MrERF039 transcription factor plays an active role in the cold response of Medicago ruthenica as a sugar molecular switch.

Cold stress severely restricts plant development, causing significant agricultural losses. We found a critical transcription factor network in Medicago ruthenica was involved in plant adaptation to low-temperature. APETALA2/ethylene responsive factor (AP2/ERF) transcription factor MrERF039 was transcriptionally induced by cold stress in M. ruthenica. Overexpression of MrERF039 significantly increased the glucose and maltose content, thereby improving the tolerance of M. ruthenica. MrERF039 could bind to the DRE cis-acting element in the MrCAS15A promoter. Additionally, the methyl group of the 14th amino acid in MrERF039 was required for binding. Transcriptome analysis showed that MrERF039 acted as a sugar molecular switch, regulating numerous sugar transporters and sugar metabolism-related genes. In addition, we found that MrERF039 could directly regulate ß-amylase gene, UDP glycosyltransferase gene, and C2H2 zinc finger protein gene expression. In conclusion, these findings suggest that high expression of MrERF039 can significantly improve the cold tolerance of M. ruthenica root tissues during cold acclimation. Our results provide a new theoretical basis and candidate genes for breeding new legume forage varieties with high resistance.

Functional Analysis of Ion Transport Properties and Salt Tolerance Mechanisms of RtHKT1 from the Recretohalophyte Reaumuria trigyna.

Reaumuria trigyna is an endangered recretohalophyte and a small archaic feral shrub that is endemic to arid and semi-arid plateau regions of Inner Mongolia, China. Based on transcriptomic data, we isolated a high-affinity potassium transporter gene (RtHKT1) from R. trigyna, which encoded a plasma membrane-localized protein. RtHKT1 was rapidly up-regulated by high Na+ or low K+ and exhibited different tissue-specific expression patterns before and after stress treatment. Transgenic yeast showed tolerance to high Na+ or low K+, while transgenic Arabidopsis exhibited tolerance to high Na+ and sensitivity to high K+, or high Na+-low K+, confirming that Na+ tolerance in transgenic Arabidopsis depends on a sufficient external K+ concentration. Under external high Na+, high K+ and low K+ conditions, transgenic yeast accumulated more Na+-K+, Na+ and K+, while transgenic Arabidopsis accumulated less Na+-more K+, more Na+ and more Na+-K+, respectively, indicating that the ion transport properties of RtHKT1 depend on the external Na+-K+ environment. Salt stress induced up-regulation of some ion transporter genes (AtSOS1/AtHAK5/AtKUP5-6), as well as down-regulation of some genes (AtNHX1/AtAVP1/AtKUP9-12), revealing that multi-ion-transporter synergism maintains Na+/K+ homeostasis under salt stress in transgenic Arabidopsis. Overexpression of RtHKT1 enhanced K+ accumulation and prevented Na+ transport from roots to shoots, improved biomass accumulation and Chl content in salt-stressed transgenic Arabidopsis. The proline content and relative water content increased significantly, and some proline biosynthesis genes (AtP5CS1 and AtP5CS2) were also up-regulated in salt-stressed transgenic plants. These results suggest that RtHKT1 confers salt tolerance on transgenic Arabidopsis by maintaining Na+/K+ homeostasis and osmotic homeostasis.

Liquid-liquid phase separation in plants: Advances and perspectives from model species to crops.

Membraneless biomolecular condensates play important roles in both normal biological activities and responses to environmental stimuli in living organisms. Liquid‒liquid phase separation (LLPS) is an organizational mechanism that has emerged in recent years to explain the formation of biomolecular condensates. In the past decade, advances in LLPS research have contributed to breakthroughs in disease fields. By contrast, although LLPS research in plants has progressed over the past 5 years, it has been concentrated on the model plant Arabidopsis, which has limited relevance to agricultural production. In this review, we provide an overview of recently reported advances in LLPS in plants, with a particular focus on photomorphogenesis, flowering, and abiotic and biotic stress responses. We propose that many potential LLPS proteins also exist in crops and may affect crop growth, development, and stress resistance. This possibility presents a great challenge as well as an opportunity for rigorous scientific research on the biological functions and applications of LLPS in crops.

Two maize homologs of mammalian proton-coupled folate transporter, ZmMFS_1-62 and ZmMFS_1-73, are essential to salt and drought tolerance.

Folates are essential to the maintenance of normal life activities in almost all organisms. Proton-coupled folate transporter (PCFT), belonging to the major facilitator superfamily, is one of the three major folate transporter types widely studied in mammals. However, information about plant PCFTs is limited. Here, a genome-wide identification of maize PCFTs was performed, and two PCFTs, ZmMFS_1-62 and ZmMFS_1-73, were functionally investigated. Both proteins contained the typical 12 transmembrane helixes with N- and C-termini located in the cytoplasm, and were localized in the plasma membrane. Molecular docking analysis indicated their binding activity with folates via hydrogen bonding. Interference with ZmMFS_1-62 and ZmMFS_1-73 in maize seedlings through virus-induced gene silencing disrupted folate homeostasis, mainly in the roots, and reduced tolerance to drought and salt stresses. Moreover, a molecular chaperone protein, ZmHSP20, was found to interact with ZmMFS_1-62 and ZmMFS_1-73, and interference with ZmHSP20 in maize seedlings also led to folate disruption and increased sensitivity to drought and salt stresses. Overall, this is the first report of functional identification of maize PCFTs, which play essential roles in salt and drought stress tolerance, thereby linking folate metabolism with abiotic stress responses in maize.

The histone methyltransferase SUVR2 promotes DSB repair via chromatin remodeling and liquid-liquid phase separation.

Maintaining genomic integrity and stability is particularly important for stem cells, which are at the top of the cell lineage origin. Here, we discovered that the plant-specific histone methyltransferase SUVR2 maintains the genome integrity of the root tip stem cells through chromatin remodeling and liquid-liquid phase separation (LLPS) when facing DNA double-strand breaks (DSBs). The histone methyltransferase SUVR2 (MtSUVR2) has histone methyltransferase activity and catalyzes the conversion of histone H3 lysine 9 monomethylation (H3K9me1) to H3K9me2/3 in vitro and in Medicago truncatula. Under DNA damage, the proportion of heterochromatin decreased and the level of DSB damage marker γ-H2AX increased in suvr2 mutants, indicating that MtSUVR2 promotes the compaction of the chromatin structure through H3K9 methylation modification to protect DNA from damage. Interestingly, MtSUVR2 was induced by DSBs to phase separate and form droplets to localize at the damage sites, and this was confirmed by immunofluorescence and fluorescence recovery after photobleaching experiments. The IDR1 and low-complexity domain regions of MtSUVR2 determined its phase separation in the nucleus, whereas the IDR2 region determined the interaction with the homologous recombinase MtRAD51. Furthermore, we found that MtSUVR2 drove the phase separation of MtRAD51 to form "DNA repair bodies," which could enhance the stability of MtRAD51 proteins to facilitate error-free homologous recombination repair of stem cells. Taken together, our study reveals that chromatin remodeling-associated proteins participate in DNA repair through LLPS.

Transformation of a marker-free and vector-free antisense ACC oxidase gene cassette into melon via the pollen-tube pathway.

PURPOSE OF WORK: Melons have short shelf-lives due to fruit ripening caused by ethylene production. The 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene is essential for ethylene biosynthesis. As fruit ripening in other fruit crops can be deterred by down-regulation of ACC oxidase expression, we have carried out similar work to improve fruit quality and shelf-life of the melon Cucumis melo. A marker-free and vector-free antisense 1-aminocyclopropane-1-carboxylic acid oxidase construct was transformed into melon via the pollen-tube pathway. Based on phenotype analysis together with RT-PCR data, a transformation frequency of 0.7% was achieved. The transgenic fruits showed respiration rate and endogenous ethylene production level at approx. 15 and 6% of those of wild-type fruits, respectively. These fruits also demonstrated improved flesh firmness and exhibited extended shelf-life of 30 days compared to less than 12 days for the wild type fruits.

Melatonin Enhances Seed Germination and Seedling Growth of Medicago sativa Under Salinity via a Putative Melatonin Receptor MsPMTR1.

Alfalfa (Medicago sativa L.) is an important forage crop, and salt stress is a major limiting factor in its yield. Melatonin (MT) is a multi-regulatory molecule in plants. We showed that basal MT content was positively correlated with the salt tolerance degree of different alfalfa varieties. MT and its precursor 5-HT fully recovered seed germination while partially ameliorated seedling growth of salt-stressed alfalfa. The 5-HT showed some divergent effects from MT with regards to growth amelioration under salinity. Salt stress caused stunted plant growth in soil culture, while MT ameliorated it by elevating plant height, fresh weight, branching number, and chlorophyll content. Silencing of a putative MT receptor, MsPMTR1, which was shown to be membrane-localized, abolished the ameliorative effects of MT on salt-stressed alfalfa seedling growth, while overexpression of MsPMTR1 improved plant growth under salt stress. The RNA sequencing analysis showed that nine pathway genes were specifically induced by MT treatment compared with salt stress. These MT-responsive differentially expressed genes include basal metabolic pathway genes, such as "ribosome, elongation factor," "sugar and lipid metabolism," and "photosynthesis" and stress-related genes encoding "membrane integrity" related proteins, heat shock protein, peroxidase/oxidoreductase, and protease. Several abiotic stress response-related genes, such as DRE, ARF, HD-ZF, MYB, and REM were repressed by NaCl treatment while induced by MT treatment. In summary, we demonstrated the importance of MsPMTR1 in MT-mediated salt tolerance in alfalfa, and we also analyzed the regulatory mechanism of MT during alfalfa seed germination under salt stress.

Molecular characterisation and expression analysis of NAC transcription factor genes in wild Medicago falcata under abiotic stresses.

The No apical meristem-Arabidopsis transcription activation factor-Cup-shaped cotyledon (NAC) proteins play vital roles in plant development processes and responses to abiotic stress. In this study, 146 unigenes were identified as NAC genes from wild Medicago falcata L. by RNA sequencing. Among these were 30 full-length NACs, which, except for MfNAC63, MfNAC64 and MfNAC91, contained a complete DNA-binding domain and a variable transcriptional activation region. Sequence analyses of MfNACs along with their Arabidopsis thaliana (L.) Heynh. counterparts allowed these proteins to be phylogenetically classified into nine groups. MfNAC35, MfNAC88, MfNAC79, MfNAC26 and MfNAC95 were found to be stress-responsive genes. The eight MfNAC genes that were chosen for further analysis had different expression abilities in the leaves, stems and roots of M. falcata. Additionally, their expression levels were regulated by salinity, drought and cold stress, and ABA. This study will be useful for understanding the roles of MfNACs in wild M. falcata and could provide important information for the selection of candidate genes associated with stress tolerance.

Ethylene Enhances Seed Germination and Seedling Growth Under Salinity by Reducing Oxidative Stress and Promoting Chlorophyll Content via ETR2 Pathway.

Alfalfa (Medicago sativa L.) is an important forage, and salinity is a major stress factor on its yield. In this study, we show that osmotic stress retards alfalfa seedling growth, while ionic/oxidative stress reduces its seed germination. Ethylene treatment can recover the germination rate of alfalfa seeds under salt stress, while ethylene inhibitor silver thiosulfate exacerbates salt effects. ETH reduces the accumulation of MDA and H2O2 and increases POD activity. ETH and ACC improve the salt tolerance of alfalfa by increasing proline content under salt stress. In contrast, STS inhibits alfalfa seed germination by reducing POD activity. NaCl treatment reduces chlorophyll content in alfalfa leaves, while ETH and ACC can increase the chlorophyll content and promote seedling growth. ETH promotes the growth of alfalfa in saline condition by reducing the expression of MsACO and MsERF8 genes, while increases its germination rate by upregulating MsERF11 gene. Silencing of MsETR2, a putative ethylene receptor gene in alfalfa, abolishes ethylene triggered tolerance to salt stress. In summary, we show that ethylene improves salt tolerance in alfalfa via MsETR2 dependent manner, and we also analyze the regulatory mechanism of ethylene during germination of alfalfa seeds under salt stress.

Corrigendum: Ethylene Enhances Seed Germination and Seedling Growth Under Salinity by Reducing Oxidative Stress and Promoting Chlorophyll Content via ETR2 Pathway.

[This corrects the article DOI: 10.3389/fpls.2020.01066.].

miR403a and SA Are Involved in NbAGO2 Mediated Antiviral Defenses Against TMV Infection in Nicotiana benthamiana.

RNAi (RNA interference) is an important defense response against virus infection in plants. The core machinery of the RNAi pathway in plants include DCL (Dicer Like), AGO (Argonaute) and RdRp (RNA dependent RNA polymerase). Although involvement of these RNAi components in virus infection responses was demonstrated in Arabidopsis thaliana, their contribution to antiviral immunity in Nicotiana benthamiana, a model plant for plant-pathogen interaction studies, is not well understood. In this study, we investigated the role of N. benthamiana NbAGO2 gene against TMV (Tomato mosaic virus) infection. Silencing of NbAGO2 by transient expression of an hpRNA construct recovered GFP (Green fluorescent protein) expression in GFP-silenced plant, demonstrating that NbAGO2 participated in RNAi process in N. benthamiana. Expression of NbAGO2 was transcriptionally induced by both MeSA (Methylsalicylate acid) treatment and TMV infection. Down-regulation of NbAGO2 gene by amiR-NbAGO2 transient expression compromised plant resistance against TMV infection. Inhibition of endogenous miR403a, a predicted regulatory microRNA of NbAGO2, reduced TMV infection. Our study provides evidence for the antiviral role of NbAGO2 against a Tobamovirus family virus TMV in N. benthamiana, and SA (Salicylic acid) mediates this by induction of NbAGO2 expression upon TMV infection. Our data also highlighted that miR403a was involved in TMV defense by regulation of target NbAGO2 gene in N. Benthamiana.

Cloning and characterization of a Ca(2+)/H(+) exchanger from the halophyte Salicornia europaea L.

The calcium ion (Ca(2+)), which functions as a second messenger, plays an important role in plants' responses to various abiotic stresses, and Ca(2+)/H(+) exchangers (CAXs) are an important part of this process. In this study, we isolated and characterized a putative Ca(2+)/H(+) exchanger gene (SeCAX3) from Salicornia europaea L., a succulent, leafless euhalophyte. The SeCAX3 open reading frame was 1368 bp long and encoded a 455-amino-acid polypeptide that showed 67.9% similarity to AtCAX3. SeCAX3 was expressed in the shoots and roots of S. europaea. Expression of SeCAX3 was up-regulated by Ca(2+), Na(+), sorbitol, Li(+), abscisic acid, and cold treatments in shoots, but down-regulated by Ca(2+), sorbitol, abscisic acid, and cold treatments in roots. When SeCAX3 was transformed into a Ca-sensitive yeast strain, the transformed cells were able to grow in the presence of 200 mM Ca(2+). Furthermore, SeCAX3 conferred drought, salt, and cold tolerance in yeast. Compared with the control strains, the yeast transformants expressing SeCAX3 were able to grow well in the presence of 30 mM Li(+), 150 mM Mg(2+), or 6 mM Ba(2+). These results showed that the expression of SeCAX3 in yeast suppressed its Ca(2+) hypersensitivity and conferred tolerance to Mg(2+) and Ba(2+). Together, these findings suggest that SeCAX3 might be a Ca(2+) transporter that plays a role in regulating cation tolerance and the responses of S. europaea to various abiotic stresses.

Isolation and characterization of two Medicago falcate AP2/EREBP family transcription factor cDNA, MfDREB1 and MfDREB1s.

DREB transcription factors play an important role in tolerance to abiotic stress in high plants. In this work, two new DRE-binding protein genes MfDREB1 and MfDREB1s cDNA that encoded an AP2/EREBP type transcription factor were isolated by RT-PCR from Medicago falcate seedlings. Sequence analysis showed MfDREB1 and MfDREB1s were almost identical except that there was a 202bp fragment at the 3' end of the MfDREB1s cDNA that is absent in MfDREB1 cDNA. The MfDREB1 has a open reading frame of 651bp, which encodes 216 amino acid residues. The putative protein is deduced a predicted molecular mass of 24.6kDa and a pI of 5.95. The MfDREB1s has a open reading frame of 555bp, the putative protein is 184 amino acid long with a predicted molecular weight of 20.8kDa, pI 9.11. The Protein Blast data revealed that the two proteins can be classified as a typical member of the AP2/EREBP family of DNA-binding proteins. The comparison of the MfDREB1 cDNA and MfDREB1s cDNA with their corresponding genes in genomic DNA showed that the size and nucleotide sequence of the cDNA was identical to that of the genomic DNA. This suggested that the genomic MfDREB1 gene and MfDREB1s gene had no introns. Southern blot analysis indicated that MfDREB1 and MfDREB1s are multi-copy genes in Medicago falcate genome. Northern blot analysis indicated that the MfDREB1 and MfDREB1s genes were induced by low temperature stress.