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.     

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.     

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.     

Post-transcriptional fine-tuning of COP9 signalosome subunit biosynthesis is regulated by the c-Myc/Lin28B/let-7 pathway

The COP9 signalosome (CSN) complex controls protein degradation via the ubiquitin (Ub) proteasome system (UPS) in eukaryotes. In mammalian cells, the multimeric CSN is composed of eight subunits (CSN1 - CSN8). It regulates cullin-RING Ub ligases (CRLs), which target essential regulatory proteins for ubiquitination and subsequent degradation. Thereby, the CSN cooperates with the UPS in a variety of essential cellular functions, including DNA repair, cell cycle and differentiation. Although functions of the CSN have been elucidated, mechanisms and regulatory principles of its de novo formation are completely unknown. Here, we show that there is a fundamental mechanism that allows a coordinated expression of all CSN subunits, a prerequisite for CSN assembly. CSN subunit mRNAs are targets of miRNAs of the let-7 family suppressing CSN subunit expression in human cells. Factors that reduce or block let-7 miRNAs induce the coordinated expression of CSN subunits. For instance, over-expression of CSN1 specifically traps let-7a-1 miRNA and elevates CSN subunit levels by two- to fourfold in a coordinated manner. CSN subunit expression is also increased by specific miRNA inhibitors or by interferon (IFN)-mediated induction of STAT1 and c-Myc reducing levels of let-7 miRNAs. Activation of STAT1 by IFNα or IFNγ induces c-Myc, which increases CSN subunit expression via the Lin28B/let-7 regulatory pathway. By contrast, a let-7a-1 mimic reduces CSN subunit expression. Our data show that let-7 miRNAs control the fine-tuning and coordinated expression of subunits for CSN de novo formation, presumably a general regulatory principle for other Zomes complexes as well.     

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.     

Cytosolic ascorbate peroxidase 1 modulates ascorbic acid metabolism through cooperating with nitrogen regulatory protein P-II in tea plant under nitrogen deficiency stress

Nitrogen (N) element is essential nutrient, and affect metabolism of secondary metabolites in higher plants. Ascorbate peroxidase (APX) plays an important role in ascorbic acid (AsA) metabolism of tea plant. However, the roles of cytosolic ascorbate peroxidase 1 (CsAPX1) in AsA metabolism under N deficiency stress in tea plant remains unclear in detail. In this work, nitrogen regulatory protein P-II (CsGLB1) and CsAPX1 were identified by isobaric tags for relative and absolute quantitation (iTRAQ) from tea plant. The cell growth rates in transgenic Escherichia coli overexpressing CsAPX1 and CsGLB1 were higher than empty vector under N sufficiency condition. Phenotype of shoots and roots, AsA accumulation, and expression levels of AtAPX1 and AtGLB1 genes were changed in transgenic Arabidopsis hosting CsAPX1 under N deficiency stress. These findings suggested that cytosolic CsAPX1 acted a regulator in AsA accumulation through cooperating with GLB1 under N deficiency stress in tea plant.     

Overexpression of HEMA1 encoding glutamyl-tRNA reductase

5-Aminolevulinic acid (ALA) synthesis has been shown to be the rate limiting step of tetrapyrrole biosynthesis. Glutamyl-tRNA reductase (GluTR) is the first committed enzyme of plant ALA synthesis and is controlled by interacting regulators, such as heme and the FLU protein. Induced inactivation of the HEMA1 gene encoding GluTR by RNAi expression in tobacco resulted in a reduced activity of Mg chelatase and Fe chelatase indicating a feed-forward regulatory mechanism that links ALA synthesis posttranslationally with late enzymes of tetrapyrrole biosynthesis (Hedtke et al., 2007). Here, the regulatory impact of GluTR was investigated by overexpression of AtHEMA1 in Arabidopsis and tobacco plants. Light-dependent ALA synthesis cannot benefit from an up to 7-fold induced expression of GluTR in Arabidopsis. While constitutive AtHEMA1 overexpression in tobacco stimulates ALA synthesis by 50-90% during light-exposed growth of seedlings, no increase in heme and chlorophyll contents is observed. HEMA1 overexpression in etiolated and dark-grown Arabidopsis and tobacco seedlings leads to additional accumulation of protochlorophyllide. As excessive accumulation of GluTR does not correlate with increased ALA formation, it is hypothesized that ALA synthesis is additionally limited by other effectors that balance the allocation of ALA with the activity of enzymes of chlorophyll and heme biosynthesis.     

The ethylene response factor AtERF98 enhances tolerance to salt through the transcriptional activation of ascorbic acid synthesis in Arabidopsis

Ascorbic acid (AsA) is an important antioxidant in plants, and its biosynthesis is finely regulated through developmental and environmental cues; however, the regulatory mechanism remains unclear. In this report, the knockout and knockdown mutants of Arabidopsis AtERF98 decreased the AsA level, whereas the overexpression of AtERF98 increased it, which suggests that AtERF98 plays an important role in regulating AsA biosynthesis. AtERF98-overexpressing plants showed enhanced expression of AsA synthesis genes in the d-mannose/l-galactose (d-Man/l-Gal) pathway and the myo-inositol pathway gene MIOX4, as well as of AsA turnover genes. In contrast, AtERF98 mutants showed decreased expression of AsA synthesis genes in the d-Man/l-Gal pathway but not of the myo-inositol pathway gene or AsA turnover genes. In addition, the role of AtERF98 in regulating AsA production was significantly impaired in the d-Man/l-Gal pathway mutant vtc1-1, but the expression of the myo-inositol pathway gene or AsA turnover genes was not affected, which indicates that the regulation of AtERF98 in AsA synthesis is primarily mediated by the d-Man/l-Gal pathway. Transient expression and chromatin immunoprecipitation assays further showed that AtERF98 binds to the promoter of VTC1, which indicates that AtERF98 modulates AsA biosynthesis by directly regulating the expression of the AsA synthesis genes. Moreover, the knockout mutant aterf98-1 displayed decreased salt-induced AsA synthesis and reduced tolerance to salt. The supplementation of exogenous AsA increased the salt tolerance of aterf98-1; coincidently, the enhanced salt tolerance of AtERF98-overexpressing plants was impaired in vtc1-1. Thus, our data provide evidence that the regulation of AtERF98 in AsA biosynthesis contributes to enhanced salt tolerance in Arabidopsis.     

Engineering ascorbic acid biosynthetic pathway in Arabidopsis leaves by single and double gene transformation

Six genes, which encode enzymes involved in ascorbic acid (AsA) biosynthesis, including guanosine diphosphate (GDP)-mannose pyrophosphorylase (GMP), GDP-mannose-3′,5′-epimerase (GME), GDP-galactose guanylyltransferase (GGT), L-galactose-1-phosphate phosphatase (GPP), L-galactose dehydrogenase (GDH) and L-galactono-1,4-lactone dehydrogenase (GLDH) were transformed into Arabidopsis thaliana, to evaluate the contribution of each gene to AsA accumulation. Additionally, two combinations, GGT-GPP and GGT-GLDH, were co-transformed into Arabidopsis with a reliable double-gene transformation system. AsA content of GGT transgenic lines was 2.9-fold higher as compared to the control, and co-transformation led up to 4.1-fold AsA enhancement. These results provided further evidence that GGT is the key enzyme in plant AsA biosynthesis.     

Manipulation of the Rice L-Galactose Pathway: Evaluation of the Effects of Transgene Overexpression on Ascorbate Accumulation and Abiotic Stress Tolerance

Ascorbic acid (AsA) is the most abundant water-soluble antioxidant in plants, and it plays a crucial role in plant growth, development and abiotic stress tolerance. In the present study, six key Arabidopsis or rapeseed genes involved in AsA biosynthesis were constitutively overexpressed in an elite Japonica rice cultivar. These genes encoded the GDP-mannose pyrophosphorylase (GMP), GDP-mannose-3'',5''-epimerase (GME), GDP-L-galactose phosphorylase (GGP), L-galactose-1-phosphate phosphatase (GPP), L-galactose dehydrogenase (GDH), and L-galactono-1,4-lactone dehydrogenase (GalLDH). The effects of transgene expression on rice leaf AsA accumulation were carefully evaluated. In homozygous transgenic seedlings, AtGGP transgenic lines had the highest AsA contents (2.55-fold greater than the empty vector transgenic control), followed by the AtGME and AtGDH transgenic lines. Moreover, with the exception of the AtGPP lines, the increased AsA content also provoked an increase in the redox state (AsA/DHA ratio). To evaluate salt tolerance, AtGGP and AtGME transgenic seedlings were exposed to salt stress for one week. The relative plant height, root length and fresh weight growth rates were significantly higher for the transgenic lines compared with the control plants. Altogether, our results suggest that GGP may be a key rate-limiting step in rice AsA biosynthesis, and the plants with elevated AsA contents demonstrated enhanced tolerance for salt stress.     

The decline in ascorbic acid content is associated with cadmium toxicity of rice seedlings

Cadmium (Cd) toxicity of rice (Oryza sativa L. cv. Taichung Native 1) seedlings was evaluated by the decrease in chlorophyll content and the increase in malondialdehyde (MDA) in the second leaves of rice seedlings. CdCl₂ (5 μM) treatment was accompanied by a decrease in the contents of ascorbic acid (AsA) and AsA + dehydroascorbate (DHA) and in the ratios of AsA/DHA in leaves. However, CdCl₂ treatment resulted in an increase in DHA content in leaves. Moreover, the decrease in AsA content was prior to the occurrence of chlorosis and associated with the increase in MDA content in the leaves of seedlings treated with Cd. Pretreatment with 0.5 mM AsA or l-galactono-1,4-lactone (GalL), the biosynthetic precursor of AsA, for 6 h resulted in an increase in the contents of AsA and reduced glutathione (GSH), the ratios of AsA/DHA and GSH/oxidized glutathione, and the activities of ascorbate peroxidase (APX) and glutathione reductase (GR) in the leaves of rice seedlings. Quantitative RT-PCR was applied to quantify the mRNA levels for OsAPX and OsGR genes from rice leaves to examine the effect of AsA or GalL pretreatment on the expression of OsAPX and OsGR genes in rice leaves. The expression of OsAPX2, OsAPX3, OsAPX4, OsAPX5, OsAPX6, OsAPX7, and OsGR1 was increased by AsA or GalL pretreatment. Rice seedlings pretreated with AsA or GalL were observed to reduce the subsequent Cd-induced toxicity. Our results suggest that AsA content may play a role in regulating Cd toxicity of rice seedlings.     

Alleviation of osmotic stress in Brassica napus,B. campestris,and B. juncea by ascorbic acid application

The roles of ascorbic acid (AsA, 1 mM) under an osmotic stress [induced by 15 % (m/v) polyethylene glycol, PEG-6000] were investigated by examining morphological and physiological attributes in Brassica species. The osmotic stress reduced the fresh and dry masses, leaf relative water content (RWC), and chlorophyll (Chl) content, whereas increased the proline (Pro), malondialdehyde (MDA), and H₂O₂ content, and lipoxygenase (LOX) activity. The ascorbate content in B. napus, B. campestris, and B. juncea decreased, increased, and remained unaltered, respectively. The dehydroascorbate (DHA) content increased only in B. napus. The AsA/DHA ratio was reduced by the osmotic stress in all the species except B. juncea. The osmotic stress increased the glutathione (GSH) content only in B. juncea, but increased the glutathione disulfide (GSSG) content and decreased the GSH/GSSG ratio in all the species. The osmotic stress increased the activities of ascorbate peroxidase (APX) (except in B. napus), glutathione reductase (GR) (except in B. napus), glutathione S-transferase (GST) (except in B. juncea), and glutathione peroxidase (GPX), and decreased the activities of catalase (CAT) and monodehydroascorbate reductase (MDHAR) (only in B. campestris). The osmotic stress decreased the glyoxalase I (Gly I) and increased glyoxalase II (Gly II) activities. The application of AsA in combination with PEG improved the fresh mass, RWC, and Chl content, whereas decreased the Pro, MDA, and H₂O₂ content in comparison with PEG alone. The AsA addition improved AsA-GSH cycle components and improved the activities of all antioxidant and glyoxalase enzymes in most of the cases. So, exogenous AsA improved physiological adaptation and alleviated oxidative damage under the osmotic stress by improving the antioxidant and glyoxalase systems. According to measured parameters, B. juncea can be recognized as more drought tolerant than B. napus and B. campestris.     

Novel aspects of COP9 signalosome functions revealed through analysis of hypomorphic csn mutants

The COP9 signalosome (CSN) is a conserved eukaryotic protein complex implicated in the regulation of cullin-RING type E3 ubiquitin ligases by cleaving the small peptide RUB/Nedd8 from cullins. However, detailed analysis of CSN physiological functions in Arabidopsis has been hampered by the early seedling-lethality of csn null mutants. We and others have now identified a number of viable hypomorphic csn mutants which start to reveal novel CSN-dependent activities in adult Arabidopsis plants.¹ Here, we present a detailed comparative analysis of the csn5a-1 and csn2-5 mutants as a mean to improve understanding of CSN functions in plant cells. Our observations point to CSN-independent activities of CSN5 and suggest a role of the CSN in cytoskeleton assembly/organization.Key words: Arabidopsis, root skewing, CSN, COP9 signalosome, SCF, ubiquitin, TIR1, auxin