ZmCIPK21, A Maize CBL-Interacting Kinase,Enhances Salt Stress Tolerance in Arabidopsis thaliana

Salt stress represents an increasing threat to crop growth and yield in saline soil. In this study, we identified a maize calcineurin B-like protein-interacting protein kinase (CIPK), ZmCIPK21, which was primarily localized in the cytoplasm and the nucleus of cells and displayed enhanced expression under salt stress. Over-expression of ZmCIPK21 in wild-type Arabidopsis plants increased their tolerance to salt, as supported by the longer root lengths and improved growth. The downstream stress-response genes, including dehydration-responsive element-binding (DREB) genes were also activated in transgenic plants over-expressing ZmCIPK21. In addition, introduction of the transgenic ZmCIPK21 gene into the Arabidopsis mutant cipk1-2 rescued the salt-sensitive phenotype under high salt stress. Measurement of Na⁺ and K⁺ content in transgenic plants showed that over-expression of ZmCIPK21 decreased accumulation of Na⁺ and allowed retention of relatively high levels of K⁺, thereby enhancing plant tolerance to salt conditions.     

Overexpression of Ferredoxin,PETF, Enhances Tolerance to Heat Stress in Chlamydomonas reinhardtii

Reactive oxygen species (ROS) produced by plants in adverse environments can cause damage to organelles and trigger cell death. Removal of excess ROS can be achieved through the ascorbate scavenger pathway to prevent plant cell death. The amount of this scavenger can be regulated by ferredoxin (FDX). Chloroplastic FDXs are electron transfer proteins that perform in distributing photosynthetic reducing power. In this study, we demonstrate that overexpression of the endogenous photosynthetic FDX gene, PETF, in Chlamydomonas reinhardtii could raise the level of reduced ascorbate and diminish H₂O₂ levels under normal growth conditions. Furthermore, the overexpressing PETF transgenic Chlamydomonas lines produced low levels of H₂O₂ and exhibited protective effects that were observed through decreased chlorophyll degradation and increased cell survival under heat-stress conditions. The findings of this study suggest that overexpression of PETF can increase the efficiency of ROS scavenging in chloroplasts to confer heat tolerance. The roles of PETF in the downregulation of the ROS level offer a method for potentially improving the tolerance of crops against heat stress.     

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.     

Overexpressing of OsAMT1-3, a High Affinity Ammonium Transporter Gene,Modifies Rice Growth and Carbon-Nitrogen Metabolic Status

AMT1-3 encodes the high affinity NH₄⁺ transporter in rice roots and is predominantly expressed under nitrogen starvation. In order to evaluate the effect of AMT1-3 gene on rice growth, nitrogen absorption and metabolism, we generated AMT1-3-overexpressing plants and analyzed the growth phenotype, yield, carbon and nitrogen metabolic status, and gene expression profiles. Although AMT1-3 mRNA accumulated in transgenic plants, these plants displayed significant decreases in growth when compared to the wild-type plants. The nitrogen uptake assay using a ¹⁵N tracer revealed poor nitrogen uptake ability in AMT1-3-overexpressing plants. We found significant decreases in AMT1-3-overexpressing plant leaf carbon and nitrogen content accompanied with a higher leaf C/N ratio. Significant changes in soluble proteins and carbohydrates were also observed in AMT1-3-overexpressing plants. In addition, metabolite profile analysis demonstrated significant changes in individual sugars, organic acids and free amino acids. Gene expression analysis revealed distinct expression patterns of genes that participate in carbon and nitrogen metabolism. Additionally, the correlation between the metabolites and gene expression patterns was consistent in AMT1-3-overexpressing plants under both low and high nitrogen growth conditions. Therefore, we hypothesized that the carbon and nitrogen metabolic imbalance caused by AMT1-3 overexpressing attributed to the poor growth and yield of transgenic plants.     

The Characterization of SaPIN2b,a Plant Trichome-Localized Proteinase Inhibitor from Solanum americanum

Proteinase inhibitors play an important role in plant resistance of insects and pathogens. In this study, we characterized the serine proteinase inhibitor SaPIN2b, which is constitutively expressed in Solanum americanum trichomes and contains two conserved motifs of the proteinase inhibitor II (PIN2) family. The recombinant SaPIN2b (rSaPIN2b), which was expressed in Escherichia coli, was demonstrated to be a potent proteinase inhibitor against a panel of serine proteinases, including subtilisin A, chymotrypsin and trypsin. Moreover, rSaPIN2b also effectively inhibited the proteinase activities of midgut trypsin-like proteinases that were extracted from the devastating pest Helicoverpa armigera. Furthermore, the overexpression of SaPIN2b in transgenic tobacco plants resulted in enhanced resistance against H. armigera. Taken together, our results demonstrated that SaPIN2b is a potent serine proteinase inhibitor that may act as a protective protein in plant defense against insect attacks.     

The Glutathione Peroxidase Gene Family in Thellungiella salsuginea: Genome-Wide Identification,Classification, and Gene and Protein Expression Analysis under Stress Conditions

Glutathione peroxidases (GPX) catalyze the reduction of H₂O₂ or organic hydroperoxides to water or corresponding alcohols using reduced glutathione, which plays an essential role in ROS (reactive oxygen species) homeostasis and stress signaling. Thellungiella salsuginea (Eutrema salsugineum), a relative of Arabidopsis thaliana, displays an extremely high level of tolerance to salt, drought, cold and oxidative stresses. The enzymatic antioxidant systems may contribute to the stress tolerance of T. salsuginea. In the present study, we aimed at understanding the roles of the antioxidant enzymes in T. salsuginea by focusing on the GPX family. We identified the eight GPX genes in T. salsuginea, and the structure of the N-terminal domains indicated their putative chloroplastic, mitochondrial and cytoplasmic location. The exon-intron organization of these genes exhibited a conserved pattern among plant GPX genes. Multiple environmental stresses and hormone response related cis-acting elements were predicted in the promoters of TsGPX genes. The gene and protein expression profiles of TsGPXs in response to high level of salinity and osmotic stresses, in leaves and roots of T. salsuginea were investigated using real-time RT-PCR and western blotting analysis. Our result showed that different members of the GPX gene family were coordinately regulated under specific environmental stress conditions, and supported the important roles of TsGPXs in salt and drought stress response in T. salsuginea.     

A C2H2-Type Zinc-Finger Protein from Millettia pinnata,MpZFP1, Enhances Salt Tolerance in Transgenic Arabidopsis

C2H2 zinc finger proteins (ZFPs) play important roles in plant development and response to abiotic stresses, and have been studied extensively. However, there are few studies on ZFPs in mangroves and mangrove associates, which represent a unique plant community with robust stress tolerance. MpZFP1, which is highly induced by salt stress in the mangrove associate Millettia pinnata, was cloned and functionally characterized in this study. MpZFP1 protein contains two zinc finger domains with conserved QALGGH motifs and targets to the nucleus. The heterologous expression of MpZFP1 in Arabidopsis increased the seeds’ germination rate, seedling survival rate, and biomass accumulation under salt stress. The transgenic plants also increased the expression of stress-responsive genes, including RD22 and RD29A, and reduced the accumulation of reactive oxygen species (ROS). These results indicate that MpZFP1 is a positive regulator of plant responses to salt stress due to its activation of gene expression and efficient scavenging of ROS.     

Genome-Wide Identification of PRP Genes in Apple Genome and the Role of MdPRP6 in Response to Heat Stress

Plant proline-rich proteins (PRPs) are cell wall proteins that occur in the plant kingdom and are involved in plant development and stress response. In this study, 9 PRP genes were identified from the apple genome and a comprehensive analysis of the PRP family was conducted, including gene structures, phylogenetic analysis, chromosome mapping, and so on. The expression of MdPRPs varied among tissues and in response to different types of stresses. MdPRP4 and MdPRP7 were induced by five detected stress treatments, including heat, drought, abscisic acid, cold, and salt; the expression patterns of the others varied under different types of stress. Subcellular localization showed that MdPRPs mainly functioned in the cytoplasm, except for MdPRP1 and MdPRP5, which also functioned in the nucleus. When MdPRP6 was overexpressed in tobacco, the transgenic plants showed higher tolerance to high temperature (48 °C) compared with wild-type (WT) plants. The transgenic plants showed milder wilting, a lower accumulation of electrolyte leakage, MDA and ROS, and a higher level of chlorophyll and SOD and POD activity, indicating that MdPRP6 may be an important gene in apples for heat stress tolerance. Overall, this study suggested that MdPRPs are critically important for the ability of apple responses to stresses.     

Cloning of the Lycopene β-cyclase Gene in Nicotiana tabacum and Its Overexpression Confers Salt and Drought Tolerance

Carotenoids are important pigments in plants that play crucial roles in plant growth and in plant responses to environmental stress. Lycopene β cyclase (β-LCY) functions at the branch point of the carotenoid biosynthesis pathway, catalyzing the cyclization of lycopene. Here, a β-LCY gene from Nicotiana tabacum, designated as Ntβ-LCY1, was cloned and functionally characterized. Robust expression of Ntβ-LCY1 was found in leaves, and Ntβ-LCY1 expression was obviously induced by salt, drought, and exogenous abscisic acid treatments. Strong accumulation of carotenoids and expression of carotenoid biosynthesis genes resulted from Ntβ-LCY1 overexpression. Additionally, compared to wild-type plants, transgenic plants with overexpression showed enhanced tolerance to salt and drought stress with higher abscisic acid levels and lower levels of malondialdehyde and reactive oxygen species. Conversely, transgenic RNA interference plants had a clear albino phenotype in leaves, and some plants did not survive beyond the early developmental stages. The suppression of Ntβ-LCY1 expression led to lower expression levels of genes in the carotenoid biosynthesis pathway and to reduced accumulation of carotenoids, chlorophyll, and abscisic acid. These results indicate that Ntβ-LCY1 is not only a likely cyclization enzyme involved in carotenoid accumulation but also confers salt and drought stress tolerance in Nicotiana tabacum.     

Application of CRISPR/CasΦ2 System for Genome Editing in Plants

CRISPR/Cas system has developed a new technology to modify target genes. In this study, CasΦ2 is a newly Cas protein that we used for genome modification in Arabidopsis and tobacco. PDS and BRI1 of marker genes were chosen for targeting. CasΦ2 has the function to cleave pre-crRNA. In the presence of 10 mM Mg²⁺ irons concentration, sgRNA3 type guided CasΦ2 to edit target gene and generate mutation, and a mutant seedling of AtBRI1 gene with an expected male sterile phenotype was obtained. In the process of tobacco transformation, the gene editing activity of CasΦ2 can be activated by 100 nM Mg²⁺ irons concentration, and sgRNA1 type guided CasΦ2 to edit target gene. Mutant seedlings of NtPDS gene with an expected albino were obtained. The results indicate that CasΦ2 can effectively edit target genes under the guidance of different sgRNA type in the presence of Mg²⁺ ions. Together, our results verify that the CRISPR/CasΦ2 system is an effective and precise tool for genome editing in plants.     

Hydrogen Sulfide Alleviates Manganese Stress in Arabidopsis

Hydrogen sulfide (H₂S) has been shown to participate in various stress responses in plants, including drought, salinity, extreme temperatures, osmotic stress, and heavy metal stress. Manganese (Mn), as a necessary nutrient for plant growth, plays an important role in photosynthesis, growth, development, and enzymatic activation of plants. However, excessive Mn²⁺ in the soil can critically affect plant growth, particularly in acidic soil. In this study, the model plant Arabidopsis thaliana was used to explore the mechanism of H₂S participation and alleviation of Mn stress. First, using wild-type Arabidopsis with excessive Mn²⁺ treatment, the following factors were increased: H₂S content, the main H₂S synthetase L-cysteine desulfhydrase enzyme (AtLCD) activity, and the expression level of the AtLCD gene. Further, using the wild-type, AtLCD deletion mutant (lcd) and overexpression lines (OE5 and OE32) as materials, the phenotype of Arabidopsis seedlings was observed by exogenous application of hydrogen sulfide donor sodium hydrosulfide (NaHS) and scavenger hypotaurine (HT) under excessive Mn²⁺ treatment. The results showed that NaHS can significantly alleviate the stress caused by Mn²⁺, whereas HT aggravates this stress. The lcd mutant is more sensitive to Mn stress than the wild type, and the overexpression lines are more resistant. Moreover, the mechanism of H₂S alleviating Mn stress was determined. The Mn²⁺ content and the expression of the Mn transporter gene in the mutant were significantly higher than those of the wild-type and overexpression lines. The accumulation of reactive oxygen species was significantly reduced in NaHS-treated Arabidopsis seedlings and AtLCD overexpression lines, and the activities of various antioxidant enzymes (SOD, POD, CAT, APX) also significantly increased. In summary, H₂S is involved in the response of Arabidopsis to Mn stress and may alleviate the inhibition of Mn stress on Arabidopsis seedling growth by reducing Mn²⁺ content, reducing reactive oxygen species content, and enhancing antioxidant enzyme activity. This study provides an important basis for further study of plant resistance to heavy metal stress.