Characterisation of the MLP genes in peach postharvest cold storage and the regulatory role of PpMLP10 in the chilling stress response.

The major latex proteins/ripening-related proteins are a subfamily of the Bet v 1 protein superfamily and are commonly involved in plant development and responses to various stresses. However, the functions of MLPs in the postharvest cold storage of fruits remain uninvestigated. Herein, we identified 30 MLP genes in the peach (Prunus persica) genome that were clustered into three subgroups. Chromosomal location analysis revealed that the PpMLP genes were unevenly distributed on five of the eight peach chromosomes. Synteny analysis of the MLP genes between peach and seven other plant species (five dicotyledons and two monocotyledons) explored their evolutionary characteristics. Furthermore, the PpMLP promoters contained cis-elements for multiple hormones and stress responses. Gene expression analysis revealed that PpMLPs participated in chilling stress responses. Ectopic expression of PpMLP10 in Arabidopsis improved chilling stress tolerance by decreasing membrane damage and maintaining membrane stability. Additional research confirmed that PpWRKY2 participates in PpMLP10-mediated chilling stress by binding to its promoter. Collectively, these results suggest the role of PpMLP10 in enhancing chilling stress tolerance, which is significant for decreasing chilling injury during the postharvest cold storage of peaches.

Metabolic engineering of Escherichia coli for high-level production of the biodegradable polyester monomer 2-pyrone-4,6-dicarboxylic acid.

2-Pyrone-4,6-dicarboxylic acid (PDC), a chemically stable pseudo-aromatic dicarboxylic acid, is a promising building block compound for manufacturing biodegradable polyesters. This study aimed to construct high-performance cell factories enabling the efficient production of PDC from glucose. Firstly, the effective enzymes of the PDC biosynthetic pathway were overexpressed on the chromosome of the 3-dehydroshikimate overproducing strain. Consequently, the one-step biosynthesis of PDC from glucose was achieved. Further, the PDC production was enhanced by multi-copy integration of the key gene PsligC encoding 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase and co-expression of Vitreoscilla hemoglobin. Subsequently, the PDC production was substantially improved by redistributing the metabolic flux for cell growth and PDC biosynthesis based on dynamically downregulating the expression of pyruvate kinase. The resultant strain PDC50 produced 129.37 g/L PDC from glucose within 78 h under fed-batch fermentation conditions, with a yield of 0.528 mol/mol and an average productivity of 1.65 g/L/h. The findings of this study lay the foundation for the potential industrial production of PDC.

Epigenetic regulation of N-hydroxypipecolic acid biosynthesis by the AIPP3-PHD2-CPL2 complex.

N-Hydroxypipecolic acid (NHP) is a signaling molecule crucial for systemic acquired resistance (SAR), a systemic immune response in plants that provides long-lasting and broad-spectrum protection against secondary pathogen infections. To identify negative regulators of NHP biosynthesis, we performed a forward genetic screen to search for mutants with elevated expression of the NHP biosynthesis gene FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1). Analysis of two constitutive expression of FMO1 (cef) and one induced expression of FMO1 (ief) mutants revealed that the AIPP3-PHD2-CPL2 protein complex, which is involved in the recognition of the histone modification H3K27me3 and transcriptional repression, contributes to the negative regulation of FMO1 expression and NHP biosynthesis. Our study suggests that epigenetic regulation plays a crucial role in controlling FMO1 expression and NHP levels in plants.

Metabolic engineering of fast-growing Vibrio natriegens for efficient pyruvate production.

BACKGROUND: Pyruvate is a widely used value-added chemical which also serves as a hub of various metabolic pathways. The fastest-growing bacterium Vibrio natriegens is a promising chassis for synthetic biology applications with high substrate uptake rates. The aim of this study was to investigate if the high substrate uptake rates of V. natriegens enable pyruvate production at high productivities. RESULTS: Two prophage gene clusters and several essential genes for the biosynthesis of byproducts were first deleted. In order to promote pyruvate accumulation, the key gene aceE encoding pyruvate dehydrogenase complex E1 component was down-regulated to reduce the carbon flux into the tricarboxylic acid cycle. Afterwards, the expression of ppc gene encoding phosphoenolpyruvate carboxylase was fine-tuned to balance the cell growth and pyruvate synthesis. The resulting strain PYR32 was able to produce 54.22 g/L pyruvate from glucose within 16 h, with a yield of 1.17 mol/mol and an average productivity of 3.39 g/L/h. In addition, this strain was also able to efficiently convert sucrose or gluconate into pyruvate at high titers. CONCLUSION: A novel strain of V. natriegens was engineered which was capable to provide higher productivity in pyruvate synthesis. This study lays the foundation for the biosynthesis of pyruvate and its derivatives in fast-growing V. natriegens.

The effects of microbial fertilizers application on growth, yield and some biochemical changes in the leaves and seeds of guar (Cyamopsis tetragonoloba L.).

Guar (Cyamopsis tetragonoloba L.) is a summer legume that is becoming a crucial industrial crop because of its high gum and protein content. Thus far, the combined effects of arbuscular mycorrhizal fungi (AMF) and Bradyrhizobium on the yield and chemical composition of guar plants are not well studied. Therefore, the current investigation was designed to estimate the individual as well as the combined effects of AMF and Bradyrhizobium on plant growth, yield and nutritional quality of seeds and leaves of guar. AMF and/or Bradyrhizobium inoculation improved chemical composition of guar seeds and its morpho-physiological (plant height, fresh weight, dry weight, and yield production) traits. In addition to increased guar growth and yield production, the inoculation of AMF and/or Bradyrhizobium increased guar leaf and seed minerals, fiber, lipids, crude protein and ash contents. At primary metabolites, there were increases in sugar levels including raffinose stachyose, verbascose and galactomannan. These increases in sugar provided a route for organic acids, amino acids and fatty acids production. Interestingly, there was an increase in essential amino acids and unsaturated fatty acids. At the bioactive secondary metabolite levels, biofertilizers improved phenols and flavonoids levels and anthocyanin and polyamines biosynthesis. In line with these increases, precursors of anthocyanin (phenylalanine, p-coumaric acid, and cinnamic acid) and the levels of polyamines (diaminopropane, putrescine, cadaverine, spermidine, spermine, and agmatine) were increased. Overall, for the first time, our study shed the light on how AMF and Bradyrhizobium improved guar yield and metabolism. Our findings suggested that the combined inoculation of AMF and Bradyrhizobium is an innovative approach to improve guar growth, yield production and yield quality.

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.

BIC2, a Cryptochrome Function Inhibitor, Is Involved in the Regulation of ABA Responses in Arabidopsis.

The plant hormone ABA (abscisic acid) is able to regulate plant responses to abiotic stresses via regulating the expression of ABA response genes. BIC1 (Blue-light Inhibitor of Cryptochromes 1) and BIC2 have been identified as the inhibitors of plant cryptochrome functions, and are involved in the regulation of plant development and metabolism in Arabidopsis . In this study, we report the identification of BIC2 as a regulator of ABA responses in Arabidopsis . RT-PCR (Reverse Transcription-Polymerase Chain Reaction) results show that the expression level of BIC1 remained largely unchanged, but that of BIC2 increased significantly in response to ABA treatment. Transfection assays in Arabidopsis protoplasts show that both BIC1 and BIC2 were mainly localized in the nucleus, and were able to activate the expression of the co-transfected reporter gene. Results in seed germination and seedling greening assays show that ABA sensitivity was increased in the transgenic plants overexpressing BIC2, but increased slightly, if any, in the transgenic plants overexpressing BIC1. ABA sensitivity was also increased in the bic2 single mutants in seedling greening assays, but no further increase was observed in the bic1 bic2 double mutants. On the other hand, in root elongation assays, ABA sensitivity was decreased in the transgenic plants overexpressing BIC2, as well as the bic2 single mutants, but no further decrease was observed in the bic1 bic2 double mutants. By using qRT-PCR (quantitative RT-PCR), we further examined how BIC2 may regulate ABA responses in Arabidopsis , and found that inhibition of ABA on the expression of the ABA receptor genes PYL4 (PYR1-Like 4) and PYL5 were decreased, but promotion of ABA on the expression of the protein kinase gene SnRK2.6 (SNF1-Related Protein Kinases 2.6) was enhanced in both the bic1 bic2 double mutants and 35S:BIC2 overexpression transgenic plants. Taken together, our results suggest that BIC2 regulates ABA responses in Arabidopsis possibly by affecting the expression of ABA signaling key regulator genes.

Relevant Risk Factor and Follow-Up of Lung Nodules in Physical Examination with Low-Dose CT Screening.

Background: We aimed to explore the risk factors of lung nodules and lung cancer in physical examination population with low-dose multi-slice spiral CT (LDCT) screening, to provide basis for lung cancer screening and follow-up management after CT examination. Methods: The general data, serum tumor markers and CT images of 2,274 patients underwent LDCT in the Physical Examination Center of the Fourth Hospital of Hebei Medical University, China in 2019 were retrospectively analyzed and followed up for three years. Results: The detection rate of lung nodules was 48.42%. The detection rate of lung nodules was higher in females, those over 70, those with history of smoking, passive smoking, drinking, precious history of lung diseases and family history of malignant tumors, with statistically significant differences (P<0.05). The abnormal rate of serum tumor markers (CA199, CA125 female and CYFRA211) were higher than that in the non-nodule group, with statistically significant differences (P<0.05). Multivariate logistic regression analysis showed that gender, age, history of smoking, passive smoking, family history of malignant tumors and serum tumor markers (CYFRA211 and CA199) were independent risk factors for the occurrence of lung nodules. Conclusion: Gender female, age>35, history of smoking, passive smoking, history of drinking, history of past lung disease, family history of malignant tumors, abnormal CYFRA211 tumor markers were detected and low dose multi-slice spiral CT image showed ground-glass nodules are risk factors for lung nodules and lung cancer, which should be paid close attention to during physical examination and follow-up.

Research on Automatic Classification and Detection of Mutton Multi-Parts Based on Swin-Transformer.

In order to realize the real-time classification and detection of mutton multi-part, this paper proposes a mutton multi-part classification and detection method based on the Swin-Transformer. First, image augmentation techniques are adopted to increase the sample size of the sheep thoracic vertebrae and scapulae to overcome the problems of long-tailed distribution and non-equilibrium of the dataset. Then, the performances of three structural variants of the Swin-Transformer (Swin-T, Swin-B, and Swin-S) are compared through transfer learning, and the optimal model is obtained. On this basis, the robustness, generalization, and anti-occlusion abilities of the model are tested and analyzed using the significant multiscale features of the lumbar vertebrae and thoracic vertebrae, by simulating different lighting environments and occlusion scenarios, respectively. Furthermore, the model is compared with five methods commonly used in object detection tasks, namely Sparser-CNN, YoloV5, RetinaNet, CenterNet, and HRNet, and its real-time performance is tested under the following pixel resolutions: 576 × 576, 672 × 672, and 768 × 768. The results show that the proposed method achieves a mean average precision (mAP) of 0.943, while the mAP for the robustness, generalization, and anti-occlusion tests are 0.913, 0.857, and 0.845, respectively. Moreover, the model outperforms the five aforementioned methods, with mAP values that are higher by 0.009, 0.027, 0.041, 0.050, and 0.113, respectively. The average processing time of a single image with this model is 0.25 s, which meets the production line requirements. In summary, this study presents an efficient and intelligent mutton multi-part classification and detection method, which can provide technical support for the automatic sorting of mutton as well as for the processing of other livestock meat.

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.

Overexpression of a Cinnamyl Alcohol Dehydrogenase-Coding Gene, GsCAD1, from Wild Soybean Enhances Resistance to Soybean Mosaic Virus.

Soybean mosaic virus (SMV) is the most prevalent soybean viral disease in the world. As a critical enzyme in the secondary metabolism of plants, especially in lignin synthesis, cinnamyl alcohol dehydrogenase (CAD) is widely involved in plant growth and development, and in defense against pathogen infestation. Here, we performed RNAseq-based transcriptome analyses of a highly SMV-resistant accession (BYO-15) of wild soybean (Glycine soja) and a SMV-susceptible soybean cultivar (Williams 82), also sequenced together with a resistant plant and a susceptible plant of their hybrid descendants at the F3 generation at 7 and 14 days post-inoculation with SMV. We found that the expression of GsCAD1 (from G. soja) was significantly up-regulated in the wild soybean and the resistant F3 plant, while the GmCAD1 from the cultivated soybean (G. max) did not show a significant and persistent induction in the soybean cultivar and the susceptible F3 plant, suggesting that GsCAD1 might play an important role in SMV resistance. We cloned GsCAD1 and overexpressed it in the SMV-susceptible cultivar Williams 82, and we found that two independent GsCAD1-overexpression (OE) lines showed significantly enhanced SMV resistance compared with the non-transformed wild-type (WT) control. Intriguingly, the lignin contents in both OE lines were higher than the WT control. Further liquid chromatography (HPLC) analysis showed that the contents of salicylic acid (SA) were significantly more improved in the OE lines than that of the wild-type (WT), coinciding with the up-regulated expression of an SA marker gene. Finally, we observed that GsCAD1-overexpression affected the accumulation of SMV in leaves. Collectively, our results suggest that GsCAD1 enhances resistance to SMV in soybeans, most likely by affecting the contents of lignin and SA.

CRISPR/Cas9 Gene Editing of NtAITRs, a Family of Transcription Repressor Genes, Leads to Enhanced Drought Tolerance in Tobacco.

Tobacco is a cash crop throughout the world, and its growth and development are affected by abiotic stresses including drought stress; therefore, drought-tolerant breeding may help to improve tobacco yield and quality under drought stress conditions. Considering that the plant hormone ABA (abscisic acid) is able to regulate plant responses to abiotic stresses via activating ABA response genes, the characterization of ABA response genes may enable the identification of genes that can be used for molecular breeding to improve drought tolerance in tobacco. We report here the identification of NtAITRs (Nicotiana tabacum ABA-induced transcription repressors) as a family of novel regulators of drought tolerance in tobacco. Bioinformatics analysis shows that there are a total of eight NtAITR genes in tobacco, and all the NtAITRs have a partially conserved LxLxL motif at their C-terminus. RT-PCR results show that the expression levels of at least some NtAITRs were increased in response to ABA and drought treatments, and NtAITRs, when recruited to the Gal4 promoter via a fused GD (Gal4 DNA-binding domain), were able to repress transcription activator LD-VP activated expression of the LexA-Gal4-GUS reporter gene. Roles of NtAITRs in regulating drought tolerance in tobacco were analyzed by generating CRISPR/Cas9 gene-edited mutants. A total of three Cas9-free ntaitr12356 quintuple mutants were obtained, and drought treatment assays show that drought tolerance was increased in the ntaitr12356 quintuple mutants. On the other hand, results of seed germination and seedling greening assays show that ABA sensitivity was increased in the ntaitr12356 quintuple mutants, and the expression levels of some ABA signaling key regulator genes were altered in the ntaitr12356-c3 mutant. Taken together, our results suggest that NtAITRs are ABA-responsive genes, and that NtAITRs function as transcription repressors and negatively regulate drought tolerance in tobacco, possibly by affecting plant ABA response via affecting the expression of ABA signaling key 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.

Tropaeolum majus R2R3 MYB Transcription Factor TmPAP2 Functions as a Positive Regulator of Anthocyanin Biosynthesis.

Anthocyanins are an important group of water-soluble and non-toxic natural pigments with antioxidant and anti-inflammatory properties that can be found in flowers, vegetables, and fruits. Anthocyanin biosynthesis is regulated by several different types of transcription factors, including the WD40-repeat protein Transparent Testa Glabra 1 (TTG1), the bHLH transcription factor Transparent Testa 8 (TT8), Glabra3 (GL3), Enhancer of GL3 (EGL3), and the R2R3 MYB transcription factor Production of Anthocyanin Pigment 1 (PAP1), PAP2, MYB113, and MYB114, which are able to form MYB-bHLH-WD40 (MBW) complexes to regulate the expression of late biosynthesis genes (LBGs) in the anthocyanin biosynthesis pathway. Nasturtium (Tropaeolum majus) is an edible flower plant that offers many health benefits, as it contains numerous medicinally important ingredients, including anthocyanins. By a comparative examination of the possible anthocyanin biosynthesis regulator genes in nasturtium varieties with different anthocyanin contents, we found that TmPAP2, an R2R3 MYB transcription factor gene, is highly expressed in "Empress of India", a nasturtium variety with high anthocyanin content, while the expression of TmPAP2 in Arabidopsis led to the overproduction of anthocyanins. Protoplast transfection shows that TmPAP2 functions as a transcription activator; consistent with this finding, some of the biosynthesis genes in the general phenylpropanoid pathway and anthocyanin biosynthesis pathway were highly expressed in "Empress of India" and the 35S:TmPAP2 transgenic Arabidopsis plants. However, protoplast transfection indicates that TmPAP2 may not be able to form an MBW complex with TmGL3 and TmTTG1. These results suggest that TmPAP2 may function alone as a key regulator of anthocyanin biosynthesis in nasturtiums.

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.

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 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.

CsMYB1 integrates the regulation of trichome development and catechins biosynthesis in tea plant domestication.

Tea trichomes synthesize numerous specialized metabolites to protect plants from environmental stresses and contribute to tea flavours, but little is known about the regulation of trichome development. Here, we showed that CsMYB1 is involved in the regulation of trichome formation and galloylated cis-catechins biosynthesis in tea plants. The variations in CsMYB1 expression levels are closely correlated with trichome indexes and galloylated cis-catechins contents in tea plant populations. Genome resequencing showed that CsMYB1 may be selected in modern tea cultivars, since a 192-bp insertion in CsMYB1 promoter was found exclusively in modern tea cultivars but not in the glabrous wild tea Camellia taliensis. Several enhancers in the 192-bp insertion increased CsMYB1 transcription in modern tea cultivars that coincided with their higher galloylated cis-catechins contents and trichome indexes. Biochemical analyses and transgenic data showed that CsMYB1 interacted with CsGL3 and CsWD40 and formed a MYB-bHLH-WD40 (MBW) transcriptional complex to activate the trichome regulator genes CsGL2 and CsCPC, and the galloylated cis-catechins biosynthesis genes anthocyanidin reductase and serine carboxypeptidase-like 1A. CsMYB1 integratively regulated trichome formation and galloylated cis-catechins biosynthesis. Results suggest that CsMYB1, trichome and galloylated cis-catechins are coincidently selected during tea domestication by harsh environments for improved adaption and by breeders for better tea flavours.

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.