Transcriptional and Physiological Analyses to Assess the Effects of a Novel Biostimulant in Tomato

This work aimed to study the effects in tomato (Solanum lycopersicum L.) of foliar applications of a novel calcium-based biostimulant (SOB01) using an omics approach involving transcriptomics and physiological profiling. A calcium-chloride fertilizer (SOB02) was used as a product reference standard. Plants were grown under well-watered (WW) and water stress (WS) conditions in a growth chamber. We firstly compared the transcriptome profile of treated and untreated tomato plants using the software RStudio. Totally, 968 and 1,657 differentially expressed genes (DEGs) (adj-p-value < 0.1 and |log2(fold change)| ≥ 1) were identified after SOB01 and SOB02 leaf treatments, respectively. Expression patterns of 9 DEGs involved in nutrient metabolism and osmotic stress tolerance were validated by real-time quantitative reverse transcription PCR (RT-qPCR) analysis. Principal component analysis (PCA) on RT-qPCR results highlighted that the gene expression profiles after SOB01 treatment in different water regimes were clustering together, suggesting that the expression pattern of the analyzed genes in well water and water stress plants was similar in the presence of SOB01 treatment. Physiological analyses demonstrated that the biostimulant application increased the photosynthetic rate and the chlorophyll content under water deficiency compared to the standard fertilizer and led to a higher yield in terms of fruit dry matter and a reduction in the number of cracked fruits. In conclusion, transcriptome and physiological profiling provided comprehensive information on the biostimulant effects highlighting that SOB01 applications improved the ability of the tomato plants to mitigate the negative effects of water stress.

Introgression of SbERD4 Gene Encodes an Early-Responsive Dehydration-Stress Protein That Confers Tolerance against Different Types of Abiotic Stresses in Transgenic Tobacco

Salicornia brachiata is an extreme halophyte that commonly grows on marsh conditions and is also considered a promising resource for drought and salt-responsive genes. To unveil a glimpse of stress endurance by plants, it is of the utmost importance to develop an understanding of stress tolerance mechanisms. ‘Early Responsive to Dehydration’ (ERD) genes are defined as a group of genes involved in stress tolerance and the development of plants. To increase this understanding, parallel to this expedited thought, a novel SbERD4 gene was cloned from S. brachiata, characterized, and functionally validated in the model plant tobacco. The study showed that SbERD4 is a plasma-membrane bound protein, and its overexpression in tobacco plants improved salinity and osmotic stress tolerance. Transgenic plants showed high relative water, chlorophylls, sugars, starch, polyphenols, proline, free amino acids, and low electrolyte leakage and H2O2 content compared to control plants (wild type and vector control) under different abiotic stress conditions. Furthermore, the transcript expression of antioxidant enzyme encoding genes NtCAT, NtSOD, NtGR, and NtAPX showed higher expression in transgenic compared to wild-type and vector controls under varying stress conditions. Overall, the overexpression of a novel early responsive to dehydration stress protein 4-encoding gene (SbERD4) enhanced the tolerance of the plant against multiple abiotic stresses. In conclusion, the overexpression of the SbERD4 gene mitigates plant physiology by enduring stress tolerance and might be considered as a promising key gene for engineering salinity and drought stress tolerance in crops.

Role of Trehalose Synthesis in Ralstonia syzygii subsp. indonesiensis PW1001 in Inducing Hypersensitive Response on Eggplant (Solanum melongena cv. Senryo-nigou)

Ralstonia syzygii subsp. indonesiensis (Rsi, former name: Ralstonia solanacearum phylotype IV) PW1001, a causal agent of potato wilt disease, induces hypersensitive response (HR) on its non-host eggplant (Solanum melongena cv. Senryo-nigou). The disaccharide trehalose is involved in abiotic and biotic stress tolerance in many organisms. We found that trehalose is required for eliciting HR on eggplant by plant pathogen Rsi PW1001. In R. solanacearum, it is known that the OtsA/OtsB pathway is the dominant trehalose synthesis pathway, and otsA and otsB encode trehalose-6-phosphate (T6P) synthase and T6P phosphatase, respectively. We generated otsA and otsB mutant strains and found that these mutant strains reduced the bacterial trehalose concentration and HR induction on eggplant leaves compared to wild-type. Trehalose functions intracellularly in Rsi PW1001 because addition of exogenous trehalose did not affect the HR level and ion leakage. Requirement of trehalose in HR induction is not common in R. solanacearum species complex because mutation of otsA in Ralstonia pseudosolanacearum (former name: Ralstonia solanacearum phylotype I) RS1002 did not affect HR on the leaves of its non-host tobacco and wild eggplant Solanum torvum. Further, we also found that each otsA and otsB mutant had reduced ability to grow in a medium containing NaCl and sucrose, indicating that trehalose also has an important role in osmotic stress tolerance.

Pepper Novel Pseudo Response Regulator Protein CaPRR2 Modulates Drought and High Salt Tolerance

Plants modify their internal states to adapt to environmental stresses. Under environmental stress conditions, plants restrict their growth and development and activate defense responses. Abscisic acid (ABA) is a major phytohormone that plays a crucial role in the osmotic stress response. In osmotic stress adaptation, plants regulate stomatal closure, osmoprotectant production, and gene expression. Here, we isolated CaPRR2 – encoding a pseudo response regulator protein – from the leaves of pepper plants (Capsicum annuum). After exposure to ABA and environmental stresses, such as drought and salt stresses, CaPRR2 expression in pepper leaves was significantly altered. Under drought and salt stress conditions, CaPRR2-silenced pepper plants exhibited enhanced osmotic stress tolerance, characterized by an enhanced ABA-induced stomatal closing and high MDA and proline contents, compared to the control pepper plants. Taken together, our data indicate that CaPRR2 negatively regulates osmotic stress tolerance.

Molecular Dissection of the Gene OsGA2ox8 Conferring Osmotic Stress Tolerance in Rice

Gibberellin 2-oxidase (GA2ox) plays an important role in the GA catabolic pathway and the molecular function of the OsGA2ox genes in plant abiotic stress tolerance remains largely unknown. In this study, we functionally characterized the rice gibberellin 2-oxidase 8 (OsGA2ox8) gene. The OsGA2ox8 protein was localized in the nucleus, cell membrane, and cytoplasm, and was induced in response to various abiotic stresses and phytohormones. The overexpression of OsGA2ox8 significantly enhanced the osmotic stress tolerance of transgenic rice plants by increasing the number of osmotic regulators and antioxidants. OsGA2ox8 was differentially expressed in the shoots and roots to cope with osmotic stress. The plants overexpressing OsGA2ox8 showed reduced lengths of shoots and roots at the seedling stage, but no difference in plant height at the heading stage was observed, which may be due to the interaction of OsGA2ox8 and OsGA20ox1, implying a complex feedback regulation between GA biosynthesis and metabolism in rice. Importantly, OsGA2ox8 was able to indirectly regulate several genes associated with the anthocyanin and flavonoid biosynthetic pathway and the jasmonic acid (JA) and abscisic acid (ABA) biosynthetic pathway, and overexpression of OsGA2ox8 activated JA signal transduction by inhibiting the expression of jasmonate ZIM domain-containing proteins. These results provide a basis for a future understanding of the networks and respective phenotypic effects associated with OsGA2ox8.

Rootstocks Overexpressing StNPR1 and StDREB1 Improve Osmotic Stress Tolerance of Wild-Type Scion in Transgrafted Tobacco Plants

In grafted plants, the movement of long-distance signals from rootstocks can modulate the development and function of the scion. To understand the mechanisms by which tolerant rootstocks improve scion responses to osmotic stress (OS) conditions, mRNA transport of osmotic responsive genes (ORGs) was evaluated in a tomato/potato heterograft system. In this system, Solanum tuberosum was used as a rootstock and Solanum lycopersicum as a scion. We detected changes in the gene expression levels of 13 out of the 21 ORGs tested in the osmotically stressed plants; of these, only NPR1 transcripts were transported across the graft union under both normal and OS conditions. Importantly, OS increased the abundance of StNPR1 transcripts in the tomato scion. To examine mRNA mobility in transgrafted plants, StNPR1 and StDREB1 genes representing the mobile and non-mobile transcripts, respectively, were overexpressed in tobacco (Nicotiana tabacum). The evaluation of transgenic tobacco plants indicated that overexpression of these genes enhanced the growth and improved the physiological status of transgenic plants growing under OS conditions induced by NaCl, mannitol and polyethylene glycol (PEG). We also found that transgenic tobacco rootstocks increased the OS tolerance of the WT-scion. Indeed, WT scions on transgenic rootstocks had higher ORGs transcript levels than their counterparts on non-transgenic rootstocks. However, neither StNPR1 nor StDREB1 transcripts were transported from the transgenic rootstock to the wild-type (WT) tobacco scion, suggesting that other long-distance signals downstream these transgenes could have moved across the graft union leading to OS tolerance. Overall, our results signify the importance of StNPR1 and StDREB1 as two anticipated candidates for the development of stress-resilient crops through transgrafting technology.

OsMADS23 phosphorylated by SAPK9 confers drought and salt tolerance by regulating ABA biosynthesis in rice

Some of MADS-box transcription factors (TFs) have been shown to play essential roles in the adaptation of plant to abiotic stress. Still, the mechanisms that MADS-box proteins regulate plant stress response are not fully understood. Here, a stress-responsive MADS-box TF OsMADS23 from rice conferring the osmotic stress tolerance in plants is reported. Overexpression of OsMADS23 remarkably enhanced, but knockout of the gene greatly reduced the drought and salt tolerance in rice plants. Further, OsMADS23 was shown to promote the biosynthesis of endogenous ABA and proline by activating the transcription of target genes OsNCED2, OsNCED3, OsNCED4 and OsP5CR that are key components for ABA and proline biosynthesis, respectively. Then, the convincing evidence showed that the OsNCED2-knockout mutants had lower ABA levels and exhibited higher sensitivity to drought and oxidative stress than wild type, which is similar to osmads23 mutant. Interestingly, the SnRK2-type protein kinase SAPK9 was found to physically interact with and phosphorylate OsMADS23, and thus increase its stability and transcriptional activity. Furthermore, the activation of OsMADS23 by SAPK9-mediated phosphorylation is dependent on ABA in plants. Collectively, these findings establish a mechanism that OsMADS23 functions as a positive regulator in response to osmotic stress by regulating ABA biosynthesis, and provide a new strategy for improving drought and salt tolerance in rice.