Liangju Wang

Genome-wide identification and characterization of cadmium-responsive microRNAs and their target genes in radish (Raphanus sativus L.) roots

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that play vital regulatory roles in plant growth, development, and environmental stress responses. Cadmium (Cd) is a non-essential heavy metal that is highly toxic to living organisms. To date, a number of conserved and non-conserved miRNAs have been identified to be involved in response to Cd stress in some plant species. However, the miRNA-mediated gene regulatory networks responsive to Cd stress in radish (Raphanus sativus L.) remain largely unexplored. To dissect Cd-responsive miRNAs and their targets systematically at the global level, two small RNA libraries were constructed from Cd-treated and Cd-free roots of radish seedlings. Using Solexa sequencing technology, 93 conserved and 16 non-conserved miRNAs (representing 26 miRNA families) and 28 novel miRNAs (representing 22 miRNA families) were identified. In all, 15 known and eight novel miRNA families were significantly differently regulated under Cd stress. The expression patterns of a set of Cd-responsive miRNAs were validated by quantitative real-time PCR. Based on the radish mRNA transcriptome, 18 and 71 targets for novel and known miRNA families, respectively, were identified by the degradome sequencing approach. Furthermore, a few target transcripts including phytochelatin synthase 1 (PCS1), iron transporter protein, and ABC transporter protein were involved in plant response to Cd stress. This study represents the first transcriptome-based analysis of miRNAs and their targets responsive to Cd stress in radish roots. These findings could provide valuable information for functional characterization of miRNAs and their targets in regulatory networks responsive to Cd stress in radish.

Promotive effect of 5-aminolevulinic acid on chlorophyll, antioxidative enzymes and photosynthesis of Pakchoi (Brassica campestris ssp.chinensis var. communis Tsen et Lee)

The objective of this article is to study the effect of 5-aminolevulinic acid (ALA) and enhanced chlorophyll content, antioxidative enzymes and photosynthesis rate by foliar application of ALA. We evaluated three concentrations (control-distilled water, T1-50 mg l−1, T2-150 mg l−1, T3-250 mg l−1) of ALA and seven cultivars, “Sanchidaye” (Sa-1), “Lichuandasuomian” (Li-1), “Aijiaohuang” (Ai-1), “Qingyou” No. 4 (Qi-1), “Aikang” No. 5 (Ak-1), “Hanxiao” (Ha-1) and “Shulv” (Sl-1). “Ak-1” showed strongest response of POD (peroxidase) enzyme activity (0.4 U g−1 min−1) in 250 mg l−1 ALA solution. The highest CAT (catalase) activity (0.8 U g−1 min−1) after administration of 250 mg l−1 ALA was observed in “Li-1”. Meanwhile, highest (1.42 mg l−1) total chlorophyll content was also observed in “Ak-1”, when leaves were treated in 50 mg l−1 ALA, “Li-1” and “Ai-1” showed strongest response of specific activity of superoxide dismutase (SOD) in 50 mg l−1 and 50 mg l−1 ALA. Two hundred and fifty milligram per milliliter of ALA-treatment significantly improved the net photosynthetic rate.

Identification and characterization of novel and conserved microRNAs in radish (Raphanus sativus L.) using high-throughput sequencing.

MicroRNAs (miRNAs) are endogenous, non-coding, small RNAs that play significant regulatory roles in plant growth, development, and biotic and abiotic stress responses. To date, a great number of conserved and species-specific miRNAs have been identified in many important plant species such as Arabidopsis, rice and poplar. However, little is known about identification of miRNAs and their target genes in radish (Raphanus sativus L.). In the present study, a small RNA library from radish root was constructed and sequenced using the high-throughput Solexa sequencing. Through sequence alignment and secondary structure prediction, a total of 545 conserved miRNA families as well as 15 novel (with their miRNA* strand) and 64 potentially novel miRNAs were identified. Quantitative real-time PCR (qRT-PCR) analysis confirmed that both conserved and novel miRNAs were expressed in radish, and some of them were preferentially expressed in certain tissues. A total of 196 potential target genes were predicted for 42 novel radish miRNAs. Gene ontology (GO) analysis showed that most of the targets were involved in plant growth, development, metabolism and stress responses. This study represents a first large-scale identification and characterization of radish miRNAs and their potential target genes. These results could lead to the further identification of radish miRNAs and enhance our understanding of radish miRNA regulatory mechanisms in diverse biological and metabolic processes.

Comparative proteomic and transcriptomic approaches to address the active role of GA4 in Japanese apricot flower bud dormancy release

Hormones are closely associated with dormancy in deciduous fruit trees, and gibberellins (GAs) are known to be particularly important. In this study, we observed that GA4 treatment led to earlier bud break in Japanese apricot. To understand better the promoting effect of GA4 on the dormancy release of Japanese apricot flower buds, proteomic and transcriptomic approaches were used to analyse the mechanisms of dormancy release following GA4 treatment, based on two-dimensional gel electrophoresis (2-DE) and digital gene expression (DGE) profiling, respectively. More than 600 highly reproducible protein spots (P<0.05) were detected and, following GA4 treatment, 38 protein spots showed more than a 2-fold difference in expression, and 32 protein spots were confidently identified according to the databases. Compared with water treatment, many proteins that were associated with energy metabolism and oxidation–reduction showed significant changes after GA4 treatment, which might promote dormancy release. We observed that genes at the mRNA level associated with energy metabolism and oxidation–reduction also played an important role in this process. Analysis of the functions of the identified proteins and genes and the related metabolic pathways would provide a comprehensive proteomic and transcriptomic view of the coordination of dormancy release after GA4 treatment in Japanese apricot flower buds.

Differential gene expression profiling of Ogura CMS line and its maintainer in radish (Raphanus sativus L.)

Cytoplasmic male sterility (CMS) is a maternally inherited trait, which becomes an important way in heterosis breeding to simplify the seed production procedures and reduce the production cost. Cytological observation of pollen development stages showed a clear difference between the newly developed CMS line and its maintainer; the profiling comparisons between floral buds of CMS line and its maintainer were conducted using differential display reverse transcription PCR (DDRT-PCR) technology. Thirty genes were up-regulated and sixty genes were down-regulated in newly developed CMS line when compared with its maintainer. These genes were involved in cell wall biosynthesis and regulation, transporter and ion channel, flower development and protein metabolism, etc. Expression patterns of six genes encoding RsCAM6, RsGPI, RsPMEI, RsRac, RsCHS, and RsSTP9 were verified by RT-PCR and qRT-PCR in different development stage of floral buds and different organs of CMS line and its maintainer. The expression level of RsRac was higher in stamens and microspores of CMS line than in maintainer. In comparison, the expression levels of the other five genes in CMS line were lower compared with maintainer. This expression profile suggests that these genes played important roles in the development of the pollen and may be closely related to male sterility. The results observed in this study will contribute to understanding the mechanism of pollen abortion during CMS in radish.

Molecular characterization of the Rs-Rf 1 gene and molecular marker-assisted development of elite radish (Raphanus sativus L.) CMS lines with a functional marker for fertility restoration

Cytoplasmic male sterility (CMS) is a maternally inherited trait that is widely utilized in hybrid seed production. In this study, the Rs-Rf1 gene and its allele, the Rs-rf1 gene, were isolated from a radish CMS restorer line and maintainer, respectively. The Rs-Rf1 gene was identified as a member of the pentatricopeptide repeat family. The results of reverse transcription (RT)-PCR and quantitative RT-PCR analyses of the floral buds at different development stages were used to characterize the expression features of Rs-Rf1 and orf138, a unique transcribed gene in the mitochondrion for its role in determining male sterility in the cytoplasm. orf138 was up-regulated in the CMS line and F1 hybrid at three bud developmental stages, including meiosis and the tetroid and microspore stages, but it was not expressed in the maintainer and restorer line, suggesting that the Rs-Rf1 gene was not able to block orf138 transcription. As in the F1 plant, the transcription of Rs-Rf1 was gradually up-regulated in the restorer line with progressive stages of bud development, but it was not transcribed in the CMS line and maintainer. Different restriction sites for SspI between the allele of Rs-Rf1 and Rs-rf1 were also identified, and a guideline for using the functional marker to distinguish the genotypes of individuals with Rs-Rf1 was developed. Due to its co-dominant character, this marker could be used to discriminate the heterozygous and homozygous fertility-restoring allele. Using this functional marker, we established a system of molecular marker-assisted development of elite CMS lines in radish. This system will contribute greatly to candidate maintainer identification and accelerate the process of elite CMS line development in late-bolting radish breeding programs.