression of RD29B, RAB18 and RD29A (ABA- and drought-responsive genes). Previously, ArathEULS3 was shown as a carbohydrate-binding plant lectin. Thus, our results reveal a direct connection between abiotic stress responses and plant lectin.

Arabidopsis ABA Receptor RCAR1/PYL9 Interacts with an R2R3-Type MYB Transcription Factor, AtMYB44

Abscisic acid (ABA) signaling plays important roles in plant growth, development and adaptation to various stresses. RCAR1/PYL9 has been known as a cytoplasm and nuclear ABA receptor in Arabidopsis. To obtain further insight into the regulatory mechanism of RCAR1/PYL9, a yeast two-hybrid approach was performed to screen for RCAR1/PYL9-interacting proteins and an R2R3-type MYB transcription factor, AtMYB44, was identified. The interaction between RCAR1/PYL9 and AtMYB44 was further confirmed by glutathione S-transferase (GST) pull-down and bimolecular fluorescence complementation (BiFC) assays. Gene expression analysis showed that AtMYB44 negatively regulated the expression of ABA-responsive gene RAB18, in contrast to the opposite role reported for RCAR1/PYL9. Competitive GST pull-down assay and analysis of phosphatase activity demonstrated that AtMYB44 and ABI1 competed for binding to RCAR1/PYL9 and thereby reduced the inhibitory effect of RCAR1/PYL9 on ABI1 phosphatase activity in the presence of ABA in vitro. Furthermore, transient activation assay in protoplasts revealed AtMYB44 probably also decreased RCAR1/PYL9-mediated inhibition of ABI1 activity in vivo. Taken together, our work provides a reasonable molecular mechanism of AtMYB44 in ABA signaling.

Overexpression of FAD2 promotes seed germination and hypocotyl elongation in Brassica napus

The enzyme fatty acid desaturase 2 (FAD2) transforms oleic acid (C18:1) to linoleic acid (C18:2) in plants and as such is involved in fatty acid synthesis. It is also involved in plant development and self-defense, such as seed germination, leaf expansion and cold resistance. We have cloned the full coding region of the Brassica napusFAD2 gene and ectopically expressed it in B. napus expressing low levels of FAD2. Overexpression of FAD2 under the control of the CaMV 35S promoter resulted in an up-regulated FAD2 mRNA level in B. napus as expected. Further analysis revealed that the FAD2 transgenic lines varied greatly in terms of their physiological characteristics, such as enhanced seed germination and increased hypocotyl length, compared to non-transgenic plants, suggesting that up-regulated FAD2 can promote seed germination and hypocotyl elongation in B. napus. Our results demonstrate the possible roles of FAD2 in plant development and also provide a platform for further analysis of fatty acid synthesis in plants.

Genetic determinants of the defense response of resistant and susceptible pepper (Capsicum annuum) cultivars infected with Phytophthora capsici (Oomycetes; Pythiaceae)

Based on culture isolation and morphological observation blight-infected pepper plants in Shaanxi Province, China, we identified the pathogen causing pepper phytophthora blight as Phytophthora capsici. Varieties that differed in resistance (CM334, PBC602, and B27) were inoculated with this pathogen. The root activity of resistant CM334 variety was the highest while that of susceptible B27 variety was the lowest. Also, significant differences in the activity of POD, PAL, and β-1,3-glucanase were found; there was a positive correlation between disease resistance and activity of these three enzymes. We inhibited mycelial growth and sporangia formation of P. capsici using crude β-1,3-glucanase and PAL enzymes isolated from the resistant variety CM334 after it had been inoculated with P. capsici. These two enzymes had a synergistic effect on inhibition of P. capsici mycelial growth and sporangia formation. Expression of the defensive genes CaPO1, CaBGLU, CaBPR1, and CaRGA in the three varieties was higher in the leaves than in the roots. All three genes were upregulated in infected leaves and roots of the pepper plants, always expressing at higher levels in the resistant cultivar than in the susceptible cultivar, suggesting that the differences in resistance among the pepper genotypes involve differences in the timing and magnitude of the defense response.

AtRAE1 is involved in degradation of ABA receptor RCAR1 and negatively regulates ABA signalling in Arabidopsis

The phytohormone abscisic acid (ABA) plays an important role in regulating plant growth, development, and adaption to various environmental stresses. Regulatory components of ABA receptors (RCARs, also known as PYR/PYLs) sense ABA and initiate ABA signalling through inhibiting the activities of protein phosphatase 2C in Arabidopsis. However, the way in which ABA receptors are regulated is not well known. A DWD protein AtRAE1 (for RNA export factor 1 in Arabidopsis), which may act as a substrate receptor of CUL4-DDB1 E3 ligase, is an interacting partner of RCAR1/PYL9. The physical interaction between RCAR1 and AtRAE1 is confirmed in vitro and in vivo. Overexpression of AtRAE1 in Arabidopsis causes reduced sensitivity of plants to ABA, whereas suppression of AtRAE1 causes increased sensitivity to ABA. Analysis of protein stability demonstrates that RCAR1 is ubiquitinated and degraded in plant cells and AtRAE1 regulates the degradation speed of RCAR1. Our findings indicate that AtRAE1 likely participates in ABA signalling through regulating the degradation of ABA receptor RCAR1.

Overexpression of NaKR3 enhances salt tolerance in Arabidopsis.

Salinity is a major abiotic stress in agriculture. Here, we report that SODIUM POTASSIUM ROOT DEFECTIVE3 (NaKR3), which encodes a heavy metal-associated domain protein, is involved in salt tolerance in Arabidopsis. The results of quantitative reverse transcription-polymerase chain reaction analysis revealed that NaKR3 was induced by high salinity and osmotic stresses, but not by Cu(2+) stress. Transient expression of NaKR3-GFP in Arabidopsis protoplasts showed that the NaKR3 protein was localized in the cytosol. Transgenic Arabidopsis plants constitutively expressing NaKR3 under the control of the cauliflower mosaic virus 35S promoter exhibited increased tolerance to salt treatment. Furthermore, overexpression of NaKR3 increased the expression of SOS1 and SOS3, but decreased the accumulation of salt-induced proline. Taken together, our results indicate that NaKR3 is involved in the salt stress response in Arabidopsis.

Kill curve analysis and response of first generation Capsicum annuum L. B12 cultivar to ethyl methane sulfonate.

Pepper seeds (Capsicum annuum L.) var. B12 were mutagenized by four presoaking treatments in ten concentrations of ethyl methane sulfonate (EMS) to determine the sensitivity of the first generation (M1) to mutagens. The spectrum of mutations and induced variability for various quantitative traits, including germination, percent plant height, injury occurrence, survival ratio, first three fruits weight, and number of seeds per first fruit, were observed in the M1 generation. Our results indicated that all of the test parameters decreased with increasing EMS concentration, except for seedling injury. There were significant differences in germination ratio, LD50, plant height, percent injury, and survival ratio among the tested presoaking treatment. The LD50 was 1% EMS in seeds that were not presoaked (T1) and seeds presoaked for 12 h before treating with EMS (T3). In contrast, the LD50 was 0.5% EMS in seeds presoaked for 6 h (T2) and seeds presoaked in water for 6 h then incubated at 28°C for 12 h before EMS treatment (T4). Five dwarf plants were observed in mutagenized seeds without presoaking as compared to control seeds (at the maturity stage of the control plant).