A Novel Thylakoid Ascorbate Peroxidase from Jatrophacurcas Enhances Salt Tolerance in Transgenic Tobacco

Ascorbate peroxidase (APX) plays an important role in the metabolism of hydrogen peroxide in higher plants. In the present study, a novel APX gene (JctAPX) was cloned from Jatropha curcas L. The deduced amino acid sequence was similar to that of APX of some other plant species. JctAPX has a chloroplast transit peptide and was localized to the chloroplasts by analysis with a JctAPX-green fluorescent protein (GFP) fusion protein. Quantitative polymerase chain reaction (qPCR) analysis showed that JctAPX was constitutively expressed in different tissues from J. curcas and was upregulated by NaCl stress. To characterize its function in salt tolerance, the construct p35S: JctAPX was created and successfully introduced into tobacco by Agrobacterium-mediated transformation. Compared with wild type (WT), the transgenic plants exhibited no morphological abnormalities in the no-stress condition. However, under 200 mM NaCl treatment, JctAPX over-expressing plants showed increased tolerance to salt during seedling establishment and growth. In addition, the transgenic lines showed higher chlorophyll content and APX activity, which resulted in lower H₂O₂ content than WT when subjected to 400 mM NaCl stress. These results suggest that the increased APX activity in the chloroplasts from transformed plants increased salt tolerance by enhancing reactive oxygen species (ROS)-scavenging capacity under short-term NaCl stress conditions.     

A Chrysanthemum Heat Shock Protein Confers Tolerance to Abiotic Stress

Heat shock proteins are associated with protection against various abiotic stresses. Here, the isolation of a chrysanthemum cDNA belonging to the HSP70 family is reported. The cDNA, designated CgHSP70, encodes a 647-residue polypeptide, of estimated molecular mass 70.90 kDa and pI 5.12. A sub-cellular localization assay indicated that the cDNA product is deposited in the cytoplasm and nucleus. The performance of Arabidopsis thaliana plants constitutively expressing CgHSP70 demonstrated that the gene enhances tolerance to heat, drought and salinity. When CgHSP70 was stably over-expressed in chrysanthemum, the plants showed an increased peroxidase (POD) activity, higher proline content and inhibited malondialdehyde (MDA) content. After heat stress, drought or salinity the transgenic plants were better able to recover, demonstrating CgHSP70 positive effect.     

Lb1G04202, an Uncharacterized Protein from Recretohalophyte Limonium bicolor,Is Important in Salt Tolerance

With global increases in saline soil, it has become increasingly important to decipher salt-tolerance mechanisms and identify strategies to improve salt tolerance in crops. Halophytes complete their life cycles in environments containing ≥200 mM NaCl; these remarkable plants provide a potential source of genes for improving crop salt tolerance. Recretohalophytes such as Limonium bicolor have salt glands that secrete Na⁺ on their leaf epidermis. Here, we identified Lb1G04202, an uncharacterized gene with no conserved domains, from L. bicolor, which was highly expressed after NaCl treatment. We confirmed its expression in the salt gland by in situ hybridization, and then heterologously expressed Lb1G04202 in Arabidopsis thaliana. The transgenic lines had a higher germination rate, greater cotyledon growth percentage, and longer roots than the wild type (WT) under NaCl treatments (50, 100 and 150 mM). At the seedling stage, the transgenic lines grew better than the WT and had lower Na⁺ and malonyldialdehyde accumulation, and higher K⁺ and proline contents. This corresponded with the high expression of the key proline biosynthesis genes AtP5CS1 and AtP5CS2 under NaCl treatment. Isotonic mannitol treatment showed that Lb1G04202 overexpression significantly relieved osmotic stress. Therefore, this novel gene provides a potential target for improving salt tolerance.     

Heterologous Expression of the Melatonin-Related Gene HIOMT Improves Salt Tolerance in Malus domestica

Melatonin, a widely known indoleamine molecule that mediates various animal and plant physiological processes, is formed from N-acetyl serotonin via N-acetylserotonin methyltransferase (ASMT). ASMT is an enzyme that catalyzes melatonin synthesis in plants in the rate-determining step and is homologous to hydroxyindole-O-methyltransferase (HIOMT) melatonin synthase in animals. To date, little is known about the effect of HIOMT on salinity in apple plants. Here, we explored the melatonin physiological function in the salinity condition response by heterologous expressing the homologous human HIOMT gene in apple plants. We discovered that the expression of melatonin-related gene (MdASMT) in apple plants was induced by salinity. Most notably, compared with the wild type, three transgenic lines indicated higher melatonin levels, and the heterologous expression of HIOMT enhanced the expression of melatonin synthesis genes. The transgenic lines showed reduced salt damage symptoms, lower relative electrolyte leakage, and less total chlorophyll loss from leaves under salt stress. Meanwhile, through enhanced activity of antioxidant enzymes, transgenic lines decreased the reactive oxygen species accumulation, downregulated the expression of the abscisic acid synthesis gene (MdNCED3), accordingly reducing the accumulation of abscisic acid under salt stress. Both mechanisms regulated morphological changes in the stomata synergistically, thereby mitigating damage to the plants’ photosynthetic ability. In addition, transgenic plants also effectively stabilized their ion balance, raised the expression of salt stress–related genes, as well as alleviated osmotic stress through changes in amino acid metabolism. In summary, heterologous expression of HIOMT improved the adaptation of apple leaves to salt stress, primarily by increasing melatonin concentration, maintaining a high photosynthetic capacity, reducing reactive oxygen species accumulation, and maintaining normal ion homeostasis.     

The Absence of the AtSYT1 Function Elevates the Adverse Effect of Salt Stress on Photosynthesis in Arabidopsis

Arabidopsis thaliana SYNAPTOTAGMIN 1 (AtSYT1) was shown to be involved in responses to different environmental and biotic stresses. We investigated gas exchange and chlorophyll a fluorescence in Arabidopsis wild-type (WT, ecotype Col-0) and atsyt1 mutant plants irrigated for 48 h with 150 mM NaCl. We found that salt stress significantly decreases net photosynthetic assimilation, effective photochemical quantum yield of photosystem II (Φ_PSII), stomatal conductance and transpiration rate in both genotypes. Salt stress has a more severe impact on atsyt1 plants with increasing effect at higher illumination. Dark respiration, photochemical quenching (qP), non-photochemical quenching and Φ_PSII measured at 750 µmol m⁻² s⁻¹ photosynthetic photon flux density were significantly affected by salt in both genotypes. However, differences between mutant and WT plants were recorded only for qP and Φ_PSII. Decreased photosynthetic efficiency in atsyt1 under salt stress was accompanied by reduced chlorophyll and carotenoid and increased flavonol content in atsyt1 leaves. No differences in the abundance of key proteins participating in photosynthesis (except PsaC and PsbQ) and chlorophyll biosynthesis were found regardless of genotype or salt treatment. Microscopic analysis showed that irrigating plants with salt caused a partial closure of the stomata, and this effect was more pronounced in the mutant than in WT plants. The localization pattern of AtSYT1 was also altered by salt stress.     

CaDHN3, a Pepper (Capsicum annuum L.) Dehydrin Gene Enhances the Tolerance against Salt and Drought Stresses by Reducing ROS Accumulation

Dehydrins (DHNs) play an important role in abiotic stress tolerance in a large number of plants, but very little is known about the function of DHNs in pepper plants. Here, we isolated a Y₁SK₂-type DHN gene “CaDHN3” from pepper. To authenticate the function of CaDHN3 in salt and drought stresses, it was overexpressed in Arabidopsis and silenced in pepper through virus-induced gene silencing (VIGS). Sub-cellular localization showed that CaDHN3 was located in the nucleus and cell membrane. It was found that CaDHN3-overexpressed (OE) in Arabidopsis plants showed salt and drought tolerance phenotypic characteristics, i.e., increased the initial rooting length and germination rate, enhanced chlorophyll content, lowered the relative electrolyte leakage (REL) and malondialdehyde (MDA) content than the wild-type (WT) plants. Moreover, a substantial increase in the activities of antioxidant enzymes; including the superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and lower hydrogen peroxide (H₂O₂) contents and higher O₂^•− contents in the transgenic Arabidopsis plants. Silencing of CaDHN3 in pepper decreased the salt- and drought-stress tolerance, through a higher REL and MDA content, and there was more accumulation of reactive oxygen species (ROS) in the CaDHN3-silenced pepper plants than the control plants. Based on the yeast two-hybrid (Y2H) screening and Bimolecular Fluorescence Complementation (BiFC) results, we found that CaDHN3 interacts with CaHIRD11 protein in the plasma membrane. Correspondingly, the expressions of four osmotic-related genes were significantly up-regulated in the CaDHN3-overexpressed lines. In brief, our results manifested that CaDHN3 may play an important role in regulating the relative osmotic stress responses in plants through the ROS signaling pathway. The results of this study will provide a basis for further analyses of the function of DHN genes in pepper.