The C2H2 zinc‐finger protein SlZF3 regulates AsA synthesis and salt tolerance by interacting with CSN5B

Abiotic stresses are a major cause of crop loss. Ascorbic acid (AsA) promotes stress tolerance by scavenging reactive oxygen species (ROS), which accumulate when plants experience abiotic stress. Although the biosynthesis and metabolism of AsA are well established, the genes that regulate these pathways remain largely unexplored. Here, we report on a novel regulatory gene from tomato (Solanum lycopersicum) named SlZF3 that encodes a Cys2/His2‐type zinc‐finger protein with an EAR repression domain. The expression of SlZF3 was rapidly induced by NaCl treatments. The overexpression of SlZF3 significantly increased the levels of AsA in tomato and Arabidopsis. Consequently, the AsA‐mediated ROS‐scavenging capacity of the SlZF3‐overexpressing plants was increased, which enhanced the salt tolerance of these plants. Protein–protein interaction assays demonstrated that SlZF3 directly binds CSN5B, a key component of the COP9 signalosome. This interaction inhibited the binding of CSN5B to VTC1, a GDP‐mannose pyrophosphorylase that contributes to AsA biosynthesis. We found that the EAR domain promoted the stability of SlZF3 but was not required for the interaction between SlZF3 and CSN5B. Our findings indicate that SlZF3 simultaneously promotes the accumulation of AsA and enhances plant salt‐stress tolerance.     

Sound waves increases the ascorbic acid content of alfalfa sprouts by affecting the expression of ascorbic acid biosynthesis-related genes

Previous studies have shown that sound wave treatment can affect the expression of plant genes and improve the growth. So, we investigated the ability of sound waves to increase AsA (l-ascorbic acid) content in alfalfa (Medicago sativa) sprouts in this study. Sprouts were exposed to a range of sound wave frequencies for two 1-h periods per day for various numbers of days. Most sound wave treated sprouts had a higher AsA content than untreated sprouts. In addition, the activity level of superoxide dismutase, an enzyme with potent antioxidative properties, was increased in sound wave-treated sprouts. The AsA content varied in response to sound wave treatment. Most processing conditions, including 500 and 1000 Hz, increased AsA content by 24–50%; however, some treatment conditions caused reduced AsA content during sprout growth. Furthermore, AsA content during sprout storage was increased by most sound wave treatment conditions, with 13–36% increases observed following 800 and 1000 Hz sound wave treatments compared to untreated sprouts. To investigate the mechanisms underlying changes in AsA content, we analyzed the expression levels of AsA biosynthesis-related genes. We found that several genes, including VTC1, VTC2, VTC4, GME, L-GalDH, GLDH, MDHAR, and DHAR1, displayed differential expression in response to sound wave treatment. Therefore, sound wave treatment may be a viable method for increasing the nutritional contents of sprouted vegetables.     

Arabidopsis CSN5B Interacts with VTC1 and Modulates Ascorbic Acid Synthesis

Light regulates ascorbic acid (AsA) synthesis, which increases in the light, presumably reflecting a need for antioxidants to detoxify reactive molecules produced during photosynthesis. Here, we examine this regulation in Arabidopsis thaliana and find that alterations in the protein levels of the AsA biosynthetic enzyme GDP-Man pyrophosphorylase (VTC1) are associated with changes in AsA contents in light and darkness. To find regulatory factors involved in AsA synthesis, we identified VTC1-interacting proteins by yeast two-hybrid screening of a cDNA library from etiolated seedlings. This screen identified the photomorphogenic factor COP9 signalosome subunit 5B (CSN5B), which interacted with the N terminus of VTC1 in yeast and plants. Gel filtration profiling showed that VTC1-CSN5B also associated with the COP9 signalosome complex, and this interaction promotes ubiquitination-dependent VTC1 degradation through the 26S proteasome pathway. Consistent with this, csn5b mutants showed very high AsA levels in both light and darkness. Also, a double mutant of csn5b with the partial loss-of-function mutant vtc1-1 contained AsA levels between those of vtc1-1 and csn5b, showing that CSN5B modulates AsA synthesis by affecting VTC1. In addition, the csn5b mutant showed higher tolerance to salt, indicating that CSN5B regulation of AsA synthesis affects the response to salt stress. Together, our data reveal a regulatory role of CSN5B in light-dark regulation of AsA synthesis.     

High Temperature Inhibits Ascorbate Recycling and Light Stimulation of the Ascorbate Pool in Tomato despite Increased Expression of Biosynthesis Genes

Understanding how the fruit microclimate affects ascorbate (AsA) biosynthesis, oxidation and recycling is a great challenge in improving fruit nutritional quality. For this purpose, tomatoes at breaker stage were harvested and placed in controlled environment conditions at different temperatures (12, 17, 23, 27 and 31°C) and irradiance regimes (darkness or 150 µmol m^-2 s^-1). Fruit pericarp tissue was used to assay ascorbate, glutathione, enzymes related to oxidative stress and the AsA/glutathione cycle and follow the expression of genes coding for 5 enzymes of the AsA biosynthesis pathway (GME, VTC2, GPP, L-GalDH, GLDH). The AsA pool size in pericarp tissue was significantly higher under light at temperatures below 27°C. In addition, light promoted glutathione accumulation at low and high temperatures. At 12°C, increased AsA content was correlated with the enhanced expression of all genes of the biosynthesis pathway studied, combined with higher DHAR and MDHAR activities and increased enzymatic activities related to oxidative stress (CAT and APX). In contrast, at 31°C, MDHAR and GR activities were significantly reduced under light indicating that enzymes of the AsA/glutathione cycle may limit AsA recycling and pool size in fruit pericarp, despite enhanced expression of genes coding for AsA biosynthesis enzymes. In conclusion, this study confirms the important role of fruit microclimate in the regulation of fruit pericarp AsA content, as under oxidative conditions (12°C, light) total fruit pericarp AsA content increased up to 71%. Moreover, it reveals that light and temperature interact to regulate both AsA biosynthesis gene expression in tomato fruits and AsA oxidation and recycling.     

Overexpression of <Emphasis Type="Italic">MeDREB1D</Emphasis> confers tolerance to both drought and cold stresses in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Cassava (Manihot esculenta) is an important tropical crop with extraordinary tolerance to drought stress but few reports on it. In this study, MeDREB1D was significantly and positively induced by drought stress. Two allelic variants of the gene named MeDREB1D(R-2) and MeDREB1D(Y-3) were identified. Overexpressing MeDREB1D(R-2) and MeDREB1D(Y-3) in Arabidopsis resulted in stronger tolerance to drought and cold stresses. Under drought stress, transgenic plants had more biomass, higher survival rates and less MDA content than wild-type plants. Under cold stress, transgenic plants also had higher survival rates than wild-type plants. To further characterize the molecular function of MeDREB1D, we conducted an RNA-Seq analysis of transgenic and wild-type Arabidopsis plants. The results showed that the Arabidopsis plants overexpressing MeDREB1D led to changes in downstream genes. Several POD genes, which may play a vital role in drought and cold tolerance, were up-regulated in transgenic plants. In brief, these results suggest that MeDREB1D can simultaneously improve plant tolerance to drought and cold stresses.     

The action of 1-alkyl-1-nitrosoureas and 1-alkyl-3-nitro-1-nitrosoguanidines on the M1 generation of barley andArabidopsis thaliana (L.) heynh.

The activity of 1-methyl-1-nitrosourea (MNH), 1-ethyl-1-nitrosourea (ENH), 1-methyl-3-nitro-1-nitrosoguanidine (MNG) and 1-ethyl-3-nitro-1-nitrosoguanidine (ENG) was tested on seeds of barley andArabidopsis. The activity of nitrosoamides tested was expressed by the germination and M₁ seedling height reduction of barley and M₁ root length reduction ofArabidopsis.

1. 1)
   After the action of both nitrosoureas (MNH and ENH) the germination of barley is at the same level as that of controls, even at concentrations, leading to a maximal reduction in the height of seedlings. After the action of both nitrosoguanidines (MNG and ENG) germination decreases in parallel with the decreasing seedling height. InArabidopsis no such differences in the relation germination to root length reduction were observed after nitrosoureas and nitrosoguanidines treatment. The differences in the M₁ generation of barley andArabidopsis after nitrosoguanidines treatment may be the reason for the non-mutagenic action of MNG and ENG in barley.     
   
   

Histone methyltransferase TXR1 is required for both H3 and H3.3 lysine 27 methylation in the well-known ciliated protist <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

DNA replication elongation is tightly controlled by histone-modifying enzymes. Our previous studies showed that the histone methytransferase TXR1 (Tetrahymena Trithorax related protein 1) specifically catalyzes H3K27 monomethylation and affects DNA replication elongation in Tetrahymena thermophila. In this study, we investigated whether TXR1 has a substrate preference to the canonical H3 over the replacement variant H3.3. We demonstrated by histone mutagenesis that K27Q mutation in H3.3 further aggravated the replication stress phenotype of K27Q mutation in canonical H3, supporting H3.3 as a physiologically relevant substrate of TXR1. This result is in apparent contrast to the strong preference for canonical H3 recently reported in Arabidopsis homologues ATXR5 and ATXR6, and further corroborates the role of TXR1 in DNA replication.     

Identification and expression pattern analysis of the glucosinolate biosynthetic gene <Emphasis Type="Italic">BoCYP83B1</Emphasis> from broccoli

Glucosinolates are a branch of amino acid-derived metabolites, which are specifically found in Brassicales. In Arabidopsis, tryptophan derived indolic glucosinolates are required for plant defense against a wide range of pathogens and herbivores due to their strong antimicrobial activity and potential signaling function. An important enzyme in indolic glucosinolate biosynthesis pathway is CYP83B1, which oxidizes indole-3-acetaldoxime, a precursor of indole-3-acetic acid (IAA). In this study, we reported isolation and expression characterization of a CYP83B1 gene from Brassica oleracea L. var. italica Plenck, which we termed BoCYP83B1. Overexpression of BoCYP83B1 in Arabidopsis resulted in an altered glucosinolate profile and early flowering phenotype. By expressing the reporter gene β-glucuronidase under the control of the BoCYP83B1 promoter in Arabidopsis, we analyzed the spatial expression pattern of BoCYP83B1 under normal growth conditions as well as in response to several hormones and stresses. The BoCYP83B1 was primarily expressed in vascular tissue through the almost whole plant. It was strongly induced by methyl jasmonate, 1-amino-1-cyclopropanecarboxylic acid, salicylic acid (SA), gibberellin, and IAA, suggesting its involvement in complex signaling pathways. Mannitol, NaCl, UV, and Flagelin 22 significantly up-regulated BoCYP83B1 expression, indicating its possible role in stress response. Interestingly, the response of BoCYP83B1 to SA and NaCl showed tissue specificity. Thus, BoCYP83B1 might have different functions in different tissues.     

Ectopic expression of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) <Emphasis Type="Italic">CsTIR</Emphasis>/<Emphasis Type="Italic">AFB</Emphasis> genes enhance salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin receptors TIR1/AFBs play an essential role in a series of signaling network cascades. These F-box proteins have also been identified to participate in different stress responses via the auxin signaling pathway in Arabidopsis. Cucumber (Cucumis sativus L.) is one of the most important crops worldwide, which is also a model plant for research. In the study herein, two cucumber homologous auxin receptor F-box genes CsTIR and CsAFB were cloned and studied for the first time. The deduced amino acid sequences showed a 78% identity between CsTIR and AtTIR1 and 76% between CsAFB and AtAFB2. All these proteins share similar characteristics of an F-box domain near the N-terminus, and several Leucine-rich repeat regions in the middle. Arabidopsis plants ectopically overexpressing CsTIR or CsAFB were obtained and verified. Shorter primary roots and more lateral roots were found in these transgenic lines with auxin signaling amplified. Results showed that expression of CsTIR/AFB genes in Arabidopsis could lead to higher seeds germination rates and plant survival rates than wild-type under salt stress. The enhanced salt tolerance in transgenic plants is probably caused by maintaining root growth and controlling water loss in seedlings, and by stabilizing life-sustaining substances as well as accumulating endogenous osmoregulation substances. We proposed that CsTIR/AFB-involved auxin signal regulation might trigger auxin mediated stress adaptation response and enhance the plant salt stress resistance by osmoregulation.     

Characterization and genome-wide association mapping of resistance to leaf rust,stem rust and stripe rust in a geographically diverse collection of spring wheat landraces

The challenge posed by rapidly changing wheat rust pathogens, both in virulence and in environmental adaptation, calls for the development and application of new techniques to accelerate the process of breeding for durable resistance. To expand the resistance gene pool available for germplasm improvement, a panel of 159 landraces plus old cultivars was evaluated for seedling and adult plant resistance (APR) to over 35 Australian pathotypes of Puccinia triticina, Puccinia graminis f. sp. tritici, and Puccinia striiformis f. sp. tritici. Known seedling resistance (SR) genes for leaf rust (Lr2a, Lr3a, Lr13, Lr23, Lr16, and Lr20), stem rust (Sr12, Sr13, Sr23, Sr30, and Sr36), and stripe rust (Yr3, Yr4, Yr5, Yr9, Yr10, Yr17, and Yr27) were postulated. The APR genes identified via field assessments and marker analyses included the pleiotropic genes (Lr34/Yr18/Sr57, Lr46/Yr29/Sr58, Lr67/Yr46/Sr55, and Sr2/Lr27/Yr30), Lr68, Lr74, and uncharacterized APR. A genome-wide association analysis using linear mixed models detected 79 single nucleotide polymorphism (SNP) markers significantly associated with rust resistance, which were mapped on chromosomes 1A, 1B, 1D, 2A, 2B, 3A, 3B, 3D, 4A, 5A, 5B, 6A, 6B, 6D, 7A, 7B and 7D. SNPs associated with multiple rust resistances probably indicate the presence of new pleiotropic or closely linked genes. SNPs were mapped on chromosome positions (1AL, 1DS, 2AL, 4AS, 5BS, 6DL, and 7AL) that have not been known to carry APR genes. This study revealed the presence of a range of possibly unidentified effective seedling and APRs among the landraces, which might represent new sources of rust resistance for the ongoing effort to develop improved wheat cultivars.     

Glutathione reductase gene expression depends on chloroplast signals in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Glutathione reductase (EC 1.6.4.2) is one of the main antioxidant enzymes of the plant cell. In Arabidopsis thaliana, glutathione reductase is encoded by two genes: the gr1 gene encodes the cytosolic-peroxisomal form, and the gr2 gene encodes the chloroplast-mitochondrial form. Little is known about the regulation of expression of plant glutathione reductase genes. In the present work, we have demonstrated that gr2 (but not gr1) gene expression in Arabidopsis leaves changes depending on changes in redox state of the photosynthetic electron transport chain. Expression of both the gr1 and gr2 genes was induced by reactive oxygen species. In heterotrophic suspension cell culture of Arabidopsis, expression of both studied genes did not depend on H₂O₂ level or on changes in the redox state of the mitochondrial electron transport chain. Our data indicate that chloroplasts are involved in the regulation of the glutathione reductase gene expression in Arabidopsis.