Response of Wheat DREB Transcription Factor to Osmotic Stress Based on DNA Methylation.

Dehydration-responsive element-binding protein (DREB) plays an important role in response to osmotic stress. In this study, DREB2, DREB6 and Wdreb2 are isolated from wheat AK58, yet they belong to different types of DREB transcription factors. Under osmotic stress, the transcript expression of DREB2, DREB6 and Wdreb2 has tissue specificity and is generally higher in leaves, but their expression trends are different along with the increase of osmotic stress. Furthermore, some elements related to stresses are found in their promoters, promoters of DREB2 and Wdreb2 are slightly methylated, but DREB6's promoter is moderately methylated. Compared with the control, the level of promoter methylation in Wdreb2 is significantly lower under osmotic stress and is also lower at CG site in DREB2, yet is significantly higher at CHG and CHH sites in DREB2, which is also found at a CHG site in DREB6. The status of promoter methylation in DREB2, DREB6 and Wdreb2 also undergoes significant changes under osmotic stress; further analysis showed that promoter methylation of Wdreb2 is negatively correlated with their expression. Therefore, the results of this research suggest the different functions of DREB2, DREB6 and Wdreb2 in response to osmotic stress and demonstrate the effects of promoter methylation on the expression regulation of Wdreb2.

Isolation of differentially expressed sex genes in garden asparagus using suppression subtractive hybridization.

Garden asparagus (Asparagus officinalis L.) is a dioecious species whose male and female flowers are found in separate unisexual individuals. A region called the M-locus, located on a pair of homomorphic sex chromosomes, controls sexual dimorphism in asparagus. To date, no sex determining gene has been isolated from asparagus. To identify more genes involved in flower development in asparagus, subtractive hybridization library of male flowers in asparagus was constructed by suppression subtraction hybridization. A total of 107 expressed sequence tags (ESTs) were identified. BLASTX analysis showed that the library contained several genes that could be related to flower development. The expression patterns of seven selected genes believed to be involved in the development of asparagus male flower were further analyzed by semi-quantitative or real-time reverse-transcription polymerase chain reaction (RT-PCR). Results showed that AOEST4-5, AOEST12-40, and AOEST13-38 were strongly expressed in the male flower stage, whereas no transcript level of AOEST13-38 was detected in the female flower stage. The expression levels of AOEST13-87, AOEST13-92, AOEST13-40, and AOEST18-87 in the male flower stage were also higher than those in the female flower stage, although these transcripts were also expressed in other tissues. The identified genes can provide a strong starting point for further studies on the underlying molecular differences between the male and female flowers of asparagus.

[Role of transposons in origin and evolution of plant XY sex chromosomes].

The XY sex-determination system is crucial for plant reproduction. However, little is known about the mechanism of the origin and evolution of the XY sex chromosomes. It has been believed that a pair of autosomes is evolved to produce young sex chromosomes (neo-X chromosome and neo-Y chromosome) by loss of function or gain of function mutation, which influences the development of pistil or stamen. With the aggravation of the recombination suppression between neo-X and neo-Y and consequent expanding of the non-recombination region, the proto-sex chromosomes were finally developed to heteromorphic sex chromosomes. Accumulation of repetitive sequences and DNA methylation were probably involved in this process. Transposons, as the most abundant repetitive sequences in the genome, might be the initial motivation factors for the evolution of sex chromosome. Moreover, transposons may also increase heterochromatin expansion and recombination suppression of sex chromosome by local epigenetics modification. In this review, we summarize the function of transposon accumulation and the relationship between transposon and heterochromatization in the evolution of plant sex chromosome.

Detection of genome DNA methylation change in spinach induced by 5-azaC.

DNA methylation has been implicated in the regulation of gene expression, genome imprinting, and chromatin remodeling in eukaryotes. In this study, we analyzed possible alterations in levels and patterns of cytosine methylation in male and female spinach plants after treatment with demethylation agent 5-azacytidine (5-azaC) using two methods: (1) direct determination of 5-methylcytidine (5 mC) amounts in genomic DNA by high-performance liquid chromatography (HPLC) separation and quantification of nucleosides and (2) methylation-sensitive inter-simple sequence repeat (MS-ISSR) technique. HPLC analysis revealed that the DNA methylation events in male and female spinach leaves markedly decreased upon 30 µM 5-azaC treatment, and the methylation level gradually decreased with the increase in 5-azaC concentration. To study the altered DNA methylation patterns in spinach after 5-azaC treatment, untreated and 500 µM 5-azaC-treated samples were analyzed by MS-ISSR assay. A total of 385 informative profiles were resolved using 35 ISSR primer sets. MS-ISSR analysis showed various altered methylation patterns between untreated and 5-azaC-treated spinach plants. These alterations were mainly demethylation events, which were largely consistent with the HPLC results. Both HPLC and MS-ISSR analyses showed that the changes in DNA methylation levels and patterns were similar in male and female spinach leaves, which implies that sex was not the main factor influencing DNA methylation levels and patterns in the vegetative organs of spinach. This study could provide a molecular basis of the altered DNA methylation induced by 5-azaC, and lay a foundation for further investigation of the relationship between methylation and sex determination and development in this dioecious plant spinach.

Comparative analysis of gene expression by microarray analysis of male and female flowers of Asparagus officinalis.

To identify rapidly a number of genes probably involved in sex determination and differentiation of the dioecious plant Asparagus officinalis, gene expression profiles in early flower development for male and female plants were investigated by microarray assay with 8,665 probes. In total, 638 male-biased and 543 female-biased genes were identified. These genes with biased-expression for male and female were involved in a variety of processes associated with molecular functions, cellular components, and biological processes, suggesting that a complex mechanism underlies the sex development of asparagus. Among the differentially expressed genes involved in the reproductive process, a number of genes associated with floral development were identified. Reverse transcription-PCR was performed for validation, and the results were largely consistent with those obtained by microarray analysis. The findings of this study might contribute to understanding of the molecular mechanisms of sex determination and differentiation in dioecious asparagus and provide a foundation for further studies of this plant.

Microdissection and painting of the Y chromosome in spinach (Spinacia oleracea).

Spinach has long been used as a model for genetic and physiological studies of sex determination and expression. Although trisomic analysis from a cross between diploid and triploid plants identified the XY chromosome as the largest chromosome, no direct evidence has been provided to support this at the molecular level. In this study, the largest chromosomes of spinach from mitotic metaphase spreads were microdissected using glass needles. Degenerate oligonucleotide primed polymerase chain reaction was used to amplify the dissected chromosomes. The amplified products from the Y chromosome were identified using the male-specific marker T11A. For the first time, the largest spinach chromosome was confirmed to be a sex chromosome at the molecular level. PCR products from the isolated chromosomes were used in an in situ probe mixture for painting the Y chromosome. The fluorescence signals were mainly distributed on all chromosomes and four pair of weaker punctate fluorescence signal sites were observed on the terminal region of two pair of autosomes. These findings provide a foundation for the study of sex chromosome evolution in spinach.

[Preliminary study on molecular mechanism of lotus mutants induced by ion implantation].

Ion implantation, as a new biophysically mutagenic technique, has shown a great potential for horticultural plant breeding. Up to date, little is known about the mutation mechanism of ion implantation at the DNA level. To reveal the mutation effect of Fe+ ion implantation on Baiyangdian red lotus, the random amplified polymorphic DNA (RAPD) was used, and then the bands of mutants and the control in the radiation-sensitive sites were cloned to be sequenced for comparing their DNA sequences. The results indicated that the total base mutation rate of mutants was 0.87%, and there was different in the six mutants. The types of base changes included base transition, transversion, deletion, and insertion. Among the 159 base changes detected, the frequency of single base substitutions (61.01%) was higher than that of base deletions and insertions (38.99%), and the frequency of base transitions (44.65%) was 2.7 times of that of the base transversions (16.35%). The transitions between C and T accounted for largest proportion, A→G transitions and A→T transversions were also present at high frequency. Adenine, thymine, guanine or cytosine could be replaced by any of other three bases, except that there was no C → G substitution. However, thymine was more sensitive to the irradiation than other bases. In our study, we found many purine bases around the purine mutational sites, and many pyrimidine bases around the pyrimidine mutational sites. These will further help us to understand the mechanism of mutagenesis by ion implantation.

[Role of repetitive sequence and heterochromatize in recombination suppression of plant sex chromosomes].

Suppression of recombination is the prerequisite for plant sex chromosome evolution from a pair of autosomes. Recombination suppression around the locus controlling sex determination results in sex chromosome degeneration and differentiation. Important events such as repetitive sequence accumulation, heterochromatize, and DNA methylation have relation to recombination suppression. Accumulation of repetitive DNA sequence, including transposable elements and satellite DNA, leads to primitive sex chromosome differentiated on morphological and molecular structure, and also gives rise to chromosome heterochromatize, and thus recombination between sex chromosomes was suppressed. Here, we re-viewed the advances in this field, meanwhile, the function of DNA methylation in recombination suppression was analyzed.

[Molecular identification on Waxy genes in wheat using multiple-PCR].

Multiple-PCR was conducted to establish a stable PCR system for identifying the three Wx genes in wheat. Two pairs of primers were employed to amplify Wx-A1, Wx-B1, and Wx-D1 genes of wheat, with the target sequences of 230 bp/265 bp, 854 bp, and 204 bp, respectively. The results showed that Wx-A1, Wx-B1, and Wx-D1 can be detected simultaneously in a single reaction. This method proved to be repeatable and low cost for evaluation of wheat quality properties in breeding program. This multiple-PCR technique can be efficiently used in marker-assisted selection for Wx genes, which will improve selection procedure for waxy wheat.

Rapid cloning and bioinformatic analysis of spinach Y chromosome-specific EST sequences.

The genome of spinach single chromosome complement is about 1000 Mbp, which is the model material to study the molecular mechanisms of plant sex differentiation. The cytological study showed that the biggest spinach chromosome (chromosome 1) was taken as spinach sex chromosome. It had three alleles of sex-related X,X(m) and Y. Many researchers have been trying to clone the sex-determining genes and investigated the molecular mechanism of spinach sex differentiation. However,there are no successful cloned reports about these genes. A new technology combining chromosome microdissection with hybridization-specific amplification (HSA) was adopted. The spinach Y chromosome degenerate oligonucleotide primed-PCR (DOP-PCR) products were hybridized with cDNA of the male spinach flowers in florescence. The female spinach genome was taken as blocker and cDNA library specifically expressed in Y chromosome was constructed. Moreover, expressed sequence tag (EST) sequences in cDNA library were cloned, sequenced and bioinformatics was analysed. There were 63 valid EST sequences obtained in this study. The fragment size was between 53 and 486 bp. BLASTn homologous alignment indicated that 12 EST sequences had homologous sequences of nucleic acids, the rest were new sequences. BLASTx homologous alignment indicated that 16 EST sequences had homologous protein-encoding nucleic acid sequence. The spinach Y chromosome-specific EST sequences laid the foundation for cloning the functional genes, specifically expressed in spinach Y chromosome. Meanwhile, the establishment of the technology system in the research provided a reference for rapid cloning of other biological sex chromosome-specific EST sequences.

Boron influences pollen germination and pollen tube growth in Picea meyeri.

To study the role of boron in pollen germination and pollen tube growth of Picea meyeri Rehd. et Wils., pollen grains were cultured in standard medium or boron-deficient medium. Effects of boron on the localization of pectins and callose in the walls of pollen tubes were observed by laser scanning confocal microscopy after staining with aniline blue or immunolabeling with antibodies JIM5 and JIM7. Changes in the structures of pectins and phenolics were investigated by fourier transform infrared (FTIR) microspectroscopy. Pollen germination in boron-deficient medium ranged from 18 to 24%, whereas pollen germination in standard medium reached 61%. Callose accumulated in the tip-regions of pollen tubes cultured in boron-deficient medium, but not in standard medium. Immunolabeling with antibody JIM5 revealed that acidic pectin preferentially accumulated in the tip regions of pollen tubes cultured in boron-deficient medium, whereas acidic pectin was weakly distributed along the entire lengths of pollen tubes cultured in standard medium. Esterified pectin, detected by immunolabeling with antibody JIM7, showed a similar distribution pattern in pollen tubes in both the boron-deficient and standard treatments. The FTIR spectra indicated slight increases in contents of phenolics and carboxylic acids and a substantial decrease in the content of saturated esters in boron-deficient pollen tubes compared with normal pollen tubes. The FTIR spectra confirmed that boron deficiency enhanced acidic pectin accumulation in pollen tubes, which may be associated with the increased content of carboxylic acid. We conclude that boron has a regulatory role in pollen germination and pollen tube growth.

Analysis of transposable elements in the genome of Asparagus officinalis from high coverage sequence data.

Asparagus officinalis is an economically and nutritionally important vegetable crop that is widely cultivated and is used as a model dioecious species to study plant sex determination and sex chromosome evolution. To improve our understanding of its genome composition, especially with respect to transposable elements (TEs), which make up the majority of the genome, we performed Illumina HiSeq2000 sequencing of both male and female asparagus genomes followed by bioinformatics analysis. We generated 17 Gb of sequence (12×coverage) and assembled them into 163,406 scaffolds with a total cumulated length of 400 Mbp, which represent about 30% of asparagus genome. Overall, TEs masked about 53% of the A. officinalis assembly. Majority of the identified TEs belonged to LTR retrotransposons, which constitute about 28% of genomic DNA, with Ty1/copia elements being more diverse and accumulated to higher copy numbers than Ty3/gypsy. Compared with LTR retrotransposons, non-LTR retrotransposons and DNA transposons were relatively rare. In addition, comparison of the abundance of the TE groups between male and female genomes showed that the overall TE composition was highly similar, with only slight differences in the abundance of several TE groups, which is consistent with the relatively recent origin of asparagus sex chromosomes. This study greatly improves our knowledge of the repetitive sequence construction of asparagus, which facilitates the identification of TEs responsible for the early evolution of plant sex chromosomes and is helpful for further studies on this dioecious plant.