Bowei Jia

A Glycine Soja 14-3-3 Protein GsGF14o Participates in Stomatal and Root Hair Development and Drought Tolerance in Arabidopsis thaliana

It is well established that 14-3-3 proteins are key regulators of multiple stress signal transduction cascades. However, the biological functions of soybean 14-3-3 proteins, especially in plant drought response, are not yet known. In this study, we characterized a Glycine soja 14-3-3 gene, GsGF14o, which is involved in plant development and drought response. GsGF14o expression was greatly induced by drought stress, as evidenced by the quantitative real-time PCR and β-glucuronidase (GUS) activity analysis. GsGF14o overexpression in Arabidopsis thaliana resulted in decreased drought tolerance during seed germination and seedling growth. Furthermore, silencing of AtGF14µ, the most homologous 14-3-3 gene of GsGF14o, led to enhanced drought tolerance at both the seed germination and seedling stage. Unexpectedly, GsGF14o transgenic lines showed reduced water loss and transpiration rates compared with wild-type plants, which was demonstrated to be the consequence of the decreased stomatal size. At the same time, the smaller stomata due to GsGF14o overexpression led to a relatively slow net photosynthesis rate, which led to a growth penalty under drought stress. We further demonstrated that GsGF14o overexpression caused deficits in root hair formation and development, and thereby reduced the water intake capacity of the transgenic root system. In addition, GsGF14o overexpression down-regulated the transcript levels of drought-responsive marker genes. Finally, we also investigated the tissue-specific accumulation of GsGF14o by using a GUS activity assay. Collectively, the results presented here confirm that GsGF14o plays a dual role in drought stress responses through its involvement in the regulation of stomatal size and root hair development.

A Glycine soja methionine sulfoxide reductase B5a interacts with the Ca(2+) /CAM-binding kinase GsCBRLK and activates ROS signaling under carbonate alkaline stress.

Although research has extensively illustrated the molecular basis of plant responses to salt and high-pH stresses, knowledge on carbonate alkaline stress is poor and the specific responsive mechanism remains elusive. We have previously characterized a Glycine soja Ca(2+) /CAM-dependent kinase GsCBRLK that could increase salt tolerance. Here, we characterize a methionine sulfoxide reductase (MSR) B protein GsMSRB5a as a GsCBRLK interactor by using Y2H and BiFc assays. Further analyses showed that the N-terminal variable domain of GsCBRLK contributed to the GsMSRB5a interaction. Y2H assays also revealed the interaction specificity of GsCBRLK with the wild soybean MSRB subfamily proteins, and determined that the BoxI/BoxII-containing regions within GsMSRBs were responsible for their interaction. Furthermore, we also illustrated that the N-terminal basic regions in GsMSRBs functioned as transit peptides, which targeted themselves into chloroplasts and thereby prevented their interaction with GsCBRLK. Nevertheless, deletion of these regions allowed them to localize on the plasma membrane (PM) and interact with GsCBRLK. In addition, we also showed that GsMSRB5a and GsCBRLK displayed overlapping tissue expression specificity and coincident expression patterns under carbonate alkaline stress. Phenotypic experiments demonstrated that GsMSRB5a and GsCBRLK overexpression in Arabidopsis enhanced carbonate alkaline stress tolerance. Further investigations elucidated that GsMSRB5a and GsCBRLK inhibited reactive oxygen species (ROS) accumulation by modifying the expression of ROS signaling, biosynthesis and scavenging genes. Summarily, our results demonstrated that GsCBRLK and GsMSRB5a interacted with each other, and activated ROS signaling under carbonate alkaline stress.

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.

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.

Overexpression of OsmiR156k leads to reduced tolerance to cold stress in rice (Oryza Sativa)

The microRNA156 (miR156) family ‘has’ been well demonstrated to regulate plant growth and development. However, no reports focused on the roles of miR156s in environmental stress responses. In previous studies, we identified 18 cold stress responsive microRNAs in rice by using microarray analysis. Here, in the present study, we focused on the biological function of one of these cold responsive microRNAs, OsmiR156k. We generated the transgenic rice overexpressing OsmiR156k under the control of CaMV35S promoter, and verified the presence of OsmiR156k by using Southern blot analysis. We found that overexpression of OsmiR156k inhibited the seedling growth at the very early seedling stage under cold stress. Furthermore, OsmiR156k overexpression decreased plant cold tolerance at the young seedling growth stage, as evidenced by lower survival rates, chlorophyll contents and proline contents. As expected, we also suggested the down-regulated expression of the cold stress responsive genes, 01g22249 and OsP5CS, and OsmiR156k-tagated SPL genes, SPL3, SPL14 and SPL17, in the OsmiR156k transgenic lines. Taken together, our findings suggest that overexpression of OsmiR156k decreased the tolerance to cold stress in rice.

Functional characterization of a Glycine soja Ca 2+ ATPase in salt–alkaline stress responses

It is widely accepted that Ca2+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 Ca2+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 Ca2+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 Ca2+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 Ca2+ATPase from wild soybean, GsACA1, which could positively regulate plant tolerance to both carbonate alkaline and neutral salt stresses.

A 14-3-3 Family Protein from Wild Soybean (Glycine Soja) Regulates ABA Sensitivity in Arabidopsis

It is widely accepted that the 14-3-3 family proteins are key regulators of multiple stress signal transduction cascades. By conducting genome-wide analysis, researchers have identified the soybean 14-3-3 family proteins; however, until now, there is still no direct genetic evidence showing the involvement of soybean 14-3-3s in ABA responses. Hence, in this study, based on the latest Glycine max genome on Phytozome v10.3, we initially analyzed the evolutionary relationship, genome organization, gene structure and duplication, and three-dimensional structure of soybean 14-3-3 family proteins systematically. Our results suggested that soybean 14-3-3 family was highly evolutionary conserved and possessed segmental duplication in evolution. Then, based on our previous functional characterization of a Glycine soja 14-3-3 protein GsGF14o in drought stress responses, we further investigated the expression characteristics of GsGF14o in detail, and demonstrated its positive roles in ABA sensitivity. Quantitative real-time PCR analyses in Glycine soja seedlings and GUS activity assays in PGsGF14O:GUS transgenic Arabidopsis showed that GsGF14o expression was moderately and rapidly induced by ABA treatment. As expected, GsGF14o overexpression in Arabidopsis augmented the ABA inhibition of seed germination and seedling growth, promoted the ABA induced stomata closure, and up-regulated the expression levels of ABA induced genes. Moreover, through yeast two hybrid analyses, we further demonstrated that GsGF14o physically interacted with the AREB/ABF transcription factors in yeast cells. Taken together, results presented in this study strongly suggested that GsGF14o played an important role in regulation of ABA sensitivity in Arabidopsis.

GsCHX19 .3, a member of cation/H + exchanger superfamily from wild soybean contributes to high salinity and carbonate alkaline tolerance

Cation/H+ exchangers (CHX) are characterized to be involved in plant growth, development and stress responses. Although soybean genome sequencing has been completed, the CHX family hasn’t yet been systematically analyzed, especially in wild soybean. Here, through Hidden Markov Model search against Glycine soja proteome, 34 GsCHXs were identified and phylogenetically clustered into five groups. Members within each group showed high conservation in motif architecture. Interestingly, according to our previous RNA-seq data, only Group IVa members exhibited highly induced expression under carbonate alkaline stress. Among them, GsCHX19.3 displayed the greatest up-regulation in response to carbonate alkaline stress, which was further confirmed by quantitative real-time PCR analysis. We also observed the ubiquitous expression of GsCHX19.3 in different tissues and its localization on plasma membrane. Moreover, we found that GsCHX19.3 expression in AXT4K, a yeast mutant lacking four ion transporters conferred resistance to low K+ at alkali pH, as well as carbonate stress. Consistently, in Arabidopsis, GsCHX19.3 overexpression increased plant tolerance both to high salt and carbonate alkaline stresses. Furthermore, we also confirmed that GsCHX19.3 transgenic lines showed lower Na+ concentration but higher K+/Na+ values under salt-alkaline stress. Taken together, our findings indicated that GsCHX19.3 contributed to high salinity and carbonate alkaline tolerance.

The opposite roles of OsmiR408 in cold and drought stress responses in Oryza sativa

MiR408 is a conserved miRNA family in plants. Although AtmiR408 is generally regarded as participating in stress responses, it still remains obscure whether OsmiR408 modulates tolerance to environmental stress. In the current study, expression of Pre-OsmiR408 and OsmiR408 was found to be induced by cold stress, but repressed by drought stress in the rice cultivar “Kongyu 131”. By comparing the wild type and OsmiR408 transgenic lines, we found that OsmiR408 overexpression conferred enhanced cold tolerance at both the early seedling stage and the young seedling stage. On the other hand, the OsmiR408 transgenic lines exhibited decreased drought tolerance, which is further verified by greater water loss. We also predicted the putative target genes of OsmiR408 and verified the decreased expression of seven targets in OsmiR408 transgenic lines, including four phytocyanins and three atypical target genes. Among them, Os09g29390, a phytocyanin gene, and Os01g53880, an auxin responsive Aux/IAA gene, were down-regulated by cold treatment, which is opposite to the cold-induced expression of OsmiR408. Taken together, our results suggest opposite roles of OsmiR408 in plant responses to cold and drought stresses.

Ectopic Expression of GsSRK in Medicago sativa Reveals Its Involvement in Plant Architecture and Salt Stress Responses

Receptor-like kinases (RLK) play fundamental roles in plant growth and stress responses. Compared with other RLKs, little information is provided concerning the S-locus LecRLK subfamily, which is characterized by an extracellular G-type lectin domain and an S-locus-glycop domain. Until now, the function of the G-type lectin domain is still unknown. In a previous research, we identified a Glycine soja S-locus LecRLK gene GsSRK, which conferred increased salt stress tolerance in transgenic Arabidopsis. In this study, to investigate the role of the G-type lectin domain and to breed transgenic alfalfa with superior salt stress tolerance, we transformed the full-length GsSRK (GsSRK-f) and a truncated version of GsSRK (GsSRK-t) deleting the G-type lectin domain into alfalfa. Our results showed that overexpression of GsSRK-t, but not GsSRK-f, resulted in changes of plant architecture, as evidenced by more branches but shorter shoots of GsSRK-t transgenic alfalfa, indicating a potential role of the extracellular G-type lectin domain in regulating plant architecture. Furthermore, we also found that transgenic alfalfa overexpressing either GsSRK-f or GsSRK-t showed increased salt stress tolerance, and GsSRK-t transgenic alfalfa displayed better growth (more branches and higher fresh weight) than GsSRK-f lines under salt stress. In addition, our results suggested that both GsSRK-f and GsSRK-t were involved in ion homeostasis, ROS scavenging, and osmotic regulation. Under salt stress, the Na+ content in the transgenic lines was significantly lower, while the K+ content was slightly higher than that in WT. Moreover, the transgenic lines displayed reduced ion leakage and MDA content, but increased SOD activity and proline content than WT. Notably, no obvious difference in these physiological indices was observed between GsSRK-f and GsSRK-t transgenic lines, implying that deletion of the GsSRK G-type lectin domain does not affect its physiological function in salt stress responses. In conclusion, results in this research reveal the dual role of GsSRK in regulating both plant architecture and salt stress responses.