A Sugarcane G-Protein-Coupled Receptor, ShGPCR1, Confers Tolerance to Multiple Abiotic Stresses

Sugarcane (Saccharum spp.) is a prominent source of sugar and serves as bioenergy/biomass feedstock globally. Multiple biotic and abiotic stresses, including drought, salinity, and cold, adversely affect sugarcane yield. G-protein-coupled receptors (GPCRs) are components of G-protein-mediated signaling affecting plant growth, development, and stress responses. Here, we identified a GPCR-like protein (ShGPCR1) from sugarcane and energy cane (Saccharum spp. hybrids) and characterized its function in conferring tolerance to multiple abiotic stresses. ShGPCR1 protein sequence contained nine predicted transmembrane (TM) domains connected by four extracellular and four intracellular loops, which could interact with various ligands and heterotrimeric G proteins in the cells. ShGPCR1 sequence displayed other signature features of a GPCR, such as a putative guanidine triphosphate (GTP)-binding domain, as well as multiple myristoylation and protein phosphorylation sites, presumably important for its biochemical function. Expression of ShGPCR1 was upregulated by drought, salinity, and cold stresses. Subcellular imaging and calcium (Ca2+) measurements revealed that ShGPCR1 predominantly localized to the plasma membrane and enhanced intracellular Ca2+ levels in response to GTP, respectively. Furthermore, constitutive overexpression of ShGPCR1 in sugarcane conferred tolerance to the three stressors. The stress-tolerance phenotype of the transgenic lines corresponded with activation of multiple drought-, salinity-, and cold-stress marker genes, such as Saccharum spp. LATE EMBRYOGENESIS ABUNDANT, DEHYDRIN, DROUGHT RESPONSIVE 4, GALACTINOL SYNTHASE, ETHYLENE RESPONSIVE FACTOR 3, SALT OVERLY SENSITIVE 1, VACUOLAR Na+/H+ ANTIPORTER 1, NAM/ATAF1/2/CUC2, COLD RESPONSIVE FACTOR 2, and ALCOHOL DEHYDROGENASE 3. We suggest that ShGPCR1 plays a key role in conferring tolerance to multiple abiotic stresses, and the engineered lines may be useful to enhance sugarcane production in marginal environments with fewer resources.

New Insights on the Role of Connexins and Gap Junctions Channels in Adipose Tissue and Obesity

Due to the inability to curb the excessive increase in the prevalence of obesity and overweight, it is necessary to comprehend in more detail the factors involved in the pathophysiology and to appreciate more clearly the biochemical and molecular mechanisms of obesity. Thus, understanding the biological regulation of adipose tissue is of fundamental relevance. Connexin, a protein that forms intercellular membrane channels of gap junctions and unopposed hemichannels, plays a key role in adipogenesis and in the maintenance of adipose tissue homeostasis. The expression and function of Connexin 43 (Cx43) during the different stages of the adipogenesis are differentially regulated. Moreover, it has been shown that cell–cell communication decreases dramatically upon differentiation into adipocytes. Furthermore, inhibition of Cx43 degradation or constitutive overexpression of Cx43 blocks adipocyte differentiation. In the first events of adipogenesis, the connexin is highly phosphorylated, which is likely associated with enhanced Gap Junction (GJ) communication. In an intermediate state of adipocyte differentiation, Cx43 phosphorylation decreases, as it is displaced from the membrane and degraded through the proteasome; thus, Cx43 total protein is reduced. Cx is involved in cardiac disease as well as in obesity-related cardiovascular diseases. Different studies suggest that obesity together with a high-fat diet are related to the production of remodeling factors associated with expression and distribution of Cx43 in the atrium.

Overexpression of NtCBL5A Leads to Necrotic Lesions by Enhancing Na+ Sensitivity of Tobacco Leaves Under Salt Stress

Many tobacco (Nicotiana tabacum) cultivars are salt-tolerant and thus are potential model plants to study the mechanisms of salt stress tolerance. The CALCINEURIN B-LIKE PROTEIN (CBL) is a vital family of plant calcium sensor proteins that can transmit Ca2+ signals triggered by environmental stimuli including salt stress. Therefore, assessing the potential of NtCBL for genetic improvement of salt stress is valuable. In our studies on NtCBL members, constitutive overexpression of NtCBL5A was found to cause salt supersensitivity with necrotic lesions on leaves. NtCBL5A-overexpressing (OE) leaves tended to curl and accumulated high levels of reactive oxygen species (ROS) under salt stress. The supersensitivity of NtCBL5A-OE leaves was specifically induced by Na+, but not by Cl−, osmotic stress, or drought stress. Ion content measurements indicated that NtCBL5A-OE leaves showed sensitivity to the Na+ accumulation levels that wild-type leaves could tolerate. Furthermore, transcriptome profiling showed that many immune response-related genes are significantly upregulated and photosynthetic machinery-related genes are significantly downregulated in salt-stressed NtCBL5A-OE leaves. In addition, the expression of several cation homeostasis-related genes was also affected in salt-stressed NtCBL5A-OE leaves. In conclusion, the constitutive overexpression of NtCBL5A interferes with the normal salt stress response of tobacco plants and leads to Na+-dependent leaf necrosis by enhancing the sensitivity of transgenic leaves to Na+. This Na+ sensitivity of NtCBL5A-OE leaves might result from the abnormal Na+ compartmentalization, plant photosynthesis, and plant immune response triggered by the constitutive overexpression of NtCBL5A. Identifying genes and pathways involved in this unusual salt stress response can provide new insights into the salt stress response of tobacco plants.

The Exocytosis Associated SNAP25-Type Protein, SlSNAP33, Increases Salt Stress Tolerance by Modulating Endocytosis in Tomato

In plants, vesicular trafficking is crucial for the response and survival to environmental challenges. The active trafficking of vesicles is essential to maintain cell homeostasis during salt stress. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are regulatory proteins of vesicular trafficking. They mediate membrane fusion and guarantee cargo delivery to the correct cellular compartments. SNAREs from the Qbc subfamily are the best-characterized plasma membrane SNAREs, where they control exocytosis during cell division and defense response. The Solanum lycopersicum gene SlSNAP33.2 encodes a Qbc-SNARE protein and is induced under salt stress conditions. SlSNAP33.2 localizes on the plasma membrane of root cells of Arabidopsis thaliana. In order to study its role in endocytosis and salt stress response, we overexpressed the SlSNAP33.2 cDNA in a tomato cultivar. Constitutive overexpression promoted endocytosis along with the accumulation of sodium (Na+) in the vacuoles. It also protected the plant from cell damage by decreasing the accumulation of hydrogen peroxide (H2O2) in the cytoplasm of stressed root cells. Subsequently, the higher level of SlSNAP33.2 conferred tolerance to salt stress in tomato plants. The analysis of physiological and biochemical parameters such as relative water content, the efficiency of the photosystem II, performance index, chlorophyll, and MDA contents showed that tomato plants overexpressing SlSNAP33.2 displayed a better performance under salt stress than wild type plants. These results reveal a role for SlSNAP33.2 in the endocytosis pathway involved in plant response to salt stress. This research shows that SlSNAP33.2 can be an effective tool for the genetic improvement of crop plants.

Characterization of Interactions between the Soybean Salt-Stress Responsive Membrane-Intrinsic Proteins GmPIP1 and GmPIP2

Salt tolerance is an important trait in soybean cultivation and breeding. Plant responses to salt stress include physiological and biochemical changes that affect the movement of water across the plasma membrane. Plasma membrane intrinsic proteins (PIPs) localize to the plasma membrane and regulate the water and solutes flow. In this study, quantitative real-time PCR and yeast two-hybridization were engaged to analyze the early gene expression profiles and interactions of a set of soybean PIPs (GmPIPs) in response to salt stress. A total of 20 GmPIPs-encoding genes had varied expression profiles after salt stress. Among them, 13 genes exhibited a downregulated expression pattern, including GmPIP1;6, the constitutive overexpression of which could improve soybean salt tolerance, and its close homologs GmPIP1;7 and 1;5. Three genes showed upregulated patterns, including the GmPIP1;6 close homolog GmPIP1;4, when four genes with earlier increased and then decreased expression patterns. GmPIP1;5 and GmPIP1;6 could both physically interact strongly with GmPIP2;2, GmPIP2;4, GmPIP2;6, GmPIP2;8, GmPIP2;9, GmPIP2;11, and GmPIP2;13. Definite interactions between GmPIP1;6 and GmPIP1;7 were detected and GmPIP2;9 performed homo-interaction. The interactions of GmPIP1;5 with GmPIP2;11 and 2;13, GmPIP1;6 with GmPIP2;9, 2;11 and GmPIP2;13, and GmPIP2;9 with itself were strengthened upon salt stress rather than osmotic stress. Taken together, we inferred that GmPIP1 type and GmPIP2 type could associate with each other to synergistically function in the plant cell; a salt-stress environment could promote part of their interactions. This result provided new clues to further understand the soybean PIP–isoform interactions, which lead to potentially functional homo- and heterotetramers for salt tolerance.

Temperature-Inducible Transgenic EDS1 and PAD4 in Arabidopsis Confer an Enhanced Disease Resistance at Elevated Temperature

Temperature is one of the most important environmental factors greatly affecting plant disease development. High temperature favors outbreaks of many plant diseases, which threaten food security and turn to be a big issue along with climate change and global warming. Here, we found that concurrent constitutive expression of the key immune regulators EDS1 and PAD4 in Arabidopsis significantly enhanced resistance to virulent bacterial pathogen Pseudomonas syringae pv. tomato at elevated temperature; however, autoimmunity-related growth retardation was also observed on these plants at a normal temperature. To balance this growth-defense trade-off, we generated transgenic plants dual expressing EDS1 and PAD4 genes under the control of a thermo-sensitive promoter from the HSP70 gene, whose expression is highly induced at an elevated temperature. Unlike constitutive overexpression lines, the proHSP70-EP transgenic lines exhibited enhanced resistance to bacterial pathogens at an elevated temperature without growth defects at normal condition. Thus, this study provides a potential strategy for genetic manipulation of plants to deal with the simultaneous abiotic and biotic stresses.

Arabidopsis thaliana Myb59 Gene Is Involved in the Response to Heterodera schachtii Infestation, and Its Overexpression Disturbs Regular Development of Nematode-Induced Syncytia

Transcription factors are proteins that directly bind to regulatory sequences of genes to modulate and adjust plants’ responses to different stimuli including biotic and abiotic stresses. Sedentary plant parasitic nematodes, such as beet cyst nematode, Heterodera schachtii, have developed molecular tools to reprogram plant cell metabolism via the sophisticated manipulation of genes expression, to allow root invasion and the induction of a sequence of structural and physiological changes in plant tissues, leading to the formation of permanent feeding sites composed of modified plant cells (commonly called a syncytium). Here, we report on the AtMYB59 gene encoding putative MYB transcription factor that is downregulated in syncytia, as confirmed by RT-PCR and a promoter pMyb59::GUS activity assays. The constitutive overexpression of AtMYB59 led to the reduction in A. thaliana susceptibility, as indicated by decreased numbers of developed females, and to the disturbed development of nematode-induced syncytia. In contrast, mutant lines with a silenced expression of AtMYB59 were more susceptible to this parasite. The involvement of ABA in the modulation of AtMYB59 gene transcription appears feasible by several ABA-responsive cis regulatory elements, which were identified in silico in the gene promoter sequence, and experimental assays showed the induction of AtMYB59 transcription after ABA treatment. Based on these results, we suggest that AtMYB59 plays an important role in the successful parasitism of H. schachtii on A. thaliana roots.

Poplar aquaporin PIP1;1 promotes Arabidopsis growth and development

Background Root hydraulic conductance is primarily determined by the conductance of living tissues to radial water flow. Plasma membrane intrinsic proteins (PIPs) in root cortical cells are important for plants to take up water and are believed to be directly involved in cell growth. Results In this study, we found that constitutive overexpression of the poplar root-specific gene PtoPIP1;1 in Arabidopsis accelerated bolting and flowering. At the early stage of the developmental process, PtoPIP1;1 OE Arabidopsis exhibited faster cell growth in both leaves and roots. The turgor pressure of plants was correspondingly increased in PtoPIP1;1 OE Arabidopsis, and the water status was changed. At the same time, the expression levels of flowering-related genes (CRY1, CRY2 and FCA) and hub genes in the regulatory networks underlying floral timing (FT and SOC1) were significantly upregulated in OE plants, while the floral repressor FLC gene was significantly downregulated. Conclusions Taken together, the results of our study indicate that constitutive overexpression of PtoPIP1;1 in Arabidopsis accelerates bolting and flowering through faster cell growth in both the leaf and root at an early stage of the developmental process. The autonomous pathway of flowering regulation may be executed by monitoring developmental age. The increase in turgor and changes in water status with PtoPIP1;1 overexpression play a role in promoting cell growth.

Metabolic engineering provides insight into the regulation of thiamin biosynthesis in plants

Thiamin (or thiamine) is a water-soluble B-vitamin (B1), which is required, in the form of thiamin pyrophosphate (TPP), as an essential cofactor in crucial carbon metabolism reactions in all forms of life. To ensure adequate metabolic functioning, humans rely on a sufficient dietary supply of thiamin. Increasing thiamin levels in plants via metabolic engineering is a powerful strategy to alleviate vitamin B1 malnutrition and thus improve global human health. These engineering strategies rely on comprehensive knowledge of plant thiamin metabolism and its regulation. Here, multiple metabolic engineering strategies were examined in the model plant Arabidopsis thaliana. This was achieved by constitutive overexpression of the three biosynthesis genes responsible for B1 synthesis, HMP-P synthase (THIC), HET-P synthase (THI1) and HMP-P kinase/TMP pyrophosphorylase (TH1), either separate or in combination. By monitoring the levels of thiamin, its phosphorylated entities, and its biosynthetic intermediates, we gained insight into the effect of either strategy on thiamin biosynthesis. Moreover, expression analysis of thiamin biosynthesis genes showed the plant’s intriguing ability to respond to alterations in the pathway. Overall, we revealed the necessity to balance the pyrimidine and thiazole branches of thiamin biosynthesis and assessed its biosynthetic intermediates. Furthermore, the accumulation of non-phosphorylated intermediates demonstrated the inefficiency of endogenous thiamin salvage mechanisms. These results serve as guidelines in the development of novel thiamin metabolic engineering strategies.