Gaisheng Zhang

De Novo Assembly and Transcriptome Analysis of Wheat with Male Sterility Induced by the Chemical Hybridizing Agent SQ-1

Wheat (Triticum aestivum L.), one of the world’s most important food crops, is a strictly autogamous (self-pollinating) species with exclusively perfect flowers. Male sterility induced by chemical hybridizing agents has increasingly attracted attention as a tool for hybrid seed production in wheat; however, the molecular mechanisms of male sterility induced by the agent SQ-1 remain poorly understood due to limited whole transcriptome data. Therefore, a comparative analysis of wheat anther transcriptomes for male fertile wheat and SQ-1–induced male sterile wheat was carried out using next-generation sequencing technology. In all, 42,634,123 sequence reads were generated and were assembled into 82,356 high-quality unigenes with an average length of 724 bp. Of these, 1,088 unigenes were significantly differentially expressed in the fertile and sterile wheat anthers, including 643 up-regulated unigenes and 445 down-regulated unigenes. The differentially expressed unigenes with functional annotations were mapped onto 60 pathways using the Kyoto Encyclopedia of Genes and Genomes database. They were mainly involved in coding for the components of ribosomes, photosynthesis, respiration, purine and pyrimidine metabolism, amino acid metabolism, glutathione metabolism, RNA transport and signal transduction, reactive oxygen species metabolism, mRNA surveillance pathways, protein processing in the endoplasmic reticulum, protein export, and ubiquitin-mediated proteolysis. This study is the first to provide a systematic overview comparing wheat anther transcriptomes of male fertile wheat with those of SQ-1–induced male sterile wheat and is a valuable source of data for future research in SQ-1–induced wheat male sterility.

Abnormal Development of Tapetum and Microspores Induced by Chemical Hybridization Agent SQ-1 in Wheat

Chemical hybridization agent (CHA)-induced male sterility is an important tool in crop heterosis. To demonstrate that CHA-SQ-1-induced male sterility is associated with abnormal tapetal and microspore development, the cytology of CHA-SQ-1-treated plant anthers at various developmental stages was studied by light microscopy, scanning and transmission electron microscopy, in situ terminal deoxynucleotidyl transferasemediated dUTP nick end-labelling (TUNEL) assay and DAPI staining. The results indicated that the SQ-1-treated plants underwent premature tapetal programmed cell death (PCD), which was initiated at the early-uninucleate stage of microspore development and continued until the tapetal cells were completely degraded; the process of microspore development was then blocked. Microspores with low-viability (fluorescein diacetate staining) were aborted. The study suggests that premature tapetal PCD is the main cause of pollen abortion. Furthermore, it determines the starting period and a key factor in CHA-SQ-1-induced male sterility at the cell level, and provides cytological evidence to further study the mechanism between PCD and male sterility.

The Over-Expression of an Arabidopsis B3 Transcription Factor, ABS2/NGAL1, Leads to the Loss of Flower Petals

Transcriptional regulations are involved in many aspects of plant development and are mainly achieved through the actions of transcription factors (TF). To investigate the mechanisms of plant development, we carried out genetic screens for mutants with abnormal shoot development. Taking an activation tagging approach, we isolated a gain-of-function mutant abs2-1D (abnormal shoot 2-1D). abs2-1D showed pleiotropic growth defects at both the vegetative and reproductive developmental stages. We cloned ABS2 and it encodes a RAV sub-family of plant B3 type of transcriptional factors. Phylogenetic analysis showed that ABS2 was closely related to NGATHA (NGA) genes that are involved in flower development and was previously named NGATHA-Like 1 (NGAL1). NGAL1 was expressed mainly in the root and the filament of the stamen in flower tissues and sub-cellular localization assay revealed that NGAL1 accumulated in the nucleus. Interestingly, over-expression of NGAL1 driven by the constitutive 35S promoter led to transgenic plants with conspicuous flower defects, particularly a loss-of-petal phenotype. A loss-of-function ngal1-1 mutant did not show obvious phenotype, suggesting the existence of redundant activities and also the utility of gain-of-function genetic screens. Our results show that the over-expression of NGAL1 is capable of altering flower petal development, as well as shoot development.

Programmed cell death, antioxidant response and oxidative stress in wheat flag leaves induced by chemical hybridization agent SQ-1

Abstract Male sterility induced by a chemical hybridization agent (CHA) is an important tool for utilizing crop heterosis. Leaves, especially the flag leaves, as CHA initial recipients play a decisive role in inducing male sterility. To investigate effects of different treatment times of CHA-SQ-1 used, morphological, biochemical and physiological responses of wheat flag leaves were detected in this study. CHA induced programmed cell death (PCD) as shown in terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) and DNA laddering analysis. In the early phase, CHA-SQ-1 trig-gered organelle changes and PCD in wheat leaves accompanied by excess production of reactive oxygen species (O2 and H2O2) and down-regulation of the activities of superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (POD). Meanwhile, leaf cell DNAs showed ladder-like patterns on agarose gel, indicating that CHA-SQ-1 led to the activation of the responsible endonuclease. The oxidative stress assays showed that lipid peroxidation was strongly activated and photosynthesis was obviously inhibited in SQ-1-induced leaves. However, CHA contents in wheat leaves gradually reduced along with the time CHA-SQ-1 applied. Young flags returned to an oxidative/antioxidative balance and ultimately developed into mature green leaves. These results provide explanation of the relations between PCD and anther abortion and practical application of CHA for hybrid breeding.

Testing the fecundity advantage hypothesis with Sitobion avenae, Rhopalosiphum padi, and Schizaphis graminum (Hemiptera: Aphididae) feeding on ten wheat accessions

The fecundity advantage hypothesis suggests that females with a large body size produce more offspring than smaller females. We tested this hypothesis by exploring the correlations between life-history traits of three aphid species feeding on ten wheat accessions at three levels of analysis with respect to the host plant: overall, inter-accession, and intra-accession. We found that fecundity was significantly correlated with mean relative growth rate (MRGR), weight gain, and development time, and that the faster aphid develops the greater body and fecundity, depending on aphid species, wheat accession, and analyses level. Larger aphids of all three species produced more offspring overall; this held true for Sitobion avenae and Schizaphis graminum at the inter-accession level, and for S. avenae, Rhopalosiphum padi, and S. graminum for three, five, and eight accessions respectively at the intra-accession level. Only one correlation, between intrinsic rates of natural increase (rm) and MRGR, was significant for all aphid species at all three analysis levels. A more accurate statement of the fecundity advantage hypothesis is that cereal aphids with greater MRGR generally maintain higher rm on wheat. Our results also provide a method for exploring relationships between individual life-history traits and population dynamics for insects on host plants.

Resistance of Wheat Accessions to the English Grain Aphid Sitobion avenae

The English grain aphid, Sitobion avenae, is a major pest species of wheat crops; however, certain varieties may have stronger resistance to infestation than others. Here, we investigated 3 classical resistance mechanisms (antixenosis, antibiosis, and tolerance) by 14 wheat varieties/lines to S. avenae under laboratory and field conditions. Under laboratory conditions, alatae given the choice between 2 wheat varieties, strongly discriminated against certain varieties. Specifically, the ‘Amigo’ variety had the lowest palatability to S. avenae alatae of all varieties. ‘Tm’ (Triticum monococcum), ‘Astron,’ ‘Xanthus,’ ‘Ww2730,’ and ‘Batis’ varieties also had lower palatability than other varieties. Thus, these accessions may use antibiosis as the resistant mechanism. In contrast, under field conditions, there were no significant differences in the number of alatae detected on the 14 wheat varieties. One synthetic line (98-10-30, a cross between of Triticum aestivum (var. Chris) and Triticum turgidum (var. durum) hybridization) had low aphid numbers but high yield loss, indicating that it has high antibiosis, but poor tolerance. In comparison, ‘Amigo,’ ‘Xiaoyan22,’ and some ‘186Tm’ samples had high aphid numbers but low yield loss rates, indicating they have low antibiosis, but good tolerance. Aphid population size and wheat yield loss rates greatly varied in different fields and years for ‘98-10-35,’ ‘Xiaoyan22,’ ‘Tp,’ ‘Tam200,’ ‘PI high,’ and other ‘186Tm’ samples, which were hybrid offspring of T. aestivum and wheat related species. Thus, these germplasm should be considered for use in future studies. Overall, S. avenae is best adapted to ‘Xinong1376,’ because it was the most palatable variety, with the greatest yield loss rates of all 14 wheat varieties. However, individual varieties/lines influenced aphid populations differently in different years. Therefore, we strongly recommend a combination of laboratory and long-term field experiments in targeted planting regions to identify varieties/lines that consistently show high resistance to S. avenae infestation.

Previous infestation with Psammotettix alienus on spring wheat seedlings decreased the fitness of Sitobion avenae in a subsequent infestation

Previous feeding by herbivores may influence the performance of other herbivores by altering the morphological and physiological properties of the shared host. The present study aimed to examine the fitness of the apterous aphid Sitobion avenae (Fabricius) (Hemiptera: Aphididae) when fed spring wheat that was previously attacked by the leafhopper Psammotettix alienus Dahlbom (Hemiptera: Cicadellidae), with an evaluation of the differences in aphid weight (WD), development time (D), mean relative growth rate (MRGR), daily production of nymphs (MDNP) and intrinsic rate of population increase (rm) under controlled greenhouse conditions. For the S. avenae reared on the two spring wheat cultivars that were previously infested with P. alienus, the WD and the MRGR decreased significantly. The S. avenae reared on the previously infested cv. Leguan had significantly prolonged D values and a lowered rm, whereas these parameters did not differ significantly when S. avenae was reared on the cv. Bobwhite. Overall, the fitness of the aphid S. avenae was adversely affected when reared on the two spring wheat cultivars that were previously infested with P. alienus.

Biological and Morphological Features Associated with English Grain Aphid and Bird Cherry-Oat Aphid Tolerance in Winter Wheat Line XN98-10-35

The English grain aphid, Sitobion avenae Fabricius (Hemiptera: Aphididae), and bird cherry-oat aphid, Rhopalosiphum padi L. (Hemiptera: Aphididae), are two common herbivore pests that have devastating effects on wheat production worldwide. To tolerate herbivores, plants coevolved various biological and morphological features. The present study focused on the characterization of the S. avenae- and R. padi-tolerant features in the winter wheat line XN98-10-35 under field conditions. The yield loss experiment revealed that XN98-10-35 presented strong tolerance to S. avenae and R. padi feeding, as we observed a small 100-kernel weight decrease induced by S. avenae and R. padi. Examination of the ultrastructure of the flag leaves revealed that XN98-10-35 exhibited a greater number of stomata and a larger stomata size than the controls. In comparative gene expression profiling tests, the expression levels of the candidate genes SaEST1 and SaEST2, which are putative photosystem I assembly protein Ycf3 and vegetative cell wall gp1-like protein, were up-regulated several-fold in the flag leaves of XN98-10-35. Additionally, genes associated with sucrose synthase and starch synthase were up-regulated in the flag leaves of XN98-10-35. These findings increase our understanding of the molecular mechanism underlying tolerance to S. avenae and R. padi infestations in XN98-10-35 and will allow rapid breeding of tolerant cultivars.

Changes in DNA methylation are associated with heterogeneous cytoplasm suppression of the multi-ovary gene in wheat (Triticum aestivum)

Abstract. Variety DUOII is a multi-ovary line of common wheat (Triticum aestivum L.) that has two or three pistils and three stamens. The multi-ovary trait is controlled by a dominant gene, the expression of which can be suppressed by the special heterogeneous cytoplasm of line TeZhiI (TZI). TZI has the nucleus of common wheat and the cytoplasm of Aegilops. DUOII (♀) × TZI (♂) shows the multi-ovary trait, whereas TZI (♀) × DUOII (♂) shows the mono-ovary trait. DNA methylation affects gene expression and plays a crucial role in organ and tissue differentiation. In order to study the relationship between DNA methylation and the suppression of the multi-ovary gene, we used methylation-sensitive amplification polymorphisms (MSAP) to assess the DNA methylation status of the reciprocal crosses. Genome-wide, 14 584 CCGG sites were detected and the overall methylation levels were 31.10% and 30.76% in the respective crosses DUOII × TZI and TZI × DUOII. Compared with DUOII × TZI, TZI × DUOII showed 672 sites (4.61%) in which methylation–demethylation processes occurred. The results showed that the special heterogeneous cytoplasm significantly changed DNA methylation, and this might have suppressed the multi-ovary gene. The results provide insight into the changing patterns of DNA methylation in the suppression of the multi-ovary gene, and provide essential background for further studies on the underlying mechanisms of heterogeneous cytoplasm suppression of the expression of the multi-ovary gene in wheat.

Chloroplast structure and DNA methylation polymorphisms in an albino mutant of wheat (Triticum aestivum) cv. Xinong 1376

Abstract. DNA methylation plays an important role in regulating plant development, including organ and tissue differentiation, which may determine variations in agronomic traits. However, no reports exist for the regulation of leaf colour in wheat. The present study investigated the chloroplast structure and epigenetic mechanisms regulating leaf colour in an albino mutant of wheat (Triticum aestivum L.) cv. Xinong 1376. Structural analysis was performed by scanning and transmission electron microscopy, and epigenetic modifications were detected by methylation-sensitive amplification polymorphism (MSAP) analysis. Mesophyll cells of green leaves showed a well-ordered arrangement and they were filled with chloroplasts with intact lamellar structures and thylakoid membranes. By contrast, mesophyll cells of red and white leaves were disorganised and contained only a few plastids or chloroplasts with no lamellar structures or thylakoid membranes. Comparison of MSAP profiles revealed that white or red leaves had higher levels of cytosine methylation and showed changes in polymorphic loci compared with green leaves (4.35% and 4.10%, respectively). We sequenced 150 DNA fragments that were differentially displayed in MSAP patterns of white or red and green leaves of the Xinong 1376 albino mutant. A further BLAST search of 77 cloned sequences located them in coding regions. Most of these sequences were found to be involved in processes such as signal transduction, transcription regulation, post-transcriptional processing, DNA modification and repair, transport, biosynthesis of cellulose, photosynthesis, protein ubiquitination, stress responses, and retroposition. Expression analysis demonstrated a decrease in the transcription of two methylated genes, psaA and psbD, which are involved in the photosystem. Although the DNA methylation changes and leaf colour changes were not directly associated, these results may indicate that methylation of specific genes is an active and rapid epigenetic response to variation of leaf colour in the Xinong 1376 albino mutant, further elucidating the mechanism of variation in leaf colour.