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Abscisic acid (ABA) transport plays an important role in systemic plant responses to environmental factors. However, it remains largely unclear about the precise regulation of ABA transporters in plants. In this study, we show that the C-terminally encoded peptide receptor 2 (CEPR2) directly interacts with the ABA transporter NRT1.2/NPF4.6. Genetic and phenotypic analyses revealed that NRT1.2/NPF4.6 positively regulates ABA response and that NRT1.2/NPF4.6 is epistatically and negatively regulated by CEPR2. Further biochemical assays demonstrated that CEPR2 phosphorylates NRT1.2/NPF4.6 at serine 292 to promote its degradation under normal conditions. However, ABA treatment and non-phosphorylation at serine 292 prevented the degradation of NRT1.2/NPF4.6, indicating that ABA inhibits the phosphorylation of this residue. Transport assays in yeast and Xenopus oocytes revealed that non-phosphorylated NRT1.2/NPF4.6 had high levels of ABA import activity, whereas phosphorylated NRT1.2/NPF4.6 did not import ABA. Analyses of complemented nrt1.2 mutants that mimicked non-phosphorylated and phosphorylated NRT1.2/NPF4.6 confirmed that non-phosphorylated NRT1.2S292A had high stability and ABA import activity in planta. Additional experiments showed that NRT1.2/NPF4.6 was degraded via the 26S proteasome and vacuolar degradation pathways. Furthermore, we found that three E2 ubiquitin-conjugating enzymes, UBC32, UBC33, and UBC34, interact with NRT1.2/NPF4.6 in the endoplasmic reticulum and mediate its ubiquitination. NRT1.2/NPF4.6 is epistatically and negatively regulated by UBC32, UBC33, and UBC34 in planta. Taken together, these results suggest that the stability and ABA import activity of NRT1.2/NPF4.6 are precisely regulated by its phosphorylation and degradation in response to environmental stress.

Numerous studies have demonstrated the function of salinity or jasmonic acid (JA) in plant growth and senescence. This study evaluated how the combination of salinity and methyl jasmonate (MeJA) (SaM) worked as a novel stress and then regulated plant growth in Arabidopsis. Firstly, we found that compared with MeJA or NaCl treatment alone, SaM would significantly intensified plant growth inhibition and senescence in wild-type (WT) seedlings, and these phenotypes could be partially compromised after SaM stress in JA-insensitive mutants. Meanwhile, genes involved in JA signaling and Senescence Associated Gene 13 (SAG13) were dramatically increased by SaM stress than that by MeJA or NaCl alone in WT. Moreover, a group of secondary metabolite - indolic glucosinolates (IGs) showed obvious over-accumulation after SaM treatment than that after each single one in WT, and the seedlings treated with IGs' metabolites performed similar inhibited growth and chlorotic leaves phenotypes compared with those caused by SaM stress. All these indicated the toxicity of IGs and their metabolites would prevent the growth progress of plants. Therefore, we concluded that SaM worked as a novel stress and intensified plant growth inhibition and senescence, which was dependent on JA-dependent and -independent signaling pathways.

Mitogen-activated protein kinase (MAPK) cascades are conserved and vital signaling components in the responses to various ambient stresses. Here, we report the identification of MAPKKK18, a drought resistance associated MAPK kinase kinase in Arabidopsis. The mapkkk18 knockout mutants displayed hypersensitivity to drought stress, whereas overaccumulation of MAPKKK18 in transgenic Arabidopsis plants significantly enhanced the resistance to drought. Expression pattern analysis revealed that the inducible expression of MAPKKK18 by osmotic stress was ABA and the canonical ABA signaling pathway dependent. Furthermore, MAPKKK18 mainly exerted its regulatory roles via downstream MAPKK3. These findings uncovered important roles for MAPKKK18 in drought resistance and expanded our understanding of the MAPK pathways in modulating abiotic stress responses.

MicroRNAs (miRNAs) have emerged as a class of regulators of gene expression through posttranscriptional degradation or translational repression in living cells. Increasing evidence points to the important relationship between miRNAs and environmental stress responses, but the regulatory mechanisms in plants are poorly understood. Here, we found that Arabidopsis thaliana intronic miR400 was cotranscribed with its host gene (At1g32583) and downregulated by heat treatment. Intriguingly, an alternative splicing (AS) event that occurred in the intron (306 bp) where MIR400 was located was specifically induced by heat stress. A 100 bp fragment was excised, and the remaining 206 bp intron containing MIR400 transcripts was retained in the host gene. The stress-induced AS event thus resulted in greater accumulation of miR400 primary transcripts and a low level of mature miR400. Together, these results provide the direct evidence that AS acts as a regulatory mechanism linking miRNAs and environmental stress in plants.

Following an initial clean-up step on silica column, high-speed counter-current chromatography (HSCCC) was used to purify gingerols from an extract of the dried rhizome of Zingiber officinale. The sample was separated with petroleum ether–ethyl acetate–methanol–water (1:0.2:0.5:0.7, v/v) and petroleum ether–ethyl acetate–methanol–water (1:0.2:0.7:0.5, v/v) in a stepwise elution and yielded 132 mg of 6-gingerol, 31 mg of 8-gingerol and 61 mg of 10-gingerol from 360 mg of pre-purified sample. The purity of each compound was over 98% as determined by HPLC. The structures of the three compounds have been identified by LC-ESI-MS, 1 H NMR and 13 C NMR.

The mir169 family is a large and conserved miRNA family in many plant species. The Arabidopsis genome contains 14 members of the mir169 family, including three tandem clusters that were transcribed as polycistrons. To investigate the regulation of mir169 expression, all the 11 promoter sequences were cloned and fused to GUS and introduced into Arabidopsis to generate pMir169:GUS transgenic plants. Histochemical GUS staining and activity assay in different tissues and developmental stages of the transgenic plants revealed unique and overlapping as well as spatio-temporal expression patterns among the family members. The endogenous transcript level of each member detected by semi-quantitative RT-PCR is largely accordant with the GUS expression profiles. Interestingly, the tandem arrayed gene pairs mir169i/j, mir169k/l and mir169m/n show greatly overlapping expression patterns. Further comparative analysis of their upstream sequences revealed significant sequence similarities, indicating that the three gene pairs may result from a duplication event. Additionally, when the transgenic plants were exposed to various abiotic stresses, a subset of family members were observed to be regulated by certain treatments, especially ABA and heat. Our results provide valuable information for functional analysis of the Arabidopsis mir169 family members as ubiquitous regulators in both developmental processes and stimuli pathways.

Helicobacter pylori (H. pylori) is established as the etiologic agent of chronic active gastritis, peptic ulcer, gastric cancer and mucosa-associated lymphoid tissue lymphoma. The development of a vaccine against H. pylori has become a priority to prevent and cure H. pylori infection. The UreB (urease B) subunit is the most effective and common immunogen of all strains of H. pylori and may stimulate the immunoresponse protecting the human body against the challenge of H. pylori. To date no report has documented an edible carrot vaccine against H. pylori. We transformed the gene of UreB into carrot by Agrobacterium-mediated transformation and the regenerated carrot plants demonstrated that the expressed UreB protein accounted for 25mug/g roots and was effective to induce immune response in mice. These results suggest that the UreB transgenic carrot can be potentially used as an edible vaccine for controlling H. pylori.

Four flavonoids, apigenin-7-O-neohesperidoside, luteolin-7-O-glucoside, apigenin-7-O-glucoside and kaempferol-7-O-glucoside have been isolated and purified for the first time from the flowers of Paeonia suffruticosa by high-speed counter-current chromatography with a two-phase solvent system composed of ethyl acetate-ethanol-acetic acid-water (4:1:0.25:5, v/v). Then, 5 mg apigenin-7-O-neohesperidoside, 4 mg luteolin-7-O-glucoside, 9 mg apigenin-7-O-glucoside and 2.5 mg kaempferol-7-O-glucoside could be obtained after injecting 40 mg sample and their purities were 94, 97, 97 and 96%, respectively. All these constituents were identified by mass spectrometry and nuclear magnetic resonance.

Psoralen and isopsoralen were extracted from Fructus Psoraleae (Psoralea corylitolia L.) by supercritical CO2. The effect of various parameters, i.e., pressure, temperature and sample particle size on yield was investigated with an analytical-scale supercritical fluid extraction (SFE) system to find the optimal conditions. The process was then scaled up by 50 times with a preparative SFE system under the optimized conditions of pressure (26 MPa), temperature (60 degrees C) and a sample particle size of 40-60 mesh. The yield of the preparative SFE was 9.1% and the combined yield of psoralen and isopsoralen was 2.5 mg/g of dry seeds. Psoralen and isopsoralen in the extract were separated and purified by high-speed counter-current chromatography with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:0.7:1:0.8, v/v), and the fractions were analyzed by HPLC, MS, 1HNMR and 13C NMR. The structures of the products were further confirmed by comparison with authentic samples (National Institute of the Control of Pharmaceutical and Biological Products, Beijing, China).

High-speed counter-current chromatography was used to isolate and purify honokiol and magnolol from cortex Magnoliae Officinalis ( Magnolia officinalis Rehd. et Wils.), a plant used in the traditional Chinese medicine. A crude sample, 150 mg, was successfully separated with a two-phase solvent system composed of n -hexane–ethyl acetate–methanol–water (1:0.4:1:0.4, v/v), and the fractions were analyzed by high-performance liquid chromatography. The separation produced 80 and 45 mg of honokiol and magnolol with purities of 99.2 and 98.2%, respectively, in 2.5 h.