Regulation of plant resistance to salt stress by the SnRK1-dependent splicing factor SRRM1L.

Most splicing factors are extensively phosphorylated but their physiological functions in plant salt resistance are still elusive. We found that phosphorylation by SnRK1 kinase is essential for SRRM1L nuclear speckle formation and its splicing factor activity in plant cells. In Arabidopsis, loss-of-function of SRRM1L leads to the occurrence of alternative pre-mRNA splicing events and compromises plant resistance to salt stress. In Arabidopsis srrm1l mutant line, we identified an intron-retention Nuclear factor Y subunit A 10 (NFYA10) mRNA variant by RNA-Seq and found phosphorylation-dependent RNA-binding of SRRM1L is indispensable for its alternative splicing activity. In the wild-type Arabidopsis, salt stress can activate SnRK1 to phosphorylate SRRM1L, triggering enrichment of functional NFYA10.1 variant to enhance plant salt resistance. By contrast, the Arabidopsis srrm1l mutant accumulates nonfunctional NFYA10.3 variant, sensitizing plants to salt stress. In summary, this work deciphered the molecular mechanisms and physiological functions of SnRK1-SRRM1L-NFYA10 module, shedding light on a regulatory pathway to fine-tune plant adaptation to abiotic stress at the post-transcriptional and post-translational levels.

Unravelling Species Diversity and Pathogenicity of Fusarium spp. Associated with Soybean Leaf and Root in Heilongjiang Province, China.

Soybean (Glycine max L.) holds significant global importance and is extensively cultivated in Heilongjiang Province, China. Soybean can be infected by Fusarium species, causing root rot, seed decay, stem rot, and leaf blight. In 2021 to 2022, a field survey of soybean diseases was carried out in 11 regions of Heilongjiang Province, and 186 soybean leaves with leaf blight symptoms and 123 soybean roots with root rot symptoms were collected. Unexpectedly, a considerable number of Fusarium isolates were obtained not only from root samples but also from leaf samples. A total of 584 Fusarium isolates (416 from leaves and 168 from roots) were obtained and identified as 18 Fusarium species based on morphological features and multilocus phylogenetic analyses with tef1 and rpb2 sequences. Fusarium graminearum and Fusarium sp. 1 in FOSC were the dominant species within soybean leaf and root samples, respectively. Pathogenicity tests were conducted for all Fusarium isolates on both soybean leaves and roots. Results showed that F. graminearum, F. ipomoeae, F. citri, F. compactum, F. flagelliforme, F. acuminatum, and F. sporotrichioides were pathogenic to both soybean leaves and roots. F. solani, F. avenaceum, F. pentaseptatum, F. serpentinum, F. annulatum, and Fusarium sp. 1 in FOSC were pathogenic to soybean roots, not to leaves. To our knowledge, this is the first study to thoroughly investigate soybean-associated Fusarium populations in leaves and roots in Heilongjiang Province.

Quantitative Phosphoproteomic Analysis Provides Insights into the Sodium Bicarbonate Responsiveness of Glycine max.

Sodium bicarbonate stress caused by NaHCO3 is one of the most severe abiotic stresses affecting agricultural production worldwide. However, little attention has been given to the molecular mechanisms underlying plant responses to sodium bicarbonate stress. To understand phosphorylation events in signaling pathways triggered by sodium bicarbonate stress, TMT-labeling-based quantitative phosphoproteomic analyses were performed on soybean leaf and root tissues under 50 mM NaHCO3 treatment. In the present study, a total of 7856 phosphopeptides were identified from cultivated soybeans (Glycine max L. Merr.), representing 3468 phosphoprotein groups, in which 2427 phosphoprotein groups were newly identified. These phosphoprotein groups contained 6326 unique high-probability phosphosites (UHPs), of which 77.2% were newly identified, increasing the current soybean phosphosite database size by 43.4%. Among the phosphopeptides found in this study, we determined 67 phosphopeptides (representing 63 phosphoprotein groups) from leaf tissue and 554 phosphopeptides (representing 487 phosphoprotein groups) from root tissue that showed significant changes in phosphorylation levels under sodium bicarbonate stress (fold change >1.2 or <0.83, respectively; p < 0.05). Localization prediction showed that most phosphoproteins localized in the nucleus for both leaf and root tissues. GO and KEGG enrichment analyses showed quite different enriched functional terms between leaf and root tissues, and more pathways were enriched in the root tissue than in the leaf tissue. Moreover, a total of 53 different protein kinases and 7 protein phosphatases were identified from the differentially expressed phosphoproteins (DEPs). A protein kinase/phosphatase interactor analysis showed that the interacting proteins were mainly involved in/with transporters/membrane trafficking, transcriptional level regulation, protein level regulation, signaling/stress response, and miscellaneous functions. The results presented in this study reveal insights into the function of post-translational modification in plant responses to sodium bicarbonate stress.

The Role of GmSnRK1-GmNodH Module in Regulating Soybean Nodulation Capacity.

SnRK1 protein kinase plays hub roles in plant carbon and nitrogen metabolism. However, the function of SnRK1 in legume nodulation and symbiotic nitrogen fixation is still elusive. In this study, we identified GmNodH, a putative sulfotransferase, as an interacting protein of GmSnRK1 by yeast two-hybrid screen. The qRT-PCR assays indicate that GmNodH gene is highly expressed in soybean roots and could be induced by rhizobial infection and nitrate stress. Fluorescence microscopic analyses showed that GmNodH was colocalized with GsSnRK1 on plasma membrane. The physical interaction between GmNodH and GmSnRK1 was further verified by using split-luciferase complementary assay and pull-down approaches. In vitro phosphorylation assay showed that GmSnRK1 could phosphorylate GmNodH at Ser193. To dissect the function and genetic relationship of GmSnRK1 and GmNodH in soybean, we co-expressed the wild-type and mutated GmSnRK1 and GmNodH genes in soybean hairy roots and found that co-expression of GmSnRK1/GmNodH genes significantly promoted soybean nodulation rates and the expression levels of nodulation-related GmNF5α and GmNSP1 genes. Taken together, this study provides the first biological evidence that GmSnRK1 may interact with and phosphorylate GmNodH to synergistically regulate soybean nodulation.

Epicoccum spp. Causing Maize Leaf Spot in Heilongjiang Province, China.

Maize leaf spot occurs worldwide and affects maize production. Maize can be infected by several pathogens causing leaf spot, such as Bipolaris zeicola, Bipolaris maydis, Curvularia species, Alternaria species, etc. In the current study, 30 Epicoccum isolates recovered from symptomatic maize leaves were identified based on morphological characteristics, pathogenicity, and multilocus sequence analyses of nuLSU, ITS, tub2, and rpb2. These maize isolates were grouped into five Epicoccum species, including E. nigrum, E. layuense, E. sorghinum, E. latusicollum, and E. pneumoniae. Pathogenicity tests showed that all five Epicoccum species could produce small ellipse- and spindle-shaped spots on maize leaves. The lesion center was grayish yellow to dark gray and surrounded by a chlorotic area. Furthermore, the Epicoccum isolates exhibited high pathogenicity to 20 main maize varieties of Heilongjiang Province but showed different sensitivities to the commonly used fungicides carbendazim and tebuconazole. In addition, these Epicoccum isolates showed different production capacity of pectinase, cellulase, protease, amylase, laccase, and gelatinase, but all showed high lipase activity. This is the first report globally of E. layuense, E. latusicollum, and E. pneumoniae as causal agents of maize leaf spot. E. pneumoniae was first reported as a plant pathogen.

Quantitative phosphoproteomics reveals the role of wild soybean GsSnRK1 as a metabolic regulator under drought and alkali stresses.

Drought and alkali stresses cause detrimental effects on plant growth and development. SnRK1 protein kinases act as key energy and stress sensors by phosphorylation-mediated signaling in the regulation of plant defense reactions against adverse environments. To understand SnRK1-dependent phosphorylation events in signaling pathways triggered by abiotic factors, we employed quantitative phosphoproteomics to compare the global changes in phosphopeptides and phosphoproteins in 2kinm mutant Arabidopsis (SnRK1.1 T-DNA knockout and SnRK1.2 knockdown by ß-estradiol-induced RNAi) complemented with wild soybean GsSnRK1(wt) or dominant negative mutant GsSnRK1(K49M) in response to drought and alkali stresses. Among 4014 phosphopeptides (representing 2380 phosphoproteins) identified in this study, we finalized 74 phosphopeptides (representing 61 phosphoproteins), and 75 phosphopeptides (representing 57 phosphoproteins) showing significant changes in phosphorylation levels under drought and alkali treatments respectively. Function enrichment and protein-protein interaction analyses indicated that the differentially-expressed phosphoproteins (DPs) under drought and alkali stresses were mainly involved in signaling transduction, stress response, carbohydrate and energy metabolism, transport and membrane trafficking, RNA splicing and processing, DNA binding and gene expression, and protein synthesis/folding/degradation. These results provide assistance to identify bona fide and novel SnRK1 phosphorylation substrates and shed new light on the biological functions of SnRK1 kinase in responses to abiotic stresses. SIGNIFICANCE: These results provide assistance to identify novel SnRK1 phosphorylation substrates and regulatory proteins, and shed new light on investigating the potential roles of reversible phosphorylation in plant responses to abiotic stresses.

Agromyces mariniharenae sp. nov., a novel indole-acetic acid producing actinobacterium isolated from marine sand.

A Gram-positive, aerobic, heterotrophic, non-endospore-forming, rod-shaped and indole-acetic acid-producing strain, designated NEAU-184T, was isolated from marine sand collected in Sanya, PR China, and its taxonomic position was investigated using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequence data indicated that strain NEAU-184T should be assigned to the genus Agromyces and formed a distinct branch with its closest neighbour, Agromyces iriomotensis NBRC 106452T (99.1 %). 2,4-Diaminobutyric acid, d-alanine, d-glutamic acid and glycine were detected in cell-wall hydrolysate and glucose, rhamnose and xylose were detected in whole-cell hydrolysate. The polar lipids were found to contain diphosphatidylglycerol, glycolipid, phosphatidylglycerol and two unidentified lipids. The major menaquinone was MK-12 and the minor menaquinones were MK-13 and MK-11. The predominant fatty acids were anteiso-C17 : 0, anteiso-C15 : 0 and iso-C16 : 0. The DNA G+C content was 71.5 mol%. Furthermore, the strain could be clearly distinguished from its closely related type strains by the combination of DNA-DNA hybridization results and some phenotypic characteristics. Meanwhile, the strain has the ability to produce indole-acetic acid (0.334mg ml-1). Therefore, strain NEAU-184T represents a novel species of the genus Agromyces, for which the name Agromyces mariniharenae sp. nov. is proposed, with strain NEAU-184T (=CGMCC 4.7505T=JCM 32546T) as the type strain.

Transcriptome sequencing of wild soybean revealed gene expression dynamics under low nitrogen stress.

Nitrogen is one of the essential elements for plant growth. Wild soybeans (Glycine soja) have strong abilities to survive in harsh and barren environments, and hence become ideal plant model for studying plant adaptability to low nitrogen (LN) conditions. In this study, we analyzed and compared the transcriptomes of wild soybean subjected to LN treatments. We totally identified 1095 (681 up and 414 down) and 5490 (2998 up and 2492 down) differentially expressed genes (DEGs) in the aerial parts (leaf and stem, LS) and roots, respectively. Gene ontology classification analysis revealed that the categories related to LN stress (including oxidation reduction, transcriptional regulation, membrane, and protein phosphorylation) were highly enriched among DEGs. In addition, a total of 784 transcription factor (TF) and 84 transporter protein (TP) genes were determined in LS DEGs, of which some TF genes (NAC1, NAC35, ZFP1, CIM1, and WRKY25) and TP genes like NRT2.5 (nitrate transporter) and ABCC12 (ABC transporter) were widely upregulated under LN stress. Nevertheless, a total of 3859 TF and 370 TP genes were identified in root DEGs, of which some TF genes (NAC6, NAC14, MYB29, MYB92, bZIP62, bZIP72, WRKY60, WRKY58) and TP genes like NRT2.4 and HAK5 (potassium transporter) were upregulated under LN stress. These findings suggest that the identified DEGs may play vital roles in plant responses to LN stress, providing important genetic resources for further functional dissection of plant molecular mechanisms to LN stress.

Isolation and screening of stress-resistant endophytic fungus strains from wild and cultivated soybeans in cold region of China.

In this study, we firstly reported the large-scale screening and isolation of endophytic fungi from nine wild and six cultivated soybeans in the cold regions of China. We totally isolated 302 endophytic fungal strains, of which 215 strains are isolated from the wild soybeans and 87 are identified from cultivated soybeans. Among these endophytic fungal strains, in the roots, stems, and leaves, 24.17% were isolated from roots, 28.8% were isolated from stems, and 47.01% were isolated from leaves, respectively. Most endophytic fungal strains isolated from the wild soybean roots were the species of Fusarium genus, and the fungal strains in the stems were the species of ascomycetes and Fusarium fungi, whereas most strains in the leaves were Alternaria fungi. To analyze the taxonomy of the obtained samples, we sequenced and compared their rDNA internal transcribed spacer (ITS) sequences. The data showed that 6 strains are putatively novel strains exhibiting ≤ 97% homology with the known strains. We next measured the secondary metabolites produced by the different strains and we found 11 strains exhibited high-performance synthesis of triterpenoids, phenols, and polysaccharides. Furthermore, we characterized their tolerance to abiotic stresses. The results indicated that 4 strains exhibited high tolerance to cadmium, and some strains exhibited resistance to acid, and alkali. The results of the study could facilitate the further exploration of the diversity of plant endophytic fungi and the potential applications of the fungi to practical agriculture and medicine industries. KEY POINTS: • 302 endophytic fungal strains isolated from wild soybean and cultivated soybean • 11 strains had high contents of triterpenoids, phenols, and polysaccharides • 4 strains exhibited high Cd tolerance, and a few strains with strong tolerance to acid and alkali solution.

Functional identification of BpMYB21 and BpMYB61 transcription factors responding to MeJA and SA in birch triterpenoid synthesis.

BACKGROUND: Triterpenoids from birch (Betula platyphylla Suk.) exert antitumor and anti-HIV activities. Due to the complexity of plant secondary metabolic pathways, triterpene compounds in plants is not always determined by a single gene; they may be controlled by polygene quantitative traits. Secondary metabolism related to terpenoids involves tissue specificity and localisation of key biosynthetic enzymes. Terpene synthesis is influenced by light, hormones and other signals, as well as upstream transcription factor regulation. RESULTS: Anchor Herein, we identified and characterised two birch MYB transcription factors (TFs) that regulate triterpenoid biosynthesis. BpMYB21 and BpMYB61 are R2R3 TFs that positively and negatively regulate responses to methyl-jasmonate (MeJA) and salicyclic acid (SA), respectively. Expression of BpMYB21 and BpMYB61 was elevated in leaves and stems more than roots during July/August in Harbin, China. BpMYB21 expression was increased by abscisic acid (ABA), MeJA, SA and gibberellins (GAs). BpMYB61 expression in leaves and BpMYB21 expression in stems was reduced by ABA, MeJA and SA, while GAs, ethylene, and injury increased BpMYB61 expression. BpMYB21 was localised in nuclei, while BpMYB61 was detected in cell membranes and nuclei. Promoters for both BpMYB21 (1302 bp) and BpMYB61 (850 bp) were active. BpMYB21 and BpMYB61 were ligated into pYES3, introduced into AnchorINVScl (yeast strain without exogenous genes), INVScl-pYES2-SSAnchorAnchor (transgenic yeast strain harbouring the SS gene from birch), and INVScl-pYES2-SE (transgenic yeast strain harbouring the SE gene from birch), and the squalene content was highest in AnchorINVScl-pYES-MYB21-SS (transgenic yeast strain harbouring SS and MYB21 genes) and INVScl-pYES3-MYB61 (transgenic yeast strain harbouring the MYB61 gene). In BpMYB21 transgenic birch key triterpenoid synthesis genes were up-regulated, and in BpMYB61 transgenic birch AnchorFPS (farnesyl pyrophosphate synthase) and SS (squalene synthase) were up-regulated, but HMGR (3-hydroxy-3-methylglutaryl coenzyme a reductase), BPWAnchor (lupeol synthase), SE (squalene epoxidase) and BPY (b-amyrin synthase) were down-regulated. Both BpMYB21 and BpMYB61 specifically activate SE and BPX (cycloartenol synthase synthesis) promoters. CONCLUSIONS: These findings support further functional characterisation of R2R3-MYB genes, and illuminate the regulatory role of BpMYB21 and BpMYB61 in the synthesis of birch triterpenoids.

Genome-wide identification and expression analyses of nitrate transporter family genes in wild soybean (Glycine soja).

Nitrate transporters (NRTs) are important channel proteins facilitating cross-membrane movement of small molecules like NO3- which is a critical nutrient for all life. However, the classification and evolution of nitrate transporters in the legume plants are still elusive. In this study, we surveyed the wild soybean (G. soja) genomic databases and identified 120 GsNRT1 and 5 GsNRT2 encoding genes. Phylogenetic analyses show that GsNRT1 subfamily is consisted of eight clades (NPF1 to NPF8), while GsNRT2 subfamily has only one clade. Gene chromosomal location and evolutionary historic analyses indicate that GsNRT genes are unevenly distributed on 19 out of 20 G. soja chromosomes and segmental duplications may take a major part in the expansion of GsNRT family. Investigations of gene structure and protein motif compositions suggest that GsNRT family members are highly conserved in structures of both gene and protein levels. In addition, we analyzed the spatial expression patterns of representative GsNRT genes and their responses to exogenous nitrogen and carbon supplies and different abiotic stresses. The qRT-PCR data indicated that 16 selected GsNRT genes showed various expression levels in the roots, stems, leaves, and pods of young G. soja plants, and these genes were regulated by not only nitrogen and carbohydrate nutrients but also NaCl, NaHCO3, abscisic acid (ABA), and salicylic acid (SA). These results suggest that GsNRT genes may be involved in the regulation of plant growth, development, and adaptation to environmental stresses, and the study will shed light on functional dissection of plant nitrate transporter proteins in the future.

Expression characteristics and function of CAS and a new beta-amyrin synthase in triterpenoid synthesis in birch (Betula platyphylla Suk.).

Triterpenoids produced by the secondary metabolism of Betula platyphylla Suk. exhibit important pharmacological activities, such as tumor inhibition, anti-HIV, and defense against pathogens, but the yield of natural synthesis is low, which is insufficient to meet people's needs. In this study, we identified two OSC genes of birch, named as BpCAS and Bpß-AS, respectively. The expression of BpCAS and Bpß-AS were higher levels in roots and in stems, respectively, and they induced expression in response to methyl jasmonate (MeJA), gibberellin (GA3), abscisic acid (ABA), ethylene and mechanical damage. The function of the two genes in the triterpene synthesis of birch was identified by reverse genetics. The inhibition of Bpß-AS gene positively regulates synthesis of betulinic acid. BpCAS interference can significantly promote the upregulation of lupeol synthase gene (BPW) and ß-amyrin synthase gene(BPY), and conversion of 2,3-oxidosqualene to the downstream products betulinic acid and oleanolic acid. This study provided a basis for the genetic improvement of triterpenoid synthesis in birch through genetic engineering. The obtained transgenic birch and suspension cells served as material resources for birch triterpenoid applications in further.

GsSnRK1 interplays with transcription factor GsERF7 from wild soybean to regulate soybean stress resistance.

Although the function and regulation of SnRK1 have been studied in various plants, its molecular mechanisms in response to abiotic stresses are still elusive. In this work, we identified an AP2/ERF domain-containing protein (designated GsERF7) interacting with GsSnRK1 from a wild soybean cDNA library. GsERF7 gene expressed dominantly in wild soybean roots and was responsive to ethylene, salt, and alkaline. GsERF7 bound GCC cis-acting element and could be phosphorylated on S36 by GsSnRK1. GsERF7 phosphorylation facilitated its translocation from cytoplasm to nucleus and enhanced its transactivation activity. When coexpressed in the hairy roots of soybean seedlings, GsSnRK1(wt) and GsERF7(wt) promoted plants to generate higher tolerance to salt and alkaline stresses than their mutated species, suggesting that GsSnRK1 may function as a biochemical and genetic upstream kinase of GsERF7 to regulate plant adaptation to environmental stresses. Furthermore, the altered expression patterns of representative abiotic stress-responsive and hormone-synthetic genes were determined in transgenic soybean hairy roots after stress treatments. These results will aid our understanding of molecular mechanism of how SnRK1 kinase plays a cardinal role in regulating plant stress resistances through activating the biological functions of downstream factors.

Cloning and expression of BpMYC4 and BpbHLH9 genes and the role of BpbHLH9 in triterpenoid synthesis in birch.

BACKGROUND: Birch (Betula platyphylla Suk.) contains triterpenoids with anti-HIV and anti-tumor pharmacological activities. However, the natural abundance of these triterpenoids is low, and their chemical synthesis is costly. Transcription factors have the ability to regulate the metabolite pathways of triterpenoids via multi-gene control, thereby improving metabolite yield. Thus, transcription factors have the potential to facilitate the production of birch triterpenoids. Plant bHLH (basic helix-loop-helix) transcription factors play important roles in stress response and secondary metabolism. RESULTS: In this study, we cloned two genes, BpMYC4 and BpbHLH9, that encode bHLH transcription factors in Betula platyphylla Suk. The open reading frame (ORF) of BpMYC4 was 1452 bp and encoded 483 amino acids, while the ORF of BpbHLH9 was 1140 bp and encoded 379 amino acids. The proteins of BpMYC4 and BpbHLH9 were localized in the cell membrane and nucleus. The tissue-specific expression patterns revealed that BpMYC4 expression in leaves was similar to that in the stem and higher than in the roots. The expression of BpbHLH9 was higher in the leaves than in the root and stem. The expressions of BpMYC4 and BpbHLH9 increased after treatment with abscisic acid, methyl jasmonate, and gibberellin and decreased after treatment with ethephon. The promoters of BpMYC4 and BpbHLH9 were isolated using a genome walking approach, and 900-bp and 1064-bp promoter sequences were obtained for BpMYC4 and BpbHLH9, respectively. The ORF of BpbHLH9 was ligated into yeast expression plasmid pYES3 and introduced into INVScl and INVScl1-pYES2-SS yeast strains. The squalene and total triterpenoid contents in the different INVScl1 transformants decreased in the following order INVScl1-pYES-SS-bHLH9 > INVScl1-pYES3-bHLH9 > INVScl1-pYES2- BpSS > INVScl-pYES2. In BpbHLH9 transgenic birch, the relative expression of the genes that encodes for enzymes critical for triterpenoid synthesis showed a different level of up-regulation compair with wild birch(control), and the contents of betulinic acid, oleanolic acid and betulin in bHLH9-8 transgenic birch were increased by 11.35%, 88.34% and 23.02% compared to in wild birch, respectively. CONCLUSIONS: Our results showed that the modulation of BpbHLH9 by different hormones affected triterpenoid synthesis and triterpenoid contents. This is the first report of the cloning of BpbHLH9, and the findings are important for understanding the regulatory role of BpbHLH9 in the synthesis of birch triterpenoids.

GsERF6, an ethylene-responsive factor from Glycine soja, mediates the regulation of plant bicarbonate tolerance in Arabidopsis.

MAIN CONCLUSION: This is an original study focus on ERF gene response to alkaline stress. GsERF6 functions as transcription factor and significantly enhanced plant tolerance to bicarbonate (HCO 3 (-) ) in transgenic Arabidopsis . Alkaline stress is one of the most harmful, but little studied environmental factors, which negatively affects plant growth, development and yield. The cause of alkaline stress is mainly due to the damaging consequence of high concentration of the bicarbonate ion, high-pH, and osmotic shock to plants. The AP2/ERF family genes encode plant-specific transcription factors involved in diverse environmental stresses. However, little is known about their physiological functions, especially in alkaline stress responses. In this study, we functionally characterized a novel ERF subfamily gene, GsERF6 from alkaline-tolerant wild soybean (Glycine soja). In wild soybean, GsERF6 was rapidly induced by NaHCO3 treatment, and its overexpression in Arabidopsis enhanced transgenic plant tolerance to NaHCO3 challenge. Interestingly, GsERF6 transgenic lines also displayed increased tolerance to KHCO3 treatment, but not to high pH stress, implicating that GsERF6 may participate specifically in bicarbonate stress responses. We also found that GsERF6 overexpression up-regulated the transcription levels of bicarbonate-stress-inducible genes such as NADP-ME, H (+)-Ppase and H (+)-ATPase, as well as downstream stress-tolerant genes such as RD29A, COR47 and KINI. GsERF6 overexpression and NaHCO3 stress also altered the expression patterns of plant hormone synthesis and hormone-responsive genes. Conjointly, our results suggested that GsERF6 is a positive regulator of plant alkaline stress by increasing bicarbonate ionic resistance specifically, providing a new insight into the regulation of gene expression under alkaline conditions.

Functional characterization of a Glycine soja Ca(2+)ATPase in salt-alkaline stress responses.

It is widely accepted that Ca(2+)ATPase family proteins play important roles in plant environmental stress responses. However, up to now, most researches are limited in the reference plants Arabidopsis and rice. The function of Ca(2+)ATPases from non-reference plants was rarely reported, especially its regulatory role in carbonate alkaline stress responses. Hence, in this study, we identified the P-type II Ca(2+)ATPase family genes in soybean genome, determined their chromosomal location and gene architecture, and analyzed their amino acid sequence and evolutionary relationship. Based on above results, we pointed out the existence of gene duplication for soybean Ca(2+)ATPases. Then, we investigated the expression profiles of the ACA subfamily genes in wild soybean (Glycine soja) under carbonate alkaline stress, and functionally characterized one representative gene GsACA1 by using transgenic alfalfa. Our results suggested that GsACA1 overexpression in alfalfa obviously increased plant tolerance to both carbonate alkaline and neutral salt stresses, as evidenced by lower levels of membrane permeability and MDA content, but higher levels of SOD activity, proline concentration and chlorophyll content under stress conditions. Taken together, for the first time, we reported a P-type II Ca(2+)ATPase from wild soybean, GsACA1, which could positively regulate plant tolerance to both carbonate alkaline and neutral salt stresses.

Overexpression of GsGSTU13 and SCMRP in Medicago sativa confers increased salt-alkaline tolerance and methionine content.

Tau-class glutathione S-transferases (GSTUs) are ubiquitous proteins encoded by a large gene family in plants, which play important roles in combating different environmental stresses. In previous studies, we constructed a Glycine soja transcriptional profile, and identified three GSTUs (GsGSTU13, GsGSTU14 and GsGSTU19) as potential salt-alkaline stress-responsive genes. Two of them, GsGSTU14 and GsGSTU19, have been shown to positively regulate plant salt-alkaline tolerance. In this study, we further demonstrated the positive function of GsGSTU13 in plant salt-alkaline stress responses by overexpressing it in Medicago sativa. Stress tolerance tests suggested that GsGSTU13 transgenic lines showed better growth and physiological indicators than wild alfalfa (cv. Zhaodong) under alkaline stress. Considering the shortage of methionine in alfalfa, we then co-transformed GsGSTU13 into two main alfalfa cultivars in Heilongjiang Province (cv. Zhaodong and cv. Nongjing No. 1) together with SCMRP, a synthesized gene that could improve the methionine content. We found that GsGSTU13/SCMRP transgenic alfalfa displayed not only higher methionine content but also higher tolerance to alkaline and salt stresses, respectively. Taken together, our results demonstrate that GsGSTU13 acts as a positive regulator in plant responses to salt and alkaline stresses, and can be used as a good candidate for generation of salt-alkaline tolerant crops.

Molecular cloning and promoter analysis of squalene synthase and squalene epoxidase genes from Betula platyphylla.

Betula platyphylla is a rich repository of pharmacologically active secondary metabolites known as birch triterpenoids (TBP). Here, we cloned the squalene synthase (SS) and squalene epoxidase genetic (SE) sequences from B. platyphylla that encode the key enzymes that are involved in triterpenoid biosynthesis and analyzed the conserved domains and phylogenetics of their corresponding proteins. The full-length sequence of BpSS is 1588 bp with a poly-A tail, which contained an open reading frame (ORF) of 1241 bp that encoded a protein of 413 amino acids. Additionally, the BpSE full-length sequence of 2040 bp with a poly-A tail was also obtained, which contained an ORF of 1581 bp encoding a protein of 526 amino acids. Their organ-specific expression patterns in 4-week-old tissue culture seedlings of B. platyphylla were detected by real-time PCR and showed that they were all highly expressed in leaves, as compared to stem and root tissues. Additionaly, both BpSS and BpSE were enhanced following stimulation with ethephon and MeJA. The expression of BpSS was enhanced by ABA, whereas BpSE was not. The SA treatment did not affect the BpSS and BpSE transcripts notably. Using a genome walking approach, promoter sequences of 965 and 1193 bp, respectively, for BpSS and BpSE were isolated, and they revealed several key cis-regulatory elements known to be involved in the response to phytohormone and abiotic plant stress. We also found that the BpSS protein is localized in the cytoplasm. Opening reading frames of BpSS and BpSE were ligated into yeast expression plasmid pYES2 under control of GAL1 promoter and introduced into the yeast INVScl1 strain. The transformants were cultured for 12 h, the squalene content of galactose-induced BpSS expression yeast cells was 13.2 times of control (empty vector control yeast cells) by high-performance liquid chromatography (HPLC) test method. And, the squalene epoxidase activity of induced BpSE expression yeast cell was about 11.8 times of control. These indicated that we cloned birch BpSS and BpSE that were indeed involved in the synthesis of triteropenoids. This is the first report wherein SS and SE from B. platyphylla were cloned and may be of significant interest to understand the regulatory role of SS and SE in the triterpenoids biosynthesis of B. platyphylla. This is the first report wherein SS and SE from B. platyphylla were cloned and may be of significant interest to understand the regulatory role of SS and SE in the biosynthesis of birch triterpenoids.

GsCML27, a Gene Encoding a Calcium-Binding Ef-Hand Protein from Glycine soja, Plays Differential Roles in Plant Responses to Bicarbonate, Salt and Osmotic Stresses.

Calcium, as the most widely accepted messenger, plays an important role in plant stress responses through calcium-dependent signaling pathways. The calmodulin-like family genes (CMLs) encode Ca2+ sensors and function in signaling transduction in response to environmental stimuli. However, until now, the function of plant CML proteins, especially soybean CMLs, is largely unknown. Here, we isolated a Glycine soja CML protein GsCML27, with four conserved EF-hands domains, and identified it as a calcium-binding protein through far-UV CD spectroscopy. We further found that expression of GsCML27 was induced by bicarbonate, salt and osmotic stresses. Interestingly, ectopic expression of GsCML27 in Arabidopsis enhanced plant tolerance to bicarbonate stress, but decreased the salt and osmotic tolerance during the seed germination and early growth stages. Furthermore, we found that ectopic expression of GsCML27 decreases salt tolerance through modifying both the cellular ionic (Na+, K+) content and the osmotic stress regulation. GsCML27 ectopic expression also decreased the expression levels of osmotic stress-responsive genes. Moreover, we also showed that GsCML27 localized in the whole cell, including cytoplasm, plasma membrane and nucleus in Arabidopsis protoplasts and onion epidermal cells, and displayed high expression in roots and embryos. Together, these data present evidence that GsCML27 as a Ca2+-binding EF-hand protein plays a role in plant responses to bicarbonate, salt and osmotic stresses.

Ectopic Expression of a Glycine soja myo-Inositol Oxygenase Gene (GsMIOX1a) in Arabidopsis Enhances Tolerance to Alkaline Stress.

Myo-inositol participates in various aspects of plant physiology, and myo-inositol oxygenase is the key enzyme of the myo-inositol oxygenation pathway. Previous studies indicated that myo-inositol oxygenase may play a role in plant responses to abiotic stresses. In this study, we focused on the functional characterization of GsMIOX1a, a remarkable alkaline stress-responsive gene of Glycine soja 07256, based on RNA-seq data. Using quantitative real-time PCR, we demonstrated that GsMIOX1a is rapidly induced by alkaline stress and expressed predominantly in flowers. We also elucidated the positive function of GsMIOX1a in the alkaline response in the wild type, atmiox1 mutant as well as GsMIOX1a-overexpressing Arabidopsis. We determined that atmiox1 mutant decreased Arabidopsis tolerance to alkaline stress, whereas GsMIOX1a overexpression increased tolerance. Moreover, the expression levels of some alkaline stress-responsive and inducible marker genes, including H+-Ppase, NADP-ME, KIN1 and RD29B, were also up-regulated in GsMIOX1a overexpression lines compared with the wild type and atmiox1 mutant. Together, these results suggest that the GsMIOX1a gene positively regulates plant tolerance to alkaline stress. This is the first report to demonstrate that ectopic expression of myo-inositol oxygenase improves alkaline tolerance in plants.