Luqing Zheng

RNA-Seq Analysis of Rice Roots Reveals the Involvement of Post-Transcriptional Regulation in Response to Cadmium Stress

Widely-spread cadmium (Cd) pollution in the soil threatens both crop production and human health. How plants deal with the excess Cd are largely unknown. To evaluate the molecular mechanism by which plants respond to Cd stress, rice seedlings were treated with two concentrations of Cd and subjected to deep RNA sequencing. Comprehensive RNA-Seq analysis of rice roots under two gradients of Cd treatment revealed 1169 Cd toxicity-responsive genes. These genes were involved in the reactive oxygen species scavenging system, stress response, cell wall formation, ion transport, and signal transduction. Nine out of 93 predicted long non-coding RNAs (lncRNAs) were detected as Cd-responsive lncRNAs due to their high correlation with the Cd stress response. In addition, we analyzed alternative splicing (AS) events under different Cd concentrations. Four hundred and seventy-six differential alternatively spliced genes with 542 aberrant splicing events were identified. GO analysis indicated that these genes were highly enriched in oxidation reduction and cellular response to chemical stimulus. Real-time qRT-PCR validation analysis strengthened the reliability of our RNA-Seq results. The results suggest that post-transcriptional AS regulation may also be involved in plant responses to high Cd stress.

Transcriptional and physiological analyses identify a regulatory role for hydrogen peroxide in the lignin biosynthesis of copper-stressed rice roots

AimsInduction of lignin biosynthesis is an adaptive response of plants subjected to many abiotic stresses. In this study, we examined the response of lignin biosynthesis to copper (Cu) stress, with a particular focus on the regulatory mechanism.MethodsWe performed a transcriptomic analysis of rice (Oryza sativa L.) roots, and the microarray data on lignin biosynthesis pathway genes were corroborated by quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis. Physiological analyses of rice seedlings treated with Cu(II) sulfate (CuSO4) were used to confirm the relationship between excess Cu and lignin biosynthesis. In addition, we examined the role of hydrogen peroxide (H2O2) in Cu-induced lignin biosynthesis through pretreatments with an NADPH oxidase inhibitor (diphenyleneiodonium, DPI) and a H2O2 scavenger (dimethylthiourea, DMTU).ResultsLignin biosynthesis pathway genes were upregulated under Cu stress. The lignin content of rice roots increased significantly with increasing concentrations and durations of Cu treatment; elevations in root lignin content were correlated with marked inhibitions in root growth. Pretreatments with DPI and DMTU inhibited the activities of Cu-induced lignin polymerization enzymes (peroxidase, POD and laccase, LAC) and lignin accumulation in rice roots. Conversely, exogenous H2O2 increased the root lignin content.ConclusionsRice roots under Cu stress accumulate lignin through enhanced polymerization of lignin monolignol, a mechanism that requires Cu stress induced H2O2.

Comprehensive Analysis of Rice Laccase Gene (OsLAC) Family and Ectopic Expression of OsLAC10 Enhances Tolerance to Copper Stress in Arabidopsis

Laccases are encoded by a multigene family and widely distributed in plant genomes where they play roles oxidizing monolignols to produce higher-order lignin involved in plant development and stress responses. We identified 30 laccase genes (OsLACs) from rice, which can be divided into five subfamilies, mostly expressed during early development of the endosperm, growing roots, and stems. OsLACs can be induced by hormones, salt, drought, and heavy metals stresses. The expression level of OsLAC10 increased 1200-fold after treatment with 20 μM Cu for 12 h. The laccase activities of OsLAC10 were confirmed in an Escherichia coli expression system. Lignin accumulation increased in the roots of Arabidopsis over-expressing OsLAC10 (OsLAC10-OX) compared to wild-type controls. After growth on 1/2 Murashige and Skoog (MS) medium containing toxic levels of Cu for seven days, roots of the OsLAC10-OX lines were significantly longer than those of the wild type. Compared to control plants, the Cu concentration decreased significantly in roots of the OsLAC10-OX line under hydroponic conditions. These results provided insights into the evolutionary expansion and functional divergence of OsLAC family. In addition, OsLAC10 is likely involved in lignin biosynthesis, and reduces the uptake of Cu into roots required for Arabidopsis to develop tolerance to Cu.

Cloning and characterization of the Oryza sativa wall-associated kinase gene OsWAK11 and its transcriptional response to abiotic stresses

AimsTo examine heavy metal-induced regulatory mechanisms at the transcriptional level, a cell wall-associated receptor kinase (WAK) gene, OsWAK11 and its upstream promoter region (−946/+28) were isolated from Oryza sativa. OsWAK11 expression in response to abiotic stress was examined using a β-glucuronidase (GUS) gene fusion.MethodsSemi-quantitative RT-PCR was used to analyze expression of the OsWAK11 gene. Histochemical detection of GUS was conducted by X-gluc staining methods, and fluorometric measurements of GUS activity were made with 4-methyl umbelliferyl glucuronide (MUG) substrate.ResultsThe WAK promoter (−946/+28) responded to aluminum chloride, sodium chloride, and copper (II) sulfate with 3.0-, 2.2-, or 6.4-fold induction of GUS activity, respectively. Sodium nitroprusside and wounding treatment stimulated GUS activity. A histochemical analysis revealed strong GUS staining in the hypocotyls, cotyledons, first leaf, and petiole of cotyledons in transgenic tobacco seedlings. Strong GUS staining was also observed in the stigma and ovary of mature flowers, but not in the stamens.ConclusionOsWAK11 expression is regulated by aluminum, sodium, and copper. The GUS expression observed in transgenic tobacco carrying WAK11 promoter demonstrated significant tissue-specificity. The OsWAK11 promoter was strongly upregulated in response to metals and wounding.

Lignins: Biosynthesis and Biological Functions in Plants

Lignin is one of the main components of plant cell wall and it is a natural phenolic polymer with high molecular weight, complex composition and structure. Lignin biosynthesis extensively contributes to plant growth, tissue/organ development, lodging resistance and the responses to a variety of biotic and abiotic stresses. In the present review, we systematically introduce the biosynthesis of lignin and its regulation by genetic modification and summarize the main biological functions of lignin in plants and their applications. We hope this review will give an in-depth understanding of the important roles of lignin biosynthesis in various plants’ biological processes and provide a theoretical basis for the genetic improvement of lignin content and composition in energy plants and crops.

Screening for Cd-Safe Cultivars of Chinese Cabbage and a Preliminary Study on the Mechanisms of Cd Accumulation

With the rapid progress of industrialization, the effects of environmental contamination on plant toxicity, and subsequently on human health, is a growing concern. For example, the heavy metal pollution of soil such as that caused by cadmium (Cd) is a serious threat. Therefore, screening for pollution-safe edible plants is an essential approach for growing plants under heavy metal-contaminated soils. In the current study, 35 Chinese cabbage (Brassica pekinensis L.) cultivars were selected with the aim of screening for Cd-safe cultivars (CSCs), analyzing their safety, and exploring the mechanism of Cd accumulation. Our field-culture experiments revealed that the Cd content in the edible parts of the cultivars were varied and were determined to possibly be CSCs. Hydroponics experiments were used to simulate six different degrees of soil contamination (high and low Cd concentrations) on possible CSCs. The results indicated a significant difference (p < 0.05) in Cd concentration in the cultivars, and verified the safety of these possible CSCs. The analyses of the transport coefficient and expression levels showed that the differences in Cd accumulation among the Chinese cabbage cultivars were related to the expression of genes involved in absorption and transport rather than a root-to-shoot translocation limitation.

Alternative Splicing Plays a Critical Role in Maintaining Mineral Nutrient Homeostasis in Rice (Oryza sativa)

Genome-wide RNA-seq and mutant analyses reveal the importance of alternative splicing in the mineral deficiency response and in mineral uptake and remobilization in rice. Alternative splicing (AS) of pre-mRNAs promotes transcriptome and proteome diversity and plays important roles in a wide range of biological processes. However, the role of AS in maintaining mineral nutrient homeostasis in plants is largely unknown. To clarify this role, we obtained whole transcriptome RNA sequencing data from rice (Oryza sativa) roots grown in the presence or absence of several mineral nutrients (Fe, Zn, Cu, Mn, and P). Our systematic analysis revealed 13,291 alternatively spliced genes, representing ∼53.3% of the multiexon genes in the rice genome. As the overlap between differentially expressed genes and differentially alternatively spliced genes is small, a molecular understanding of the plant’s response to mineral deficiency is limited by analyzing differentially expressed genes alone. We found that the targets of AS are highly nutrient-specific. To verify the role of AS in mineral nutrition, we characterized mutants in genes encoding Ser/Arg (SR) proteins that function in AS. We identified several SR proteins as critical regulators of Zn, Mn, and P nutrition and showed that three SR protein-encoding genes regulate P uptake and remobilization between leaves and shoots of rice, demonstrating that AS has a key role in regulating mineral nutrient homeostasis in rice.