Zongliang Xia

Combined Linkage and Association Mapping Reveals QTL and Candidate Genes for Plant and Ear Height in Maize

Plant height (PH) and ear height (EH) are two very important agronomic traits related to the population density and lodging in maize. In order to better understand of the genetic basis of nature variation in PH and EH, two bi-parental populations and one genome-wide association study (GWAS) population were used to map quantitative trait loci (QTL) for both traits. Phenotypic data analysis revealed a wide normal distribution and high heritability for PH and EH in the three populations, which indicated that maize height is a highly polygenic trait. A total of 21 QTL for PH and EH in three common genomic regions (bin 1.05, 5.04/05, and 6.04/05) were identified by QTL mapping in the two bi-parental populations under multiple environments. Additionally, 41 single nucleotide polymorphisms (SNPs) were identified for PH and EH by GWAS, of which 29 SNPs were located in 19 unique candidate gene regions. Most of the candidate genes were related to plant growth and development. One QTL on Chromosome 1 was further verified in a near-isogenic line (NIL) population, and GWAS identified a C2H2 zinc finger family protein that maybe the candidate gene for this QTL. These results revealed that nature variation of PH and EH are strongly controlled by multiple genes with low effect and facilitated a better understanding of the underlying mechanism of height in maize.

ZmRFP1, the putative ortholog of SDIR1, encodes a RING-H2 E3 ubiquitin ligase and responds to drought stress in an ABA-dependent manner in maize

Drought is one of the most important limiting factors in crop production. To identify genes required for the drought stress response in the cereal crop maize, a gene coding for RING-finger protein (ZmRFP1), which is highly responsive to PEG-induced drought stress, was isolated by mRNA differential display and rapid amplification of cDNA ends. The ZmRFP1 encodes a protein of 280 amino acids and contains a single C(3)H(2)C(3)-type RING motif in its C-terminal region. ZmRFP1 is an ortholog of Arabidopsis SDIR1 (salt- and drought-induced RING finger 1) (66% identity to AtSDIR1).The recombinant ZmRFP1 protein purified from Escherichia coli exhibited an in vitro E3 ubiquitin ligase activity. Real-time PCR analysis indicates that the transcript levels of ZmRFP1 were higher in aerial tissues including stems, leaves, tassels and immature ears, and were markedly up-regulated by drought stress, and exogenous ABA, but not by salt, heat and cold stresses. Transient expression of the green fluorescent protein (GFP)-ZmRFP1 fusion protein in onion cells revealed a plasma membrane localization of the protein. Further analysis of ZmRFP1 transcripts between an ABA-deficient transposon mutant viviparous14 (vp14) and its isogenic wild-type line W22 showed that ZmRFP1 transcript levels were induced significantly in the wild-type line under drought stress, but not in the mutant line VP14. These results indicate that ZmRFP1 responds to drought stress in an ABA-dependent way and is likely to function in the ubiquitin conjunction pathway. The ZmRFP1 might serve as a candidate gene in genetic improvement for drought tolerance engineering in cereal crop plants.

Overexpression of the Maize psbA Gene Enhances Drought Tolerance Through Regulating Antioxidant System, Photosynthetic Capability, and Stress Defense Gene Expression in Tobacco

The psbA (encoding D1 protein) plays an important role in protecting photosystem II (PSII) from oxidative damage in higher plants. In our previous study, the role of the psbA from maize (Zea mays. L) in response to SO2 stress was characterized. To date, information about the involvement of the psbA gene in drought response is scarce. Here we found that overexpression (OE) of ZmpsbA showed increased D1 protein abundance and enhanced drought stress tolerance in tobacco. The drought-tolerant phenotypes of the OE lines were accompanied by increases of key antioxidant enzymes SOD, CAT, and POD activities, but decreases of hydrogen peroxide, malondialdehyde, and ion leakage. Further investigation showed that the OE plants had much less reductions than the wild-type in the net photosynthesis rate (Pn), stomatal conductance (Gs), and the maximal photochemical efficiency of PSII (Fv/Fm) during drought stress; indicating that OE of ZmpsbA may alleviate photosynthesis inhibition during drought. qRT-PCR analysis revealed that there was significantly increased expression of NtLEA5, NtERD10C, NtAREB, and NtCDPK2 in ZmpsbA-OE lines. Together, our results indicate that ZmpsbA improves drought tolerance in tobacco possibly by alleviating photosynthesis reduction, reducing reactive oxygen species accumulation and membrane damage, and modulating stress defense gene expression. ZmpsbA could be exploited for engineering drought-tolerant plants in molecular breeding of crops.

The Maize AAA-Type Protein SKD1 Confers Enhanced Salt and Drought Stress Tolerance in Transgenic Tobacco by Interacting with Lyst-Interacting Protein 5

ATPase associated with various cellular activities (AAA) proteins are important regulators involved in diverse cellular functions. To date, the molecular mechanisms of AAA proteins involved in response to salt and drought stresses in plants are largely unknown. In this study, a putative SKD1 (suppressor of K + transport growth defect 1) ortholog from Zea mays (ZmSKD1), which encodes a putative AAA protein, was isolated. The transcript levels of ZmSKD1 were higher in aerial tissues and were markedly up-regulated by salt or drought stress. Over-expression of ZmSKD1 in tobacco plants enhanced their tolerances not only to salt but to drought. Moreover, reactive oxygen species accumulations in ZmSKD1 transgenic lines were relative less than those in wild-type plants during salt or PEG-induced water stress. The interaction between ZmSKD1 and NtLIP5 (Lyst-Interacting Protein 5 homolog from Nicotiana tabacum) was confirmed by both yeast two-hybrid and immuno-precipitation assays; moreover, the α-helix-rich domain in the C-terminus of ZmSKD1 was identified to be required for its interaction with NtLIP5 using truncation mutations. Collectively, these data demonstrate that ZmSKD1could be involved in salt and drought stress responses and its over-expression enhances salt or drought stress tolerance possibly through interacting with LIP5 in tobacco. This study may facilitate our understandings of the biological roles of SKD1-mediated ESCRT pathway under stress conditions in higher plants and accelerate genetic improvement of crop plants tolerant to environmental stresses.

The Mechanisms of Maize Resistance to Fusarium verticillioides by Comprehensive Analysis of RNA-seq Data

Fusarium verticillioides is the most commonly reported fungal species responsible for ear rot of maize which substantially reduces grain yield. It also results in a substantial accumulation of mycotoxins that give rise to toxic response when ingested by animals and humans. For inefficient control by chemical and agronomic measures, it thus becomes more desirable to select more resistant varieties. However, the molecular mechanisms underlying the infection process remain poorly understood, which hampers the application of quantitative resistance in breeding programs. Here, we reveal the disease-resistance mechanism of the maize inbred line of BT-1 which displays high resistance to ear rot using RNA high throughput sequencing. By analyzing RNA-seq data from the BT-1 kernels before and after F. verticillioides inoculation, we found that transcript levels of genes associated with key pathways are dramatically changed compared with the control treatment. Differential gene expression in ear rot resistant and susceptible maize was confirmed by RNA microarray and qRT-PCR analyses. Further investigation suggests that the small heat shock protein family, some secondary metabolites, and the signaling pathways of abscisic acid, jasmonic acid, or salicylic acids (SA) may be involved in the pathogen-associated molecular pattern-triggered immunity against F. verticillioides. These data will not only provide new insights into the molecular resistant mechanisms against fungi invading, but may also result in the identification of key molecular factors associated with ear rot resistance in maize.

Association mapping resolving the major loci Scmv2 conferring resistance to sugarcane mosaic virus in maize

Sugarcane mosaic virus (SCMV) is one of the most damaging virus diseases in maize. Two major loci, Scmv1 and Scmv2, have been identified in many studies across a broad spectrum of germplasm by linkage mapping. In this study, we undertook a genome-wide association study (GWAS) in a maize association panel comprising 504 diverse inbred lines with over 556,000 SNP markers in three environments. Extensive phenotypic variation of resistance to SCMV was observed in the association panel. GWAS identified five genes significantly associated with resistance to SCMV. A cluster of four genes was detected near or within the Scmv2 region on chromosome 3 (bin 3.05). GRMZM2G116204, which encoded auxin binding protein and resided within the fine-mapped Scmv2 region, was predicted as the most likely candidate gene for Scmv2. In conclusion, we found that GWAS along with linkage analyses are effective approaches to the mapping of resistance to SCMV and to the subsequent identification of causal genes.

The Arabidopsis LYST INTERACTING PROTEIN 5 Acts in Regulating Abscisic Acid Signaling and Drought Response

Multivesicular bodies (MVBs) are unique endosomes containing vesicles in the lumens and play essential roles in many eukaryotic cellular processes. The Arabidopsis LYST INTERACTING PROTEIN 5 (LIP5), a positive regulator of MVB biogenesis, has critical roles in biotic and abiotic stress responses. However, whether the abscisic acid (ABA) signaling is involved in LIP5-mediated stress response is largely unknown. Here, we report that LIP5 functions in regulating ABA signaling and drought response in Arabidopsis. Analyses of a LIP5 promoter-β-glucuronidase (GUS) construct revealed substantial GUS activity in whole seedlings. The expression of LIP5 was induced by ABA and drought, and overexpression of LIP5 led to ABA hypersensitivity, enhanced stomatal closure, reduced water loss, and, therefore, increased drought tolerance. On the contrary, LIP5 knockdown mutants showed ABA-insensitive phenotypes and reduced drought tolerance; suggesting that LIP5 acts in regulating ABA response. Further analysis using a fluorescent dye revealed that ABA and water stress induced cell endocytosis or vesicle trafficking in a largely LIP5-dependent manner. Furthermore, expression of several drought- or ABA-inducible marker genes was significantly down-regulated in the lip5 mutant seedlings. Collectively, our data suggest that LIP5 positively regulates drought tolerance through ABA-mediated cell signaling.

Comparative Physiological and Molecular Analyses of Two Contrasting Flue-Cured Tobacco Genotypes under Progressive Drought Stress

Drought is a major environmental factor that limits crop growth and productivity. Flue-cured tobacco (Nicotiana tabacum) is one of the most important commercial crops worldwide and its productivity is vulnerable to drought. However, comparative analyses of physiological, biochemical and gene expression changes in flue-cured tobacco varieties differing in drought tolerance under long-term drought stress are scarce. In this study, drought stress responses of two flue-cured tobacco varieties, LJ851 and JX6007, were comparatively studied at the physiological and transcriptional levels. After exposing to progressive drought stress, the drought-tolerant LJ851 showed less growth inhibition and chlorophyll reduction than the drought-sensitive JX6007. Moreover, higher antioxidant enzyme activities and lower levels of H2O2, Malondialdehyde (MDA), and electrolyte leakage after drought stress were found in LJ851 when compared with JX6007. Further analysis showed that LJ851 plants had much less reductions than the JX6007 in the net photosynthesis rate and stomatal conductance during drought stress; indicating that LJ851 had better photosynthetic performance than JX6007 during drought. In addition, transcriptional expression analysis revealed that LJ851 exhibited significantly increased transcripts of several categories of drought-responsive genes in leaves and roots under drought conditions. Together, these results indicated that LJ851 was more drought-tolerant than JX6007 as evidenced by better photosynthetic performance, more powerful antioxidant system, and higher expression of stress defense genes during drought stress. This study will be valuable for the development of novel flue-cured tobacco varieties with improved drought tolerance by exploitation of natural genetic variations in the future.

Impairment of Sulfite Reductase Decreases Oxidative Stress Tolerance in Arabidopsis thaliana

As an essential enzyme in the sulfate assimilation reductive pathway, sulfite reductase (SiR) plays important roles in diverse metabolic processes such as sulfur homeostasis and cysteine metabolism. However, whether plant SiR is involved in oxidative stress response is largely unknown. Here, we show that SiR functions in methyl viologen (MV)-induced oxidative stress in Arabidopsis. The transcript levels of SiR were higher in leaves, immature siliques, and roots and were markedly and rapidly up-regulated by MV exposure. The SiR knock-down transgenic lines had about 60% residual transcripts and were more susceptible than wild-type when exposed to oxidative stress. The severe damage phenotypes of the SiR-impaired lines were accompanied by increases of hydrogen peroxide (H2O2), malondialdehyde (MDA), and sulfite accumulations, but less amounts of glutathione (GSH). Interestingly, application of exogenous GSH effectively rescued corresponding MV hypersensitivity in SiR-impaired plants. qRT-PCR analysis revealed that there was significantly increased expression of several sulfite metabolism-related genes in SiR-impaired lines. Noticeably, enhanced transcripts of the three APR genes were quite evident in SiR-impaired plants; suggesting that the increased sulfite in the SiR-impaired plants could be a result of the reduced SiR coupled to enhanced APR expression during oxidative stress. Together, our results indicate that SiR is involved in oxidative stress tolerance possibly by maintaining sulfite homeostasis, regulating GSH levels, and modulating sulfite metabolism-related gene expression in Arabidopsis. SiR could be exploited for engineering environmental stress-tolerant plants in molecular breeding of crops.

Sulfite Oxidase is Essential for Timely Germination of Maize Seeds upon Sulfite Exposure

Acid rain adversely affects maize seed germination by sulfite toxicity. Unfortunately, the molecular mechanisms underlying seed germination inhibition by sulfite in maize are largely unknown. Previously, the involvement of sulfite oxidase (SO) in sulfite detoxification in model plants was characterized. Here, we characterized Zea mays SO (ZmSO) in transgenic maize to investigate its role during seed germination upon sulfite exposure. ZmSO was responsive to sulfite stress at the transcriptional level during germination of maize seeds. Unlike the null mutant atso-1, ZmSO-overexpressing transgenic Arabidopsis plants were tolerant to SO2 stress and could effectively rescue the susceptible phenotype of atso-1. Silencing of ZmSO could lead to seed germination delay upon sulfite exposure, but not under normal conditions; interestingly, expressions of several seed germination-related genes encoding hydrolytic enzymes such as α-amylase, β-amylase, and glucosidase were reduced markedly in germinating seeds of ZmSO-compromised lines. This indicates that embryonic SO might alleviate the inhibitory effect of toxic sulfite by sulfite oxidation and the modulation of several hydrolytic enzymes during seed germination. Collectively, these data demonstrate that embryonic SO could be essential for timely seed germination upon sulfite exposure in maize. ZmSO might be a promising target for genetic improvement of crops tolerant to acid rain in molecular breeding programs.