Overexpression of the WRKY transcription factor gene NtWRKY65 enhances salt tolerance in tobacco (Nicotiana tabacum).

BACKGROUND: Salt stress severely inhibits plant growth, and the WRKY family transcription factors play important roles in salt stress resistance. In this study, we aimed to characterize the role of tobacco (Nicotiana tabacum) NtWRKY65 transcription factor gene in salinity tolerance. RESULTS: This study characterized the role of tobacco (Nicotiana tabacum) NtWRKY65 transcription factor gene in salinity tolerance using four NtWRKY65 overexpression lines. NtWRKY65 is localized to the nucleus, has transactivation activity, and is upregulated by NaCl treatment. Salinity treatment resulted in the overexpressing transgenic tobacco lines generating significantly longer roots, with larger leaf area, higher fresh weight, and greater chlorophyll content than those of wild type (WT) plants. Moreover, the overexpressing lines showed elevated antioxidant enzyme activity, reduced malondialdehyde content, and leaf electrolyte leakage. In addition, the Na+ content significantly decreased, and the K+/Na+ ratio was increased in the NtWRKY65 overexpression lines compared to those in the WT. These results suggest that NtWRKY65 overexpression enhances salinity tolerance in transgenic plants. RNA-Seq analysis of the NtWRKY65 overexpressing and WT plants revealed that NtWRKY65 might regulate the expression of genes involved in the salt stress response, including cell wall component metabolism, osmotic stress response, cellular oxidant detoxification, protein phosphorylation, and the auxin signaling pathway. These results were consistent with the morphological and physiological data. These findings indicate that NtWRKY65 overexpression confers enhanced salinity tolerance. CONCLUSIONS: Our results indicated that NtWRKY65 is a critical regulator of salinity tolerance in tobacco plants.

Overexpression of NtDOGL4 improves cadmium tolerance through abscisic acid signaling pathway in tobacco.

The DELAY OF GERMINATION1-LIKE (DOGL) genes play an essential role in diverse biological processes in plants. However, their exact involvement in the response to cadmium (Cd) stress via the ABA pathway remains unclear. Here, we focused on NtDOGL4, a tobacco DOGL gene whose expression is highly induced upon exposure to Cd. Overexpression of NtDOGL4 in tobacco resulted in elevated endogenous ABA levels, reduced Cd accumulation, and increased tolerance to Cd. Moreover, NtDOGL4 overexpression led to decreased accumulation of reactive oxygen species (ROS) and improved ROS scavenging capacity under Cd stress. Further analyses revealed the direct binding of the transcription factor ABSCISIC ACID-INSENSITIVE 5 (ABI5) to the NtDOGL4 promoter, positively regulating its expression in tobacco. Notably, NtDOGL4 overexpression promoted suberin formation and deposition, while suppressing the expression of Cd transporter genes in tobacco roots, as evidenced by histochemical staining, suberin fraction determination, and qRT-PCR assays. Collectively, our results demonstrate that NtDOGL4 overexpression reduces Cd accumulation, thereby improving Cd stress tolerance through the modulation of antioxidant system, transcription of Cd transporters, and suberin deposition. Notably, the NtABI5-NtDOGL4 module functions as a positive regulator in tobacco's Cd tolerance, underscoring its potential as a molecular target for developing low-Cd crops to ensure environmental safety.

NtHSP70-8b positively regulates heat tolerance and seed size in Nicotiana tabacum.

Heat stress considerably restricts the geographical distribution of crops and affects their growth, development, and productivity. HSP70 plays a critical regulatory role in plant growth response to heat stress. However, the mechanisms of this regulatory remain poorly understood. Here, an HSP70 gene, NtHSP70-8b, which is involved in the heat stress response of tobacco, was cloned and identified. The expression of NtHSP70-8b was induced by exogenous abscisic acid (ABA) treatment and abiotic stress, including heat, drought, and salt. Notably, high NtHSP70-8b expression occurred under heat stress conditions, which was consistent with the ß-glucuronidase histochemical analysis. Moreover, NtHSP70-8b overexpression markedly enhanced heat stress tolerance by changing the stomatal conductance and antioxidant capacity in tobacco leaves. qRT-PCR showed that the expression levels of ABA synthesis and response genes (NtNCED3 and NtAREB), stress defence genes (NtERD10C and NtLEA5), and other HSP genes (NtHSP90 and NtHSP26a) in NtHSP70-8b-overexpressing tobacco were high under heat stress. The interaction of NtHSP70-8b with NtHSP26a was further confirmed by a luciferase complementation imaging assay. In contrast, NtHSP70-8b knockout mutants showed significantly reduced antioxidant capacity compared to the wild type (WT) under heat stress conditions, suggesting that NtHSP70-8b acts as a positive regulator of heat stress in tobacco. Moreover, NtHSP70-8b overexpression increased the 1000-seed weight. Taken together, NtHSP70-8b is involved in the heat stress response, and NtHSP70-8b overexpression contributed to enhanced tolerance to heat stress, which is thus an essential gene with potential application value for developing heat stress-tolerant crops.

Sulfur dioxide improves the thermotolerance of maize seedlings by regulating salicylic acid biosynthesis.

Heat stress (HS) has become a serious threat to crop growth and yield. Sulfur dioxide (SO2) is being verified as a signal molecule in regulating the plant stress response. However, it is unknown whether SO2 plays a significant role in the plant heat stress response (HSR). Herein, maize seedlings were pretreated with various concentrations of SO2 and then kept at 45 °C for heat stress treatment, aiming to study the effect of SO2 pretreatment on HSR in maize by phenotypic, physiological, and biochemical analyses. It was found that SO2 pretreatment greatly improved the thermotolerance of maize seedlings. The SO2-pretreated seedlings showed 30-40% lower ROS accumulation and membrane peroxidation, but 55-110% higher activities of antioxidant enzymes than the distilled water-pretreated seedlings under heat stress. Interestingly, endogenous salicylic acid (SA) levels were increased by ∼85% in SO2-pretreated seedlings, as revealed by phytohormone analyses. Furthermore, the SA biosynthesis inhibitor paclobutrazol markedly reduced SA levels and attenuated SO2-triggered thermotolerance of maize seedlings. Meanwhile, transcripts of several SA biosynthesis and signaling, and heat stress-responsive genes in SO2-pretreated seedlings were significantly elevated under HS. These data have demonstrated that SO2 pretreatment increased endogenous SA levels, which activated the antioxidant machinery and strengthened the stress defense system, thereby improving the thermotolerance of maize seedlings under HS. Our current study provides a new strategy for mitigating heat stress damage for safe crop production.

Sulfur dioxide promotes seed germination by modulating reactive oxygen species production in maize.

Sulfur dioxide (SO2) is generally considered to be toxic to cells, but recent studies have shown that SO2 has positive roles in stress defense responses in plants. However, whether SO2 functions as a signaling molecule in the developmental process, especially in seed germination, is yet to be studied. Here, we present data supporting the role of SO2 in seed germination and possible molecular mechanisms. SO2 treatment significantly promoted the seed germination and seed vigor in maize. The germinating seeds treated with SO2 treatment exhibited higher reactive oxygen species (ROS) levels and NADPH oxidase activities. Furthermore, the specific NADPH oxidase inhibitor diphenyleneiodinium (DPI) strongly inhibited ROS accumulations, and SO2-promoted seed germination and vigor. Meanwhile, α-Amylase activity and transcripts in germinating seeds treated with SO2 were significantly elevated. These data have demonstrated that NADPH oxidase-dependent ROS production contributes to the induction of α-Amylase activity, thereby promoting seed germination upon SO2 exposure. SO2 might function as a signaling molecule in plant growth and development, especially in seed germination. This study might provide a theoretical foundation for the potential exploitation of hydrated SO2 in seed germination control in crop management.

The maize SUMO conjugating enzyme ZmSCE1b protects plants from paraquat toxicity.

Paraquat (PQ) herbicide causes damage to green plant tissues by inducing the production of toxic reactive oxygen species (ROS). SUMOylation is an important post-translational modification that enables plants to defend against multiple stresses. However, it is still unknown whether the SUMOylation is involved in PQ resistance response in crops. Herein, we showed that a maize SUMO conjugating enzyme gene (ZmSCE1b) functioned in PQ resistance. The quantitative real-time PCR (qRT-PCR) analysis revealed that this gene was significantly up-regulated upon PQ exposure. The overexpression of ZmSCE1b increased the levels of SUMO conjugates and improved PQ resistance in transgenic Arabidopsis. The ZmSCE1b-transgenic plants showed lower levels of ROS and lipid peroxidation, as well as higher antioxidant enzyme activities, upon PQ exposure. Furthermore, Western blotting showed that levels of SUMOylation in these transgenic plants were significantly elevated. In addition, the abundance of transcripts of several defense-related genes was apparently up-regulated in the over-expressing lines using qRT-PCR. Collectively, our results manifested the effect of overexpression of ZmSCE1b in improving resistance to PQ, possibly by regulating the levels of SUMO conjugates, antioxidant machinery, and expression of defense genes. Findings of this study can facilitate the understanding of the regulatory mechanisms underlying the involvement of SCE-mediated SUMOylation in PQ resistance response in crop plants. Meanwhile, ZmSCE1b could be utilized for engineering PQ-resistant crops in phytoremediation.

Overexpression of NtDOG1L-T Improves Heat Stress Tolerance by Modulation of Antioxidant Capability and Defense-, Heat-, and ABA-Related Gene Expression in Tobacco.

Drought and heat stresses are two major environmental stress factors that severely threaten crop growth and productivity. Plant delay of germination 1-like (DOG1L) family genes play important roles in various developmental processes and stress responses. In our previous study, a tobacco DOG1L gene (NtDOG1L-T) was found to regulate seedling growth and drought response. Unfortunately, the role of DOG1L genes in heat stress response is yet to be studied. Here, we present data supporting the role of DOG1L genes in heat stress and possible underlying molecular mechanisms. Transcript levels of NtDOG1L-T were rapidly induced by heat or abscisic acid (ABA) treatment. Furthermore, NtDOG1L-T promoter activity was markedly activated by ABA or heat stress, as revealed by histochemical staining in transgenic tobacco seedlings. Overexpression of NtDOG1L-T in transgenic lines improved heat stress tolerance. The NtDOG1L-T-transgenic plants exhibited lower levels of reactive oxygen species (ROS) and lipid peroxidation but higher antioxidant enzyme activities in response to heat stress. Furthermore, transcript abundance of some defense-, heat-, and ABA-responsive marker genes was significantly upregulated, as shown by reverse transcription quantitative PCR (qPCR) in these transgenic plants. In conclusion, NtDOG1L-T positively regulates heat stress tolerance possibly by modulation of antioxidant capability and defense-, heat-, and ABA-related gene expression in tobacco. This study may provide valuable resource for the potential exploitation of DOG1Ls in genetic improvement of heat stress tolerance in crops.

Transcriptome analysis of a new maize albino mutant reveals that zeta-carotene desaturase is involved in chloroplast development and retrograde signaling.

Carotenoids are a group of natural tetraterpenoid pigments with essential roles in a variety of physiological processes of plants. Although carotenoid biosynthesis has been well characterized, the genetic basis of the pathway, especially in crop plants, is largely unknown. In this study, we characterized a new albino maize mutant called albino1 (alb1), which was obtained from a Mutator mutagenized population. The alb1 mutant showed defective chloroplast development and declined photosynthetic pigments, leading to a seedling-lethal phenotype. Genetic and molecular analyses indicated that ALB1 encoded a putative ζ-carotene desaturase (ZDS) involved in carotenoid biosynthesis. Measurement of carotenoids revealed that several major carotenoid compounds downstream of the ZDS were significantly reduced in alb1 mutant, indicating that ALB1 is a functional ZDS. Further transcriptome analysis revealed that several groups of nuclear genes involved in photosynthesis, such as light-harvesting complex, pigment metabolism, and chloroplast function, were significantly down-regulated in alb1 compared with wide type. Interestingly, expression of some maize plastid-localized nuclear genes, including POR, CAO, Lhcb, and RbcS, was substantially reduced in alb1 plants. Furthermore, treatment of the inhibitor fluridone significantly rescued gene transcripts of these nucleus-encoded genes in alb1 mutant, which supported the retrograde signaling of ζ-carotene/phytofluene derived molecules. These results suggested that ALB1/ZDS might function as a regulator to coordinate nuclear photosynthetic gene expression in plastid-to-nucleus retrograde signaling during development of maize plants. Together, these results have demonstrated that ALB1/ZDS is essential for carotenoids biosynthesis and plays crucial roles in chloroplast biogenesis and development in maize.

A distinct class of plant and animal viral proteins that disrupt mitosis by directly interrupting the mitotic entry switch Wee1-Cdc25-Cdk1.

Many animal viral proteins, e.g., Vpr of HIV-1, disrupt host mitosis by directly interrupting the mitotic entry switch Wee1-Cdc25-Cdk1. However, it is unknown whether plant viruses may use this mechanism in their pathogenesis. Here, we report that the 17K protein, encoded by barley yellow dwarf viruses and related poleroviruses, delays G2/M transition and disrupts mitosis in both host (barley) and nonhost (fission yeast, Arabidopsis thaliana, and tobacco) cells through interrupting the function of Wee1-Cdc25-CDKA/Cdc2 via direct protein-protein interactions and alteration of CDKA/Cdc2 phosphorylation. When ectopically expressed, 17K disrupts the mitosis of cultured human cells, and HIV-1 Vpr inhibits plant cell growth. Furthermore, 17K and Vpr share similar secondary structural feature and common amino acid residues required for interacting with plant CDKA. Thus, our work reveals a distinct class of mitosis regulators that are conserved between plant and animal viruses and play active roles in viral pathogenesis.

The Arabidopsis APR2 positively regulates cadmium tolerance through glutathione-dependent pathway.

Cadmium (Cd) is a dangerous environmental pollutant with high toxicity to plants. The adenosine 5'-phosphosulfate reductase 2 (APR2) is the dominant APRs in Arabidopsis and plays an important role in reductive sulfate assimilation pathway. However, whether the involvement of plant APRs in Cd stress response is largely unclear. Herein, we report that APR2 functions in Cd accumulation and tolerance in Arabidopsis. The transcript levels of APR2 were markedly induced by Cd exposure. Transgenic plants overexpressing APR2 improved Cd tolerance, whereas knockout of APR2 reduced Cd tolerance. APR2-overexpressing plants with increased Cd accumulation and tolerance showed higher glutathione (GSH) and phytochelatin (PC) levels than the wild type and apr2 mutant plants, but lower H2O2 and TBARS contents upon Cd exposure. Moreover, exogenous GSH application effectively rescued Cd hypersensitivity in APR2-knockout plants. Further analysis showed that buthionine sulfoximine (BSO, an inhibitor of GSH synthesis) treatment completely eliminated the enhanced Cd tolerance phenotypes of APR2-overexpressing plants, implying that APR2-mediated enhanced Cd tolerance is GSH dependent. In addition, over-expression of the APR2 led to elevated expressions of the GSH/PC synthesis-related genes under Cd stress. Taken together, our results indicated that APR2 regulated Cd accumulation and tolerance possibly through modulating GSH-dependent antioxidant capability and Cd-chelation machinery in Arabidopsis. APR2 could be exploited for engineering heavy metal-tolerant plants in phytoremediation.

The Maize Class-I SUMO Conjugating Enzyme ZmSCE1d Is Involved in Drought Stress Response.

Post-translational modification of cellular proteins by sumoylation plays a vital role in stress responses of plants. However, the mechanisms underlying the sumoylation's involvement in stress responses in crop species remain largely unknown. Herein, a maize class-I SUMO conjugating enzyme gene (ZmSCE1d) was identified, whose expression was upregulated upon drought stress. Over-expression of ZmSCE1d in transgenic Arabidopsis plants increased SUMO conjugates and improved drought tolerance. The ZmSCE1d-transgenic plants showed higher antioxidant enzyme activities, but lower reactive oxygen species and lipid peroxidation upon drought stress. Furthermore, transcripts of several drought-responsive genes were significantly elevated, as revealed by qPCR in the transgenic lines. Taken together, these data have demonstrated that ZmSCE1d overexpression improved drought tolerance likely by regulating sumoylation levels, antioxidant capability, and drought-responsive gene expression in transgenic plants. This study may facilitate our understanding of the mechanisms underlying SCE-mediated sumoylation under drought stress and accelerate genetic improvement of crop plants tolerant to drought stress by manipulating the SUMO system.

Genome-Wide Identification and Analysis of the AP2 Transcription Factor Gene Family in Wheat (Triticum aestivum L.).

The AP2 transcription factors play important roles in regulating plant growth and development. However, limited data are available on the contributions of AP2 transcription factors in wheat (Triticum aestivum L.). In the present study, a total of 62 AP2 genes were identified in wheat from a genome-wide search against the latest wheat genome data. Phylogenetic and sequence alignment analyses divided the wheat AP2 genes into 3 clusters, euAP2, euANT, and basalANT. Chromosomal distribution, gene structure and duplication, and motif composition were subsequently investigated. The 62 TaAP2 genes were unevenly distributed on 21 chromosomes. Twenty-four homologous gene sets among A, B, and D sub-genomes were detected, which contributed to the expansion of the wheat AP2 gene family. The expression levels of TaAP2 genes were examined using the WheatExp database; most detected genes exhibited tissue-specific expression patterns. The transcript levels of 9 randomly selected TaAP2 genes were validated through qPCR analyses. Overexpression of TaAP2-10-5D, the most likely homolog of Arabidopsis ANT gene, increased organ sizes in Arabidopsis. Our results extend our knowledge of the AP2 gene family in wheat, and contribute to further functional characterization of AP2s during wheat development with the ultimate goal of improving crop production.

Overexpression of NtabDOG1L promotes plant growth and enhances drought tolerance in Nicotiana tabacum.

Drought is one of the major environmental stresses limiting crop growth and production. It is very important to exploit and utilize drought-tolerance genes to improve crop drought-resistance. In this study, we identified two homoeologs of a Nicotiana tabacum (Ntab) DELAY OF GERMINATION (DOG) 1 like gene, named as NtabDOG1L-T and NtabDOG1L-S, respectively. The NtabDOG1L genes were preferentially expressed in roots and their expression levels were induced by polyethylene glycol, high salt, cold, and abscisic acid treatments. Subcellular localization results indicated that NtabDOG1L-T was localized in the nucleus, cytoplasm and cell membrane. Overexpression of NtabDOG1L-T in tobacco resulted in roots growth enhancement in transgenic plants. Furthermore, overexpression of NtabDOG1L-T enhanced drought stress tolerance in transgenic tobacco. The transgenic tobacco lines exhibited lower leaf water loss and electrolyte leakage, lower content of malondialdehyde and reactive oxygen species (ROS), and higher antioxidant enzymes activities after drought treatment when compared with wild type (WT) plants. In addition, the expression levels of several genes encoding key antioxidant enzymes and drought-related proteins were higher in the transgenic plants than in the WT plants under drought stress. Taken together, our results showed that NtabDOG1L functions as a novel regulator that improves plant growth and drought tolerance in tobacco.

Identification of a 119-bp Promoter of the Maize Sulfite Oxidase Gene (ZmSO) That Confers High-Level Gene Expression and ABA or Drought Inducibility in Transgenic Plants.

Drought adversely affects crop growth and yields. The cloning and characterization of drought- or abscisic acid (ABA)-inducible promoters is of great significance for their utilization in the genetic improvement of crop resistance. Our previous studies have shown that maize sulfite oxidase (SO) has a sulfite-oxidizing function and is involved in the drought stress response. However, the promoter of the maize SO gene has not yet been characterized. In this study, the promoter (ZmSOPro, 1194 bp upstream region of the translation initiation site) was isolated from the maize genome. The in-silico analysis of the ZmSOPro promoter identified several cis-elements responsive to the phytohormone ABA and drought stress such as ABA-responsive element (ABRE) and MYB binding site (MBS), besides a number of core cis-acting elements, such as TATA-box and CAAT-box. A 5' RACE (rapid amplification of cDNA ends) assay identified an adenine residue as the transcription start site of the ZmSO. The ZmSOPro activity was detected by ß-glucuronidase (GUS) staining at nearly all developmental stages and in most plant organs, except for the roots in transgenic Arabidopsis. Moreover, its activity was significantly induced by ABA and drought stress. The 5'-deletion mutant analysis of the ZmSOPro in tobacco plants revealed that a 119-bp fragment in the ZmSOPro (upstream of the transcription start site) is a minimal region, which is required for its high-level expression. Moreover, the minimal ZmSOPro was significantly activated by ABA or drought stress in transgenic plants. Further mutant analysis indicated that the MBS element in the minimal ZmSOPro region (119 bp upstream of the transcription start site) is responsible for ABA and drought-stress induced expression. These results improve our understanding of the transcriptional regulation mechanism of the ZmSO gene, and the characterized 119-bp promoter fragment could be an ideal candidate for drought-tolerant gene engineering in both monocot and dicot crops.

The maize bHLH transcription factor bHLH105 confers manganese tolerance in transgenic tobacco.

Manganese (Mn) toxicity is an important limiting factor for crop production in acidic soils. The basic helix-loop-helix (bHLH) transcription factors are involved in a variety of physiological processes. However, whether the bHLHs are involved in excess Mn stress response is largely unknown. Here, we report the functional characterization of ZmbHLH105 isolated from maize (Zea mays). The transcript levels of ZmbHLH105 were higher in leaves, and were markedly up-regulated under excess Mn stress in maize. ZmbHLH105 was localized in the nucleus with transactivation activity. Ectopic expression of ZmbHLH105 enhanced Mn tolerance in Saccharomyces cerevisiae cells. ZmbHLH105-overexpressing (OE) plants showed improved excess Mn tolerance in transgenic tobacco. The stress-tolerant phenotypes of these OE tobacco lines were accompanied by increases of key antioxidant enzyme activities, but decreases of reactive oxygen species (ROS) accumulations. Importantly, the OE plants had less increases than the wild-type in toxic Mn accumulation. Moreover, the transcript levels of Mn/Fe-related transporters in the OE lines displayed remarkable decreases compared with the wild-type under Mn stress, suggesting that ZmbHLH105 reduced Mn accumulation in plants largely by repressing expression of Mn/Fe-regulated transporter genes. Taken together, these results indicate that ZmbHLH105 confers improved Mn stress tolerance possibly by regulating antioxidant machinery-mediated ROS scavenging and expression of Mn/Fe-related transporters in plants. ZmbHLH105 could be exploited for developing drought-tolerant maize varieties.

Overexpression of a maize SUMO conjugating enzyme gene (ZmSCE1e) increases Sumoylation levels and enhances salt and drought tolerance in transgenic tobacco.

As an essential regulatory process of post-translational modifications, Sumoylation has been shown to play a central role in stress responses in higher plants. However, the mechanisms underlying the involvement of the Sumoylation in stress responses in crops are largely unknown. In this study, a putative SUMO conjugating enzyme ortholog from Zea mays (ZmSCE1e) was isolated. Sequence alignments and phylogenetic analysis showed that ZmSCE1e possesses a central active domain similar to known SCE1 proteins, but is the cereal-specific isoform.The transcript levels of ZmSCE1e were markedly up-regulated by salt or drought stress. Over-expression of ZmSCE1e in tobacco plants increased levels of SUMO conjugates and enhanced their tolerances to salt and drought stresses. ZmSCE1e-transgenic plants showed higher activities of key antioxidant enzymes but lower hydrogen peroxide (H2O2) and malondialdehyde (MDA) accumulations under salt or drought stress. Furthermore, expression of several stress defense genes was significantly elevated as revealed by qPCR in the ZmSCE1e-transgenic lines. Together, these data have demonstrated that ZmSCE1e improved salt and drought tolerance likely by modulating Sumoylation levels, antioxidant capability, and stress defense gene expression in transgenic plants. This study may facilitate our understanding of the biological roles of SCE-mediated Sumoylation under stress conditions in higher plants and accelerate genetic improvement of crop plants tolerant to environmental stresses.

The Maize Sulfite Reductase Is Involved in Cold and Oxidative Stress Responses.

Sulfite reductase (SiR) functions in sulfate assimilation pathway. However, whether it is involved in stress response in crops is largely unknown. Here, the SiR ortholog from Zea mays (ZmSiR) was characterized. The recombinant ZmSiR protein was purified from E. coli. It exhibited sulfite-dependent activity and had strong affinity for sulfite. ZmSiR transcripts were markedly up-regulated by cold and methyl viologen (MV) treatments. Overexpression of ZmSiR complemented growth retardation phenotype of Arabidopsis atsir mutant. ZmSiR-overexpressing Arabidopsis plants were tolerant to severe SO2 stress and rescued the susceptible phenotype of the atsir. ZmSiR knock-down transgenic maize plants with 60% residual transcripts were more susceptible to cold or oxidative stress than wild-type. The severe damage phenotypes of the ZmSiR-compromised maize plants were accompanied by increases of sulfite and H2O2 accumulations, but less amounts of GSH. The qPCR analysis revealed that there was significantly altered expression of several key sulfur metabolism-related genes in ZmSiR-impaired maize lines under cold or MV stress. Particularly, ZmAPR2 expression was significantly elevated, suggesting that toxic sulfite accumulation in ZmSiR-impaired plants could be attributable to the reduced SiR coupled to increased ZmAPR2 expression. Together, our results indicate that ZmSiR is involved in cold and oxidative stress tolerance possibly by modulating sulfite reduction, GSH-dependent H2O2 scavenging, and sulfur-metabolism related gene expression. ZmSiR could be exploited for engineering environmental stress-tolerant varieties in molecular breeding of maize.

Combining Three Mapping Strategies to Reveal Quantitative Trait Loci and Candidate Genes for Maize Ear Length.

Ear length (EL) is an important trait in maize ( L.) because it is positively correlated with grain yield. To understand the genetic basis of natural EL variation, a F, a four-way cross and a genome-wide association study (GWAS) population were used to identify the quantitative trait loci (QTLs) and candidate EL genes. Linkage mapping identified 14 QTLs in two types of populations from multiple environments. Six of them were located in three common genomic regions considered "stable QTLs". Candidate genes for the three stable QTLs were identified by the GWAS results. These were related to auxin transport, cell proliferation, and developmental regulation. These results confirm that maize EL is under strong genetic control by many small-effect genes. They also improve our understanding of the genetic basis of maize EL.

Overexpression of the Maize Sulfite Oxidase Increases Sulfate and GSH Levels and Enhances Drought Tolerance in Transgenic Tobacco.

Sulfite oxidase (SO) plays a pivotal role in sulfite metabolism. In our previous study, sulfite-oxidizing function of the SO from Zea mays (ZmSO) was characterized. To date, the knowledge of ZmSO's involvement in abiotic stress response is scarce. In this study, we aimed to investigate the role of ZmSO in drought stress. The transcript levels of ZmSO were relatively high in leaves and immature embryos of maize plants, and were up-regulated markedly by PEG-induced water stress. Overexpression of ZmSO improved drought tolerance in tobacco. ZmSO-overexpressing transgenic plants showed higher sulfate and glutathione (GSH) levels but lower hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents under drought stress, indicating that ZmSO confers drought tolerance by enhancing GSH-dependent antioxidant system that scavenged ROS and reduced membrane injury. In addition, the transgenic plants exhibited more increased stomatal response than the wild-type (WT) to water deficit. Interestingly, application of exogenous GSH effectively alleviated growth inhibition in both WT and transgenic plants under drought conditions. qPCR analysis revealed that the expression of several sulfur metabolism-related genes was significantly elevated in the ZmSO-overexpressing lines. Taken together, these results imply that ZmSO confers enhanced drought tolerance in transgenic tobacco plants possibly through affecting stomatal regulation, GSH-dependent antioxidant system, and sulfur metabolism-related gene expression. ZmSO could be exploited for developing drought-tolerant maize varieties in molecular breeding.

The SULTR gene family in maize (Zea mays L.): Gene cloning and expression analyses under sulfate starvation and abiotic stress.

Sulfur is an essential macronutrient required for plant growth, development and stress responses. The family of sulfate transporters (SULTRs) mediates the uptake and translocation of sulfate in higher plants. However, basic knowledge of the SULTR gene family in maize (Zea mays L.) is scarce. In this study, a genome-wide bioinformatic analysis of SULTR genes in maize was conducted, and the developmental expression patterns of the genes and their responses to sulfate starvation and abiotic stress were further investigated. The ZmSULTR family includes eight putative members in the maize genome and is clustered into four groups in the phylogenetic tree. These genes displayed differential expression patterns in various organs of maize. For example, expression of ZmSULTR1;1 and ZmSULTR4;1 was high in roots, and transcript levels of ZmSULTR3;1 and ZmSULTR3;3 were high in shoots. Expression of ZmSULTR1;2, ZmSULTR2;1, ZmSULTR3;3, and ZmSULTR4;1 was high in flowers. Also, these eight genes showed differential responses to sulfate deprivation in roots and shoots of maize seedlings. Transcript levels of ZmSULTR1;1, ZmSULTR1;2, and ZmSULTR3;4 were significantly increased in roots during 12-day-sulfate starvation stress, while ZmSULTR3;3 and ZmSULTR3;5 only showed an early response pattern in shoots. In addition, dynamic transcriptional changes determined via qPCR revealed differential expression profiles of these eight ZmSULTR genes in response to environmental stresses such as salt, drought, and heat stresses. Notably, all the genes, except for ZmSULTR3;3, were induced by drought and heat stresses. However, a few genes were induced by salt stress. Physiological determination showed that two important thiol-containing compounds, cysteine and glutathione, increased significantly under these abiotic stresses. The results suggest that members of the SULTR family might function in adaptations to sulfur deficiency stress and adverse growing environments. This study will lay a foundation for better understanding the functional diversity of the SULTR family and exploring genes of interest for genetic improvement of sulfur use efficiency in cereal crop plants.