A long non-coding RNA functions as a competitive endogenous RNA to modulate TaNAC018 by acting as a decoy for tae-miR6206.

Increasing evidence indicates a strong correlation between the deposition of cuticular waxes and drought tolerance. However, the precise regulatory mechanism remains elusive. Here, we conducted a comprehensive transcriptome analysis of two wheat (Triticum aestivum) near-isogenic lines, the glaucous line G-JM38 rich in cuticular waxes and the non-glaucous line NG-JM31. We identified 85,143 protein-coding mRNAs, 4,485 lncRNAs, and 1,130 miRNAs. Using the lncRNA-miRNA-mRNA network and endogenous target mimic (eTM) prediction, we discovered that lncRNA35557 acted as an eTM for the miRNA tae-miR6206, effectively preventing tae-miR6206 from cleaving the NAC transcription factor gene TaNAC018. This lncRNA-miRNA interaction led to higher transcript abundance for TaNAC018 and enhanced drought-stress tolerance. Additionally, treatment with mannitol and abscisic acid (ABA) each influenced the levels of tae-miR6206, lncRNA35557, and TaNAC018 transcript. The ectopic expression of TaNAC018 in Arabidopsis also improved tolerance toward mannitol and ABA treatment, whereas knocking down TaNAC018 transcript levels via virus-induced gene silencing in wheat rendered seedlings more sensitive to mannitol stress. Our results indicate that lncRNA35557 functions as a competing endogenous RNA to modulate TaNAC018 expression by acting as a decoy target for tae-miR6206 in glaucous wheat, suggesting that non-coding RNA has important roles in the regulatory mechanisms responsible for wheat stress tolerance.

An efficient genetic transformation system mediated by Rhizobium rhizogenes in fruit trees based on the transgenic hairy root to shoot conversion.

Genetic transformation is a critical tool for gene editing and genetic improvement of plants. Although many model plants and crops can be genetically manipulated, genetic transformation systems for fruit trees are either lacking or perform poorly. We used Rhizobium rhizogenes to transfer the target gene into the hairy roots of Malus domestica and Actinidia chinensis. Transgenic roots were generated within 3 weeks, with a transgenic efficiency of 78.8%. Root to shoot conversion of transgenic hairy roots was achieved within 11 weeks, with a regeneration efficiency of 3.3%. Finally, the regulatory genes involved in stem cell activity were used to improve shoot regeneration efficiency. MdWOX5 exhibited the most significant effects, as it led to an improved regeneration efficiency of 20.6% and a reduced regeneration time of 9 weeks. Phenotypes of the overexpression of RUBY system mediated red roots and overexpression of MdRGF5 mediated longer root hairs were observed within 3 weeks, suggesting that the method can be used to quickly screen genes that influence root phenotype scores through root performance, such as root colour, root hair, and lateral root. Obtaining whole plants of the RUBY system and MdRGF5 overexpression lines highlights the convenience of this technology for studying gene functions in whole plants. Overall, we developed an optimized method to improve the transformation efficiency and stability of transformants in fruit trees.

Vv-circSIZ1 mediated by pre-mRNA processing machinery contributes to salt tolerance.

CircRNAs exist widely in plants, but the regulatory mechanisms for the biogenesis and function of plant circRNAs remain largely unknown. Using extensive mutagenesis of expression plasmids and genetic transformation methods, we analyzed the biogenesis and anti-salt functions of a new grape circRNA Vv-circSIZ1. We identified Vv-circSIZ1 that is mainly expressed in the cytoplasm of xylem. CircSIZ1 is species-specific, and genomic circSIZ1-forming region of seven tested species could be backspliced in Nicotiana benthamiana, but not in Arabidopsis. The retention length of Vv-circSIZ1 flanking introns was significantly positively correlated with its generation efficiency. The precise splicing of Vv-circSIZ1 does not depend on its mature exon sequence or internal intron sequences, but on the AG/GT splicing signal sites and branch site of the flanking introns. The spliceosome activity was inversely proportional to the expression level of Vv-circSIZ1. Furthermore, RNA-binding proteins can regulate the expression of Vv-circSIZ1. The overexpression of Vv-circSIZ1 improved salt tolerance of grape and N. benthamiana. Additionally, Vv-circSIZ1 could relieve the repressive effect of VvmiR3631 on its target VvVHAc1. Vv-circSIZ1 also promoted transcription of its parental gene. Overall, these results broaden our understanding of circRNAs in plants.

Intronic microRNA-directed regulation of mitochondrial reactive oxygen species enhances plant stress tolerance in Arabidopsis.

MicroRNAs (miRNAs) play crucial roles in regulating plant development and stress responses. However, the functions and mechanism of intronic miRNAs in plants are poorly understood. This study reports a stress-responsive RNA splicing mechanism for intronic miR400 production, whereby miR400 modulates reactive oxygen species (ROS) accumulation and improves plant tolerance by downregulating its target expression. To monitor the intron splicing events, we used an intronic miR400 splicing-dependent luciferase transgenic line. Luciferase activity was observed to decrease after high cadmium concentration treatment due to the retention of the miR400-containing intron, which inhibited the production of mature miR400. Furthermore, we demonstrated that under Cd treatments, Pentatricopeptide Repeat Protein 1 (PPR1), the target of miR400, acts as a positive regulator by inducing ROS accumulation. Ppr1 mutation affected the Complex III activity in the electron transport chain and RNA editing of the mitochondrial gene ccmB. This study illustrates intron splicing as a key step in intronic miR400 production and highlights the function of intronic miRNAs as a 'signal transducer' in enhancing plant stress tolerance.

Tyrosylprotein sulfotransferase suppresses ABA signaling via sulfation of SnRK2.2/2.3/2.6.

Phytohormone abscisic acid (ABA) plays vital roles in stress tolerance, while long-term overactivation of ABA signaling suppresses plant growth and development. However, the braking mechanism of ABA responses is not clear. Protein tyrosine sulfation catalyzed by tyrosylprotein sulfotransferase (TPST) is a critical post-translational modification. Through genetic screening, we identified a tpst mutant in Arabidopsis that was hypersensitive to ABA. In-depth analysis revealed that TPST could interact with and sulfate SnRK2.2/2.3/2.6, which accelerated their degradation and weakened the ABA signaling. Taken together, these findings uncovered a novel mechanism of desensitizing ABA responses via protein sulfation.

Regulation of drought tolerance in Arabidopsis involves the PLATZ4-mediated transcriptional repression of plasma membrane aquaporin PIP2;8.

Plant A/T-rich protein and zinc-binding protein (PLATZ) transcription factors play important roles in plant growth, development and abiotic stress responses. However, how PLATZ influences plant drought tolerance remains poorly understood. The present study showed that PLATZ4 increased drought tolerance in Arabidopsis thaliana by causing stomatal closure. Transcriptional profiling analysis revealed that PLATZ4 affected the expression of a set of genes involved in water and ion transport, antioxidant metabolism, small peptides and abscisic acid (ABA) signaling. Among these genes, the direct binding of PLATZ4 to the A/T-rich sequences in the plasma membrane intrinsic protein 2;8 (PIP2;8) promoter was identified. PIP2;8 consistently reduced drought tolerance in Arabidopsis through inhibiting stomatal closure. PIP2;8 was localized in the plasma membrane, exhibited water channel activity in Xenopus laevis oocytes and acted epistatically to PLATZ4 in regulating the drought stress response in Arabidopsis. PLATZ4 increased ABA sensitivity through upregulating the expression of ABSCISIC ACID INSENSITIVE 3 (ABI3), ABI4 and ABI5. The transcripts of PLATZ4 were induced to high levels in vegetative seedlings under drought and ABA treatments within 6 and 3 h, respectively. Collectively, these findings reveal that PLATZ4 positively influences plant drought tolerance through regulating the expression of PIP2;8 and genes involved in ABA signaling.

Transcription Factor SiDi19-3 Enhances Salt Tolerance of Foxtail Millet and Arabidopsis.

Salt stress is an important limiting factor of crop production. Foxtail millet (Setaria italica L.) is an important model crop for studying tolerance to various abiotic stressors. Therefore, examining the response of foxtail millet to salt stress at the molecular level is critical. Herein, we discovered that SiDi19-3 interacts with SiPLATZ12 to control salt tolerance in transgenic Arabidopsis and foxtail millet seedlings. SiDi19-3 overexpression increased the transcript levels of most Na+/H+ antiporter (NHX), salt overly sensitive (SOS), and calcineurin B-like protein (CBL) genes and improved the salt tolerance of foxtail millet and Arabidopsis. Six SiDi19 genes were isolated from foxtail millet. Compared with roots, stems, and leaves, panicles and seeds had higher transcript levels of SiDi19 genes. All of them responded to salt, alkaline, polyethylene glycol, and/or abscisic acid treatments with enhanced expression levels. These findings indicate that SiDi19-3 and other SiDi19 members regulate salt tolerance and other abiotic stress response in foxtail millet.

BREVIPEDICELLUS Positively Regulates Salt-Stress Tolerance in Arabidopsis thaliana.

Salt stress is one of the major environmental threats to plant growth and development. However, the mechanisms of plants responding to salt stress are not fully understood. Through genetic screening, we identified and characterized a salt-sensitive mutant, ses5 (sensitive to salt 5), in Arabidopsis thaliana. Positional cloning revealed that the decreased salt-tolerance of ses5 was caused by a mutation in the transcription factor BP (BREVIPEDICELLUS). BP regulates various developmental processes in plants. However, the biological function of BP in abiotic stress-signaling and tolerance are still not clear. Compared with wild-type plants, the bp mutant exhibited a much shorter primary-root and lower survival rate under salt treatment, while the BP overexpressors were more tolerant. Further analysis showed that BP could directly bind to the promoter of XTH7 (xyloglucan endotransglucosylase/hydrolase 7) and activate its expression. Resembling the bp mutant, the disruption of XTH7 gave rise to salt sensitivity. These results uncovered novel roles of BP in positively modulating salt-stress tolerance, and illustrated a putative working mechanism.

Growth or stress responses: TMK-FER balancing act.

Transmembrane kinases (TMKs) and Feronia (FER) belong to the leucine-rich repeat receptor-like kinase family. Recent studies reveal that they coordinate plant growth and stress responses by regulating the balance between acidification and alkalization and crosstalk between auxin and abscisic acid, revealing a dynamic equilibrium in the regulation of the TMK-FER module in plants.

SES1 is vital for seedling establishment and post-germination growth under high-potassium stress conditions in Arabidopsis thaliana.

Background: The potassium ion (K+) plays an important role in maintaining plant growth and development, while excess potassium in the soil can cause stress to plants. The understanding of the molecular mechanism of plant's response to high KCl stress is still limited. Methods: At the seed stage, wild type (WT) and SENSITIVE TO SALT1 (SES1) mutants were exposed to different concentrations of potassium treatments. Tolerance was assayed as we compared their performances under stress using seedling establishment rate and root length. Na+content, K+content, and K+/Na+ ratio were determined using a flame atomic absorption spectrometer. In addition, the expressions of KCl-responding genes and ER stress-related genes were also detected and analyzed using qRT-PCR. Results: SES1 mutants exhibited seedling establishment defects under high potassium concentration conditions and exogenous calcium partially restored the hypersensitivity phenotype of ses1 mutants. The expression of some K+ transporter/channel genes were higher in ses1-2, and the ratio of potassium to sodium (K+/Na+) in ses1-2 roots decreased after KCl treatment compared with WT. Further analysis showed that the ER stress marker genes were dramatically induced by high K+ treatment and much higher expression levels were detected in ses1-2, indicating ses1-2 suffers a more serious ER stress than WT, and ER stress may influence the seedling establishment of ses1-2 under high KCl conditions. Conclusion: These results strongly indicate that SES1 is a potassium tolerance relevant molecule that may be related to maintaining the seedling K+/Na+ balance under high potassium conditions during seedling establishment and post-germination growth. Our results will provide a basis for further studies on the biological roles of SES1 in modulating potassium uptake, transport, and adaptation to stress conditions.

DEMETHYLATION REGULATOR 1 regulates DNA demethylation of the nuclear and mitochondrial genomes.

Active DNA demethylation effectively modulates gene expression during plant development and in response to stress. However, little is known about the upstream regulatory factors that regulate DNA demethylation. We determined that the demethylation regulator 1 (demr1) mutant exhibits a distinct DNA methylation profile at selected loci queried by methylation-sensitive polymerase chain reaction and globally based on whole-genome bisulfite sequencing. Notably, the transcript levels of the DNA demethylase gene REPRESSOR OF SILENCING 1 (ROS1) were lower in the demr1 mutant. We established that DEMR1 directly binds to the ROS1 promoter in vivo and in vitro, and the methylation level in the DNA methylation monitoring sequence of ROS1 promoter decreased by 60% in the demr1 mutant. About 40% of the hyper-differentially methylated regions (DMRs) in the demr1 mutant were shared with the ros1-4 mutant. Genetic analysis indicated that DEMR1 acts upstream of ROS1 to positively regulate abscisic acid (ABA) signaling during seed germination and seedling establishment stages. Surprisingly, the loss of DEMR1 function also caused a rise in methylation levels of the mitochondrial genome, impaired mitochondrial structure and an early flowering phenotype. Together, our results show that DEMR1 is a novel regulator of DNA demethylation of both the nuclear and mitochondrial genomes in response to ABA and plant development in Arabidopsis.

Maize HEAT UP-REGULATED GENE 1 plays vital roles in heat stress tolerance.

Increasing temperature is one of the major threats to maize growth and yield globally. Under heat stress conditions, intracellular protein homeostasis is seriously disturbed, leading to accumulation of abnormally folded proteins, especially in the endoplasmic reticulum (ER). Molecular chaperones are vital players in the renaturation process and in preventing protein aggregation. However, heat stress tolerance-associated chaperones are not well documented in maize. Here, we characterized the biological roles of HEAT UP-REGULATED GENE 1 (ZmHUG1) in maize. ZmHUG1 encodes a heat-inducible holdase-type molecular chaperone localized in the ER. Knockout mutant of ZmHUG1 exhibited remarkably enhanced sensitivity to heat stress. Accordingly, the zmhug1 mutant showed severe ER stress under high temperature. MAIZE PRENYLATED RAB ACCEPTOR 1.C1 (ZmPRA1.C1) was identified as a client of ZmHUG1, and heat-induced aggregation of ZmPRA1.C1 was accelerated in the zmhug1 mutant. Furthermore, the expression of ZmHUG1 was rapidly transactivated by ER stress sensor BASIC LEUCINE ZIPPER DOMAIN 60 (bZIP60) when heat stress occurred. This study reveals a ZmHUG1-based thermo-protective mechanism in maize.

Genome-wide analysis of the apple family 1 glycosyltransferases identified a flavonoid-modifying UGT, MdUGT83L3, which is targeted by MdMYB88 and contributes to stress adaptation.

The plant family 1 UDP-glycosyltransferases (UGTs) are increasingly being investigated because of their contribution to plant secondary metabolism and other diverse biological roles. The apple (Malus domestica) is one of the most widely cultivated fruit trees with great economic importance. However, little is known regarding the apple UGTs. In this study, we identified 229 members of family 1 through a genome-wide analysis of the apple UGTs, which were clustered into 18 groups, from A to R. We also performed detailed analysis of 34 apple UGTs by quantitative RT-PCR, and discovered a number of stress-regulated UGTs. Among them, we characterized the role of MD09G1064900, also named MdUGT83L3, which was significantly induced by salt and cold. In vivo analysis showed that it has high activity towards cyanidin, and moderate activity towards quercetin and keampferol. Transgenic callus and regenerated apple plants overexpressing MdUGT83L3 showed enhanced tolerance to salt and cold treatments. Overexpression of MdUGT83L3 also increased anthocyanin accumulation in the callus tissues and enhanced ROS clearing upon exposure to salt and cold stresses. Furthermore, via yeast-one-hybrid assay, EMSA and CHIP analyses, we also found that MdUGT83L3 could be directly regulated by MdMYB88. Our study indicated that MdUGT83L3, under the regulation of MdMYB88, plays important roles in salt and cold stress adaptation via modulating flavonoid metabolism in apple.

Advances in the regulation of plant salt-stress tolerance by miRNA.

Salt stress significantly affects the growth, development, yield, and quality of plants. MicroRNAs (miRNAs) are involved in various stress responses via target gene regulation. Their role in regulating salt stress has also received significant attention from researchers. Various transcription factor families are the common target genes of plant miRNAs. Thus, regulating the expression of miRNAs is a novel method for developing salt-tolerant crops. This review summarizes plant miRNAs that mediate salt tolerance, specifically miRNAs that have been utilized in genetic engineering to modify plant salinity tolerance. The molecular mechanism by which miRNAs mediate salt stress tolerance merits elucidation, and this knowledge will promote the development of miRNA-mediated salt-tolerant crops and provide new strategies against increasingly severe soil salinization.

AhABI4s Negatively Regulate Salt-Stress Response in Peanut.

Soil salinity is one of the major factors that limit the area of cultivable land and yield potential of crops. The ability of salt tolerance varies with plant species. Peanut (Arachis hypogaea L.) is a moderately salt-sensitive and economically important crop, however, their biological processes involved in salt-stress response remain unclear. In this study, we investigated the role of A. hypogaea L. ABSCISIC ACID INSENSITIVE 4s (AhABI4s) in salt tolerance and elucidated its mode of action in peanuts. The results showed that the downregulation of AhABI4s via whole plant virus-induced gene silencing has enhanced the survival rate, biomass accumulation, and root/shoot ratio of peanut seedlings in response to salt-stress. Transcriptomics, quantitative proteomics, and phosphoproteomic analyses were performed using AhABI4s-silenced and Mock plants. The expression pattern of 15,247 genes, 1,900 proteins, and 2,620 phosphorylation sites were affected by silencing of AhABI4s in peanut leaf and root after sodium chloride (NaCl) treatment. Among them, 63 potential downstream target genes of ABI4 changed consistently at both transcription and translation levels, and the protein/phosphorylation levels of 31 ion transporters/channels were also affected. Electrophoretic mobility shift assays (EMSA) showed that ABI4 was able to bind to the promoters of HSP70, fructokinase (FRK), and pyruvate kinase (PK) coding genes in vitro. In addition, we also detected a binding preference of AhABI4 for CACT(G/T)GCA motif in the promoters of down-regulated genes in peanut leaf. Collectively, the potential downstream targets which were regulated at the levels of transcription and translation, binding preference, and in vivo phosphorylation sites that had been revealed in this study will provide new insight into the AhABI4s-mediated salt tolerance regulation mechanism in peanuts.

Salt responsive alternative splicing of a RING finger E3 ligase modulates the salt stress tolerance by fine-tuning the balance of COP9 signalosome subunit 5A.

Increasing evidence points to the tight relationship between alternative splicing (AS) and the salt stress response in plants. However, the mechanisms linking these two phenomena remain unclear. In this study, we have found that Salt-Responsive Alternatively Spliced gene 1 (SRAS1), encoding a RING-Type E3 ligase, generates two splicing variants: SRAS1.1 and SRAS1.2, which exhibit opposing responses to salt stress. The salt stress-responsive AS event resulted in greater accumulation of SRAS1.1 and a lower level of SRAS1.2. Comprehensive phenotype analysis showed that overexpression of SRAS1.1 made the plants more tolerant to salt stress, whereas overexpression of SRAS1.2 made them more sensitive. In addition, we successfully identified the COP9 signalosome 5A (CSN5A) as the target of SRAS1. CSN5A is an essential player in the regulation of plant development and stress. The full-length SRAS1.1 promoted degradation of CSN5A by the 26S proteasome. By contrast, SRAS1.2 protected CSN5A by competing with SRAS1.1 on the same binding site. Thus, the salt stress-triggered AS controls the ratio of SRAS1.1/SRAS1.2 and switches on and off the degradation of CSN5A to balance the plant development and salt tolerance. Together, these results provide insights that salt-responsive AS acts as post-transcriptional regulation in mediating the function of E3 ligase.

SiCEP3, a C-terminally encoded peptide from Setaria italica, promotes ABA import and signaling.

C-terminally encoded peptides (CEPs) are small peptides, typically post-translationally modified, and highly conserved in many species. CEPs are known to inhibit plant growth and development, but the mechanisms are not well understood. In this study, 14 CEPs were identified in Setaria italica and divided into two groups. The transcripts of most SiCEPs were more abundant in roots than in other detected tissues. SiCEP3, SiCEP4, and SiCEP5 were also highly expressed in panicles. Moreover, expression of all SiCEPs was induced by abiotic stresses and phytohormones. SiCEP3 overexpression and application of synthetic SiCEP3 both inhibited seedling growth. In the presence of abscisic acid (ABA), growth inhibition and ABA content in seedlings increased with the concentration of SiCEP3. Transcripts encoding eight ABA transporters and six ABA receptors were induced or repressed by synthetic SiCEP3, ABA, and their combination. Further analysis using loss-of-function mutants of Arabidopsis genes functioning as ABA transporters, receptors, and in the biosynthesis and degradation of ABA revealed that SiCEP3 promoted ABA import at least via NRT1.2 (NITRATE TRANSPORTER 1.2) and ABCG40 (ATP-BINDING CASSETTE G40). In addition, SiCEP3, ABA, or their combination inhibited the kinase activities of CEP receptors AtCEPR1/2. Taken together, our results indicated that the CEP-CEPR module mediates ABA signaling by regulating ABA transporters and ABA receptors in planta.

Identification and functional characterization of a new flavonoid synthase gene MdFLS1 from apple.

MAIN CONCLUSION: The flavonoid synthase gene MdFLS1 from apple, which possibly plays an important role in anthocyanin synthesis, accumulates in the purple-red branches of Malus 'Pink spire'. Flavonoid metabolism serves an important function in plant growth and development. In this study, we selected 20 varieties of apple lines, 10 green and ten red branches, from the plant nursery of Qingdao Agriculture Academy. Metabolite analysis revealed that large amounts of anthocyanins accumulated in the purple-red branches of M. 'Pink spire'. Real-time polymerase chain reaction showed that the expression of the flavonol synthase gene MdFLS1 was over 1500-fold higher in M. 'Pink spire' than in the other varieties. A single base A was inserted at the first three bases of the active binding site of MdFLS1 to prove that the purple-red colour of apple leaves and stems in M. 'Pink spire' may be caused by the inactivation of MdFLS1 protein. The results of in vitro enzymatic reaction revealed that the MdFLS1 protein lost its activity. MdFLS1 was expressed in Arabidopsis thaliana to explore further its functions. High-expression wild-type strains (OE1 and OE2) and high-expression strains of A-base insertion (A-OE1 and A-OE2) were obtained. Compared with the wild-type strains, the overexpression lines showed lighter tissue colour and less accumulation of anthocyanins. However, A-OE1 and A-OE2 showed no difference in colouration. In conclusion, we speculated that the MdFLS1 gene in M. 'Pink spire' cannot bind flavonoids, triggering the synthesis of anthocyanins in another branch of the flavonoid metabolic pathway and resulting in the purple-red colouration of apple leaves and stems. These results suggest that MdLS1 is a potential genetic target for breeding high-flavonoid apples in future cultivar development.

The Chloroplastic Small Heat Shock Protein Gene KvHSP26 Is Induced by Various Abiotic Stresses in Kosteletzkya virginica.

Small heat shock proteins (sHSPs) are a group of chaperone proteins existed in all organisms. The functions of sHSPs in heat and abiotic stress responses in many glycophyte plants have been studied. However, their possible roles in halophyte plants are still largely known. In this work, a putative sHSP gene KvHSP26 was cloned from K. virginica. Bioinformatics analyses revealed that KvHSP26 encoded a chloroplastic protein with the typical features of sHSPs. Amino acid sequence alignment and phylogenetic analysis demonstrated that KvHSP26 shared 30%-77% homology with other sHSPs from Arabidopsis, cotton, durian, salvia, and soybean. Quantitative real-time PCR (qPCR) assays exhibited that KvHSP26 was constitutively expressed in different tissues such as leaves, stems, and roots, with a relatively higher expression in leaves. Furthermore, expression of KvHSP26 was strongly induced by salt, heat, osmotic stress, and ABA in K. virginica. All these results suggest that KvHSP26 encodes a new sHSP, which is involved in multiple abiotic stress responses in K. virginica, and it has a great potential to be used as a candidate gene for the breeding of plants with improved tolerances to various abiotic stresses.