AtEAU1 and AtEAU2, Two EAR Motif-Containing ABA Up-Regulated Novel Transcription Repressors Regulate ABA Response in Arabidopsis.

EAR (Ethylene-responsive element binding factor-associated Amphiphilic Repression) motif-containing transcription repressors have been shown to regulate plant growth and development, and plant responses to plant hormones and environmental stresses including biotic and abiotic stresses. However, the functions of most EAR-motif-containing proteins remain largely uncharacterized. The plant hormone abscisic acid (ABA) also plays important roles in regulating plant responses to abiotic stresses via activation/repression of ABA-responsive genes. We report here the identification and functional characterization of two ABA-responsive EAR motif-containing protein genes, AtEAU1 (Arabidopsis thaliana EAR motif-containing ABAUp-regulated 1) and AtEAU2. Quantitative RT-PCR results show that the expressions of AtEAU1 and AtEAU2 were increased by ABA treatment, and were decreased in the ABA biosynthesis mutant aba1-5. Assays in transfected Arabidopsis protoplasts show that both AtEAU1 and AtEAU2 were specifically localized in the nucleus, and when recruited to the promoter region of the reporter gene by a fused DNA binding domain, repressed reporter gene expression. By using T-DNA insertion mutants and a gene-edited transgene-free mutant generated by CRISPR/Cas9 gene editing, we performed ABA sensitivity assays, and found that ABA sensitivity in the both ateau1 and ateau2 single mutants was increased in seedling greening assays. ABA sensitivity in the ateau1 ateau2 double mutants was also increased, but was largely similar to the ateau1 single mutants. On the other hand, all the mutants showed a wild type response to ABA in root elongation assays. Quantitative RT-PCR results show that the expression level of PYL4, an ABA receptor gene was increased, whereas that of ABI2, a PP2C gene was decreased in the ateau1 and ateau1 single, and the ateau1 ateau2 double mutants. In summary, our results suggest that AtEAU1 and AtEAU2 are ABA-response genes, and AtEAU1 and AtEAU2 are novel EAR motif-containing transcription repressors that negatively regulate ABA responses in Arabidopsis, likely by regulating the expression of some ABA signaling key regulator genes.

The Carboxyl-Terminus of TRANSPARENT TESTA GLABRA1 Is Critical for Its Functions in Arabidopsis.

The Arabidopsis WD40 repeat protein TRANSPARENT TESTA GLABRA1 (TTG1) regulates cell fate determination, including trichome initiation and root hair formation, as well as secondary metabolism such as flavonoid biosynthesis and seed coat mucilage production. TTG1 regulates different processes via regulating the expression of its downstream target genes by forming MYB-bHLH-WD40 (MBW) activator complexes with different R2R3 MYB and bHLH transcription factors. Here, we report the identification of the carboxyl (C)-terminus as a critical domain for TTG1's functions in Arabidopsis. We found that the ttg1Δ15aa mutant shows pleiotropic phenotypes identical to a TTG1 loss-of-function mutant. Gene sequencing indicates that a single nucleotide substitution in TTG1 led to a premature stop at the W327 residue, leading to the production of a truncated TTG1 protein with a deletion of the last 15 C-terminal amino acids. The expression of TTG1 under the control of its native promoter fully restored the ttg1Δ15aa mutant phenotypes. Consistent with these observations, the expression levels of TTG1 downstream genes such as GLABRA2 (GL2) and CAPRICE (CPC) were reduced in the ttg1Δ15aa mutant. Assays in Arabidopsis protoplast show that TTG1Δ15aa failed to interact with the bHLH transcription factor GL3, and the deletion of the last 3 C-terminal amino acids or the 339L amino acid alone fully abolished the interaction of TTG1 with GL3. Furthermore, the expression of TTG1Δ3aa under the control of TTG1 native promoter failed to restore the ttg1Δ15aa mutant phenotypes. Taken together, our results suggest that the C-terminal domain of TTG1 is required for its proper function in Arabidopsis.

Oxidative decomposition of propylene carbonate in lithium ion batteries: a DFT study.

This paper reports an in-depth mechanistic study on the oxidative decomposition of propylene carbonate in the presence of lithium salts (LiClO4, LiBF4, LiPF6, and LiAsF6) with the aid of density functional theory calculations at the B3LYP/6-311++G(d,p) level of theory. The solvent effect is accounted for by using the implicit solvation model with density method. Moreover, the rate constants for the decompositions of propylene carbonate have been investigated by using transition-state theory. The shortening of the original carbonyl C-O bond and a lengthening of the adjacent ethereal C-O bonds of propylene carbonate, which occurs as a result of oxidation, leads to the formation of acetone radical and CO2 as a primary oxidative decomposition product. The termination of the primary radical generates polycarbonate, acetone, diketone, 2-(ethan-1-ylium-1-yl)-4-methyl-1,3-dioxolan-4-ylium, and CO2. The thermodynamic and kinetic data show that the major oxidative decomposition products of propylene carbonate are independent of the type of lithium salt. However, the decomposition rate constants of propylene carbonate are highly affected by the lithium salt type. On the basis of the rate constant calculations using transition-state theory, the order of gas volume generation is: [PC-ClO4](-) > [PC-BF4](-) > [PC-AsF6](-) > [PC-PF6](-).

ASR1 and ASR2, Two Closely Related ABA-Induced Serine-Rich Transcription Repressors, Function Redundantly to Regulate ABA Responses in Arabidopsis.

The plant hormone abscisic acid (ABA) is able to regulate the expression of ABA-responsive genes via signaling transduction, and thus plays an important role in regulating plant responses to abiotic stresses. Hence, characterization of unknown ABA response genes may enable us to identify novel regulators of ABA and abiotic stress responses. By using RT-PCR analysis, we found that the expression levels of ABA-induced Serine-rich Repressor 1 (ASR1)and ASR2, two closely related unknown function genes, were increased in response to ABA treatment. Amino acid sequence analyses show that ASR1 contains an L×L×L motif and both ASR1 and ASR2 are enriched in serine. Transfection assays in Arabidopsis leaf protoplasts show that ASR1 and ASR2 were predominantly localized in the nucleus and were able to repress the expression of the reporter gene. The roles of ASRs in regulating ABA responses were examined by generating transgenic Arabidopsis plants over-expressing ASR1 and ASR2, respectively, and CRISPR/Cas9 gene-edited single and double mutants for ASR1 and ASR2. In both the seed germination and cotyledon greening assays, ABA sensitivity remained largely unchanged in the over-expression transgenic plants and the single mutants of ASR1 and ASR2, but greatly increased ABA sensitivity was observed in the asr1 asr2 double mutants. In root elongation assays, however, decreased ABA sensitivity was observed in the 35S:ASR1 and 35S:ASR2 transgenic plants, whereas increased ABA sensitivity was observed in the asr1 and asr2 single mutants, and ABA sensitivity was further increased in the asr1 asr2 double mutants. Transcriptome analysis show that the differentially expressed genes (DEGs) down-regulated in the 35S:ASR1 transgenic plant seedlings, but up-regulated in the asr1 asr2 double mutant seedlings were highly enriched in processes including responses to plant hormones and stress stimuli. Taken together, our results show that ASR1 and ASR2 are closely related ABA response genes, ASR1 and ASR2 are serine-rich novel transcription repressors, and they negatively regulate ABA responses in Arabidopsis in a redundant manner.

Antagonistic Regulation of ABA Responses by Duplicated Tandemly Repeated DUF538 Protein Genes in Arabidopsis.

The plant hormone ABA (abscisic acid) regulates plant responses to abiotic stresses by regulating the expression of ABA response genes. However, the functions of a large portion of ABA response genes have remained unclear. We report in this study the identification of ASDs (ABA-inducible signal peptide-containing DUF538 proteins), a subgroup of DUF538 proteins with a signal peptide, as the regulators of plant responses to ABA in Arabidopsis. ASDs are encoded by four closely related DUF538 genes, with ASD1/ASD2 and ASD3/ASD4 being two pairs of duplicated tandemly repeated genes. The quantitative RT-PCR (qRT-PCR) results showed that the expression levels of ASDs increased significantly in response to ABA as well as NaCl and mannitol treatments, with the exception that the expression level of ASD2 remained largely unchanged in response to NaCl treatment. The results of Arabidopsis protoplast transient transfection assays showed that ASDs were localized on the plasma membrane and in the cytosol and nucleus. When recruited to the promoter of the reporter gene via a fused GD domain, ASDs were able to slightly repress the expression of the co-transfected reporter gene. Seed germination and cotyledon greening assays showed that ABA sensitivity was increased in the transgenic plants that were over-expressing ASD1 or ASD3 but decreased in the transgenic plants that were over-expressing ASD2 or ASD4. On the other hand, ABA sensitivity was increased in the CRISPR/Cas9 gene-edited asd2 single mutants but decreased in the asd3 single mutants. A transcriptome analysis showed that differentially expressed genes in the 35S:ASD2 transgenic plant seedlings were enriched in several different processes, including in plant growth and development, the secondary metabolism, and plant hormone signaling. In summary, our results show that ASDs are ABA response genes and that ASDs are involved in the regulation of plant responses to ABA in Arabidopsis; however, ASD1/ASD3 and ASD2/ASD4 have opposite functions.

AtS40-1, a group I DUF584 protein positively regulates ABA response and salt tolerance in Arabidopsis.

Abiotic stresses such as salt and drought affect plants growth and development, whereas the plant hormone ABA is able to regulate plant responses to abiotic stresses by regulating downstream gene expression. Therefore characterization of unknown function ABA responsive genes is able to identify novel regulators of plant abiotic stress responses. We report here the characterization of AtS40-1, a Group I DUF584 protein in the regulation of ABA and salt responses in Arabidopsis. RT-PCR results show that the expression of AtS40-1 was dramatically induced by ABA, but only slightly increase, if any, was observed for other three Group I DUF584 genes including AtS40-1L, AtS40-2 and AtS40-3. Transfection assays in Arabidopsis protoplasts show that all the four Group I DUF584 proteins were predominately localized in nucleus and were able to repress the expression of the co-transfected reporter gene. The roles of AtS40-1 in regulating plant response to ABA and abiotic stress responses were analyzed, by using transgenic plants and inactivation mutants. The results show that the ABA responses were increased in the 35S:AtS40-1 transgenic plants, but decreased in the ats40-1 mutants. Similar to AtS40-1, the results indicate that AtS40-1L, the most closely related DUF584 protein to AtS40-1, positively regulates ABA responses in Arabidopsis. However, further decreased ABA responses were not observed in the ats40-1 ats40-1L double mutants. On the other hand, salt tolerance was increased in the transgenic plants overexpressing AtS40-1 or AtS40-1L, but decreased in the ats40-1 and ats40-1L mutants. Quantitative RT-PCR results show that the ABA induced expression of the ABA signaling regulator genes ABI3, ABI4 and ABA responsive gene RAB18 was decreased, where as ABA signaling gene ABI1 was increased in the ats40-1 mutants. These results suggest that AtS40-1 regulates ABA and salt responses in Arabidopsis, possibly by affecting ABA induced expression of some ABA signaling regulator genes.

AtbZIP62 Acts as a Transcription Repressor to Positively Regulate ABA Responses in Arabidopsis.

The basic region/leucine zipper (bZIP) transcription factor AtbZIP62 is involved in the regulation of plant responses to abiotic stresses, including drought and salinity stresses, NO3 transport, and basal defense in Arabidopsis. It is unclear if it plays a role in regulating plant responses to abscisic acid (ABA), a phytohormone that can regulate plant abiotic stress responses via regulating downstream ABA-responsive genes. Using RT-PCR analysis, we found that the expression level of AtbZIP62 was increased in response to exogenously applied ABA. Protoplast transfection assays show that AtbZIP62 is predominantly localized in the nucleus and functions as a transcription repressor. To examine the roles of AtbZIP62 in regulating ABA responses, we generated transgenic Arabidopsis plants overexpressing AtbZIP62 and created gene-edited atbzip62 mutants using CRISPR/Cas9. We found that in both ABA-regulated seed germination and cotyledon greening assays, the 35S:AtbZIP62 transgenic plants were hypersensitive, whereas atbzip62 mutants were hyposensitive to ABA. To examine the functional mechanisms of AtbZIP62 in regulating ABA responses, we generated Arabidopsis transgenic plants overexpressing 35S:AtbZIP62-GR, and performed transcriptome analysis to identify differentially expressed genes (DEGs) in the presence and absence of DEX, and found that DEGs are highly enriched in processes including response to abiotic stresses and response to ABA. Quantitative RT-PCR results further show that AtbZIP62 may regulate the expression of several ABA-responsive genes, including USP, ABF2, and SnRK2.7. In summary, our results show that AtbZIP62 is an ABA-responsive gene, and AtbZIP62 acts as a transcription repressor to positively regulate ABA responses in Arabidopsis.

The R2R3 MYB Transcription Factor MYB71 Regulates Abscisic Acid Response in Arabidopsis.

Abscisic acid (ABA) regulates plant responses to abiotic stresses via regulating the expression of downstream genes, yet the functions of many ABA responsive genes remain unknown. We report here the characterization of MYB71, a R2R3 MYB transcription factor in regulating ABA responses in Arabidopsis. RT-PCR results show that the expression level of MYB71 was increased in response to ABA treatment. Arabidopsis protoplasts transfection results show that MYB71 was specifically localized in nucleus and it activated the Gal4:GUS reporter gene when recruited to the Gal4 promoter by a fused DNA binding domain GD. Roles of MYB71 in regulating plant response to ABA were analyzed by generating Arabidopsis transgenic plants overexpression MYB71 and gene edited mutants of MYB71. The results show that ABA sensitivity was increased in the transgenic plants overexpression MYB71, but decreased in the MYB71 mutants. By using a DEX inducible system, we further identified genes are likely regulated by MYB71, and found that they are enriched in biological process to environmental stimuli including abiotic stresses, suggesting that MYB71 may regulate plant response to abiotic stresses. Taken together, our results suggest that MYB71 is an ABA responsive gene, and MYB71 functions as a transcription activator and it positively regulates ABA response in Arabidopsis.

AITRL, an evolutionarily conserved plant specific transcription repressor regulates ABA response in Arabidopsis.

Expression of stress response genes can be regulated by abscisic acid (ABA) dependent and ABA independent pathways. Osmotic stresses promote ABA accumulation, therefore inducing the expression of stress response genes via ABA signaling. Whereas cold and heat stresses induce the expression of stress response genes via ABA independent pathway. ABA induced transcription repressors (AITRs) are a family of novel transcription factors that play a role in ABA signaling, and Drought response gene (DRG) has previously been shown to play a role in regulating plant response to drought and freezing stresses. We report here the identification of DRG as a novel transcription factor and a regulator of ABA response in Arabidopsis. We found that the expression of DRG was induced by ABA treatment. Homologs searching identified AITR5 as the most closely related Arabidopsis protein to DRG, and homologs of DRG, including the AITR-like (AITRL) proteins in bryophytes and gymnosperms, are specifically presented in embryophytes. Therefore we renamed DRG as AITRL. Protoplast transfection assays show that AITRL functioned as a transcription repressor. In seed germination and seedling greening assays, the aitrl mutants showed an increased sensitivity to ABA. By using qRT-PCR, we show that ABA responses of some ABA signaling component genes including some PYR1-likes (PYLs), PROTEIN PHOSPHATASE 2Cs (PP2Cs) and SUCROSE NONFERMENTING 1 (SNF1)-RELATED PROTEIN KINASES 2s (SnRK2s) were reduced in the aitrl mutants. Taken together, our results suggest that AITRLs are a family of novel transcription repressors evolutionally conserved in embryophytes, and AITRL regulates ABA response in Arabidopsis by affecting ABA response of some ABA signaling component genes.

Mutation of GmAITR Genes by CRISPR/Cas9 Genome Editing Results in Enhanced Salinity Stress Tolerance in Soybean.

Breeding of stress-tolerant plants is able to improve crop yield under stress conditions, whereas CRISPR/Cas9 genome editing has been shown to be an efficient way for molecular breeding to improve agronomic traits including stress tolerance in crops. However, genes can be targeted for genome editing to enhance crop abiotic stress tolerance remained largely unidentified. We have previously identified abscisic acid (ABA)-induced transcription repressors (AITRs) as a novel family of transcription factors that are involved in the regulation of ABA signaling, and we found that knockout of the entire family of AITR genes in Arabidopsis enhanced drought and salinity tolerance without fitness costs. Considering that AITRs are conserved in angiosperms, AITRs in crops may be targeted for genome editing to improve abiotic stress tolerance. We report here that mutation of GmAITR genes by CRISPR/Cas9 genome editing leads to enhanced salinity tolerance in soybean. By using quantitative RT-PCR analysis, we found that the expression levels of GmAITRs were increased in response to ABA and salt treatments. Transfection assays in soybean protoplasts show that GmAITRs are nucleus proteins, and have transcriptional repression activities. By using CRISPR/Cas9 to target the six GmAITRs simultaneously, we successfully generated Cas9-free gmaitr36 double and gmaitr23456 quintuple mutants. We found that ABA sensitivity in these mutants was increased. Consistent with this, ABA responses of some ABA signaling key regulator genes in the gmaitr mutants were altered. In both seed germination and seedling growth assays, the gmaitr mutants showed enhanced salt tolerance. Most importantly, enhanced salinity tolerance in the mutant plants was also observed in the field experiments. These results suggest that mutation of GmAITR genes by CRISPR/Cas9 is an efficient way to improve salinity tolerance in soybean.

SlEAD1, an EAR motif-containing ABA down-regulated novel transcription repressor regulates ABA response in tomato.

EAR motif-containing proteins are able to repress gene expression, therefore play important roles in regulating plants growth and development, plant response to environmental stimuli, as well as plant hormone signal transduction. ABA is a plant hormone that regulates abiotic stress tolerance in plants via signal transduction. ABA signaling via the PYR1/PYLs/RCARs receptors, the PP2Cs phosphatases, and SnRK2s protein kinases activates the ABF/AREB/ABI5-type bZIP transcription factors, resulting in the activation/repression of ABA response genes. However, functions of many ABA response genes remained largely unknown. We report here the identification of the ABA-responsive gene SlEAD1 (Solanum lycopersicum EAR motif-containing ABA down-regulated 1) as a novel EAR motif-containing transcription repressor gene in tomato. We found that the expression of SlEAD1 was down-regulated by ABA treatment, and SlEAD1 repressed reporter gene expression in transfected protoplasts. By using CRISPR gene editing, we generated transgene-free slead1 mutants and found that the mutants produced short roots. By using seed germination and root elongation assays, we examined ABA response of the slead1 mutants and found that ABA sensitivity in the mutants was increased. By using qRT-PCR, we further show that the expression of some of the ABA biosynthesis and signaling component genes were increased in the slead1 mutants. Taken together, our results suggest that SlEAD1 is an ABA response gene, that SlEAD1 is a novel EAR motif-containing transcription repressor, and that SlEAD1 negatively regulates ABA responses in tomato possibly by repressing the expression of some ABA biosynthesis and signaling genes.

Clinical significance of detecting survivin-expressing circulating cancer cells in patients with non-small cell lung cancer.

We previously demonstrated that the detection of circulating cancer cells (CCC) expressing survivin mRNA could provide valuable information for predicting metastasis and recurrence in breast cancer. The objective of this study was to investigate the significance of detecting survivin-expressing CCC on the clinical outcomes of patients with non-small cell lung cancer (NSCLC). Peripheral blood samples collected from 143 NSCLC patients and 177 healthy volunteers were quantitatively evaluated using a technique developed in our laboratory that detected reverse transcription-polymerase chain reaction (RT-PCR) products based on a hybridisation-enzyme linked immunosorbant essay (ELISA), which we called RT-PCR ELISA. The presence of survivin-expressing CCC was detected in 63 cancer patients (44.1%) and was significantly associated with pathological T classification, nodal status, and disease stages (all P<0.001). During a follow-up period of 36 months, patients who had positive survivin expressions at the time of the initial assay test had a higher relapse rate and shorter survival time when compared to those who had negative survivin expressions (all P<0.001). Through multivariate analysis, the detection of survivin-expressing CCC was found to be an independent predictor for cancer recurrence (HR=43.5; 95% CI=2.67-70.9; P=0.008) and survival (HR=1.35; 95% CI=1.02-4.31; P=0.049). Thus, detection of survivin-expressing CCC could be used in the prediction of disease recurrence as well as in the prognosis of NSCLC.

A new role of anterograde motor Kif5b in facilitating large clathrin-coated vesicle mediated endocytosis via regulating clathrin uncoating.

Kif5b-driven anterograde transport and clathrin-mediated endocytosis (CME) are responsible for opposite intracellular trafficking, contributing to plasma membrane homeostasis. However, whether and how the two trafficking processes coordinate remain unclear. Here, we show that Kif5b directly interacts with clathrin heavy chain (CHC) at a region close to that for uncoating catalyst (Hsc70) and preferentially localizes on relatively large clathrin-coated vesicles (CCVs). Uncoating in vitro is decreased for CCVs from the cortex of kif5b conditional knockout (mutant) mouse and facilitated by adding Kif5b fragments containing CHC-binding site, while cell peripheral distribution of CHC or Hsc70 keeps unaffected by Kif5b depletion. Furthermore, cellular entry of vesicular stomatitis virus that internalizes into large CCV is inhibited by Kif5b depletion or introducing a dominant-negative Kif5b fragment. These findings showed a new role of Kif5b in regulating large CCV-mediated CME via affecting CCV uncoating, indicating Kif5b as a molecular knot connecting anterograde transport to CME.