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.     

The Calcium-Dependent Protein Kinase TaCDPK27 Positively Regulates Salt Tolerance in Wheat

As essential calcium ion (Ca²⁺) sensors in plants, calcium-dependent protein kinases (CDPKs) function in regulating the environmental adaptation of plants. However, the response mechanism of CDPKs to salt stress is not well understood. In the current study, the wheat salt-responsive gene TaCDPK27 was identified. The open reading frame (ORF) of TaCDPK27 was 1875 bp, coding 624 amino acids. The predicted molecular weight and isoelectric point were 68.905 kDa and 5.6, respectively. TaCDPK27 has the closest relationship with subgroup III members of the CDPK family of rice. Increased expression of TaCDPK27 in wheat seedling roots and leaves was triggered by 150 mM NaCl treatment. TaCDPK27 was mainly located in the cytoplasm. After NaCl treatment, some of this protein was transferred to the membrane. The inhibitory effect of TaCDPK27 silencing on the growth of wheat seedlings was slight. After exposure to 150 mM NaCl for 6 days, the NaCl stress tolerance of TaCDPK27-silenced wheat seedlings was reduced, with shorter lengths of both roots and leaves compared with those of the control seedlings. Moreover, silencing of TaCDPK27 further promoted the generation of reactive oxygen species (ROS); reduced the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT); aggravated the injury to photosystem II (PS II); and increased programmed cell death (PCD) in wheat leaves under NaCl treatment, confirming that the TaCDPK27-silenced seedlings exhibited more NaCl injury than control seedlings. Taken together, the decrease in NaCl tolerance in TaCDPK27-silenced seedlings was due to excessive ROS accumulation and subsequent aggravation of the NaCl-induced PCD. TaCDPK27 may be essential for positively regulating salt tolerance in wheat seedlings.     

Flavonone 3-hydroxylase Relieves Bacterial Leaf Blight Stress in Rice via Overaccumulation of Antioxidant Flavonoids and Induction of Defense Genes and Hormones

Efficient accumulation of flavonoids is important for increased tolerance to biotic stress. Although several plant defense mechanisms are known, the roles of many pathways, proteins, and secondary metabolites in stress tolerance are unknown. We generated a flavanone 3-hydroxylase (F3H) overexpressor rice line and inoculated Xanthomonas Oryzae pv. oryzae and compared the control and wildtype inoculated plants. In addition to promoting plant growth and developmental maintenance, the overexpression of F3H increased the accumulation of flavonoids and increased tolerance to bacterial leaf blight (BLB) stress. Moreover, leaf lesion length was higher in the infected wildtype plants compared with infected transgenics. Kaempferol and quercetin, which scavenge reactive oxygen species, overaccumulated in transgenic lines compared with wildtypes in response to pathogenic infection, detected by scanning electron microscopy and spectrophotometry. The induction of F3H altered the antioxidant system and reduced the levels of glutathione peroxidase activity and malondialdehyde (MDA) contents in the transgenic lines compared with the wildtypes. Downstream gene regulation analysis showed that the expression of F3H increased the regulation of flavonol synthase (FLS), dihydroflavonol 4-reductase (DFR), and slender rice mutant (SLR1) during BLB stress. The analysis of SA and JA signaling revealed an antagonistic interaction between both hormones and that F3H induction significantly promoted SA and inhibited JA accumulation in the transgenic lines. SA-dependent nonexpressor pathogenesis-related (NPR1) and Xa1 showed significant upregulation in the infected transgenic lines compared with the infected control and wildtype lines. Thus, the overexpression of F3H was essential for increasing BLB stress tolerance.     

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.     

Stu-miR827-Targeted StWRKY48 Transcription Factor Negatively Regulates Drought Tolerance of Potato by Increasing Leaf Stomatal Density

Stomata are specialized portals in plant leaves to modulate water loss from plants to the atmosphere by control of the transpiration, thereby determining the water-use efficiency and drought resistance of plants. Despite that the stomata developmental progression is well-understood at the molecular level, the experimental evidence that miRNA regulates stomata development is still lacking, and the underlying mechanism remains elusive. This study demonstrates the involvement of stu-miR827 in regulating the drought tolerance of potato due to its control over the leaf stomatal density. The expression analysis showed that stu-miR827 was obviously repressed by drought stresses and then rapidly increased after rewatering. Suppressing the expression of stu-miR827 transgenic potato lines showed an increase in stomatal density, correlating with a weaker drought resistance compared with wildtype potato lines. In addition, StWRKY48 was identified as the target gene of stu-miR827, and the expression of StWRKY48 was obviously induced by drought stresses and was greatly upregulated in stu-miR827 knockdown transgenic potato lines, suggesting its involvement in the drought stress response. Importantly, the expression of genes associated with stomata development, such as SDD (stomatal density and distribution) and TMM (too many mouths), was seriously suppressed in transgenic lines. Altogether, these observations demonstrated that suppression of stu-miR827 might lead to overexpression of StWRKY48, which may contribute to negatively regulating the drought adaptation of potato by increasing the stomatal density. The results may facilitate functional studies of miRNAs in the process of drought tolerance in plants.     

Overexpression of a Weed (Solanum americanum) Proteinase Inhibitor in Transgenic Tobacco Results in Increased Glandular Trichome Density and Enhanced Resistance to Helicoverpa armigera and Spodoptera litura

In this study we produced transgenic tobacco plants by overexpressing a serine proteinase inhibitor gene, SaPIN2a, from the American black nightshade Solanum americanum under the control of the CaMV 35S promoter using Agrobacterium tumefaciens-mediated transformation. SaPIN2a was properly transcribed and translated as indicated by Northern blot and Western blot analyses. Functional integrity of SaPIN2a in transgenic plants was confirmed by proteinase inhibitory activity assay. Bioassays for insect resistance showed that SaPIN2a-overexpressing transgenic tobacco plants were more resistant to cotton bollworm (Helicoverpa armigera) and tobacco cutworm (Spodoptera litura) larvae, two devastating pests of important crop plants, than the control plants. Interestingly, overexpression of SaPIN2a in transgenic tobacco plants resulted in a significant increase in glandular trichome density and a promotion of trichome branching, which could also provide an additional resistance mechanism in transgenic plants against insect pests. Therefore, SaPIN2a could be used as an alternative proteinase inhibitor for the production of insect-resistant transgenic plants.     

The Opuntia streptacantha OpsHSP18 Gene Confers Salt and Osmotic Stress Tolerance in Arabidopsis thaliana

Abiotic stress limits seed germination, plant growth, flowering and fruit quality, causing economic decrease. Small Heat Shock Proteins (sHSPs) are chaperons with roles in stress tolerance. Herein, we report the functional characterization of a cytosolic class CI sHSP (OpsHSP18) from Opuntia streptacantha during seed germination in Arabidopsis thaliana transgenic lines subjected to different stress and hormone treatments. The over-expression of the OpsHSP18 gene in A. thaliana increased the seed germination rate under salt (NaCl) and osmotic (glucose and mannitol) stress, and in ABA treatments, compared with WT. On the other hand, the over-expression of the OpsHSP18 gene enhanced tolerance to salt (150 mM NaCl) and osmotic (274 mM mannitol) stress in Arabidopsis seedlings treated during 14 and 21 days, respectively. These plants showed increased survival rates (52.00 and 73.33%, respectively) with respect to the WT (18.75 and 53.75%, respectively). Thus, our results show that OpsHSP18 gene might have an important role in abiotic stress tolerance, in particular in seed germination and survival rate of Arabidopsis plants under unfavorable conditions.     

Irrigation with Magnetized Water Alleviates the Harmful Effect of Saline–Alkaline Stress on Rice Seedlings

Saline–alkaline stress suppresses rice growth and threatens crop production. Despite substantial research on rice’s tolerance to saline–alkaline stress, fewer studies have examined the impact of magnetic water treatments on saline–alkaline-stressed rice plants. We explored the physiological and molecular mechanisms involved in saline–alkaline stress tolerance enhancement via irrigation with magnetized water using Nipponbare. The growth of Nipponbare plants was inhibited by saline–alkaline stress, but this inhibition was alleviated by irrigating the plants with magnetized water, as evidenced by greater plant height, biomass, chlorophyll content, photosynthetic rates, and root system in plants irrigated with magnetized water compared to those irrigated with non-magnetized water. Plants that were irrigated with magnetized water were able to acquire more total nitrogen. In addition, we proved that rice seedlings irrigated with magnetized water had a greater root NO₃⁻-nitrogen concentration and root NH₄⁺-nitrogen concentration than plants irrigated with non-magnetized water. These findings suggest that treatment with magnetized water could increase nitrogen uptake. To test this hypothesis, we analyzed the expression levels of genes involved in nitrogen acquisition. The expression levels of OsNRT1;1, OsNRT1;2, OsNRT2;1, OsAMT1;2, OsAMT2;1, OsAMT2;2, OsAMT2;3, OsAMT3;1, OsAMT3;2, and OsAMT3;3 were higher in plants exposed to magnetized water medium compared to those exposed to non-magnetized water media. We further demonstrated that treatment with magnetized water increases available nitrogen, NO₃⁻-nitrogen content, and NH₄⁺-nitrogen content in soil under saline–alkaline stress. Our results revealed that the increased resistance of rice seedlings to saline–alkaline stress may be attributable to a very effective nitrogen acquisition system enhanced by magnetized water.     

TaNBR1, a Novel Wheat NBR1-like Domain Gene Negatively Regulates Drought Stress Tolerance in Transgenic Arabidopsis

Drought stress is an important factor that severely affects crop yield and quality. Autophagy has a crucial role in the responses to abiotic stresses. In this study, we explore TaNBR1 in response to drought stress. Expression of the TaNBR1 gene was strongly induced by NaCl, PEG, and abscisic acid treatments. The TaNBR1 protein is localized in the Golgi apparatus and autophagosome. Transgenic Arabidopsis plants overexpressing TaNBR1 exhibited reduced drought tolerance. When subjected to drought stress, compared to the wild-type (WT) lines, the transgenic overexpressing TaNBR1 plants had a lower seed germination rate, relative water content, proline content, and reduced accumulation of antioxidant enzymes, i.e., superoxide dismutase, peroxidase, and catalase, as well as higher chlorophyll losses, malondialdehyde contents, and water loss. The transgenic plants overexpressing TaNBR1 produced much shorter roots in response to mannitol stress, in comparison to the WT plants, and they exhibited greater sensitivity to abscisic acid treatment. The expression levels of the genes related to stress in the transgenic plants were affected in response to drought stress. Our results indicate that TaNBR1 negatively regulates drought stress responses by affecting the expression of stress-related genes in Arabidopsis.