Huijun Guo

RNAseq analysis reveals pathways and candidate genes associated with salinity tolerance in a spaceflight-induced wheat mutant

Salinity stress has become an increasing threat to food security worldwide and elucidation of the mechanism for salinity tolerance is of great significance. Induced mutation, especially spaceflight mutagenesis, is one important method for crop breeding. In this study, we show that a spaceflight-induced wheat mutant, named salinity tolerance 1 (st1), is a salinity-tolerant line. We report the characteristics of transcriptomic sequence variation induced by spaceflight, and show that mutations in genes associated with sodium ion transport may directly contribute to salinity tolerance in st1. Furthermore, GO and KEGG enrichment analysis of differentially expressed genes (DEGs) between salinity-treated st1 and wild type suggested that the homeostasis of oxidation-reduction process is important for salt tolerance in st1. Through KEGG pathway analysis, “Butanoate metabolism” was identified as a new pathway for salinity responses. Additionally, key genes for salinity tolerance, such as genes encoding arginine decarboxylase, polyamine oxidase, hormones-related, were not only salt-induced in st1 but also showed higher expression in salt-treated st1 compared with salt-treated WT, indicating that these genes may play important roles in salinity tolerance in st1. This study presents valuable genetic resources for studies on transcriptome variation caused by induced mutation and the identification of salt tolerance genes in crops.

Cloning and characterization of Ku70 and Ku80 homologues involved in DNA repair process in wheat ( Triticum aestivum L.)

Error-prone repair of radiation-induced DNA double-strand breaks (DSBs) results in DNA mutation that is essential for mutation breeding. Non-homologous end joining might be the principal DSB repair mechanism in eukaryotes, which is mediated and activated by Ku protein, a heterodimer of 70 and 80 kDa subunits. In this study, on the basis of complementary DNA (cDNA), the genomic sequences of TaKu70 and TaKu80 genes in all the three genomes of wheat were characterized. Only single-nucleotide substitutions and no insertions or deletions were detected in the exons of TaKu70 and TaKu80 genes. The size of the introns exhibited a slight variation between the sequences. Yeast two-hybrid analysis demonstrated that TaKu70 and TaKu80 formed a heterodimer, and electrophoretic mobility shift assays revealed that this heterodimer bound to double-stranded DNA, but not to single-stranded DNA. The quantitative polymerase chain reaction analysis revealed that the expression of TaKu70 and TaKu80 genes was up-regulated under g-ray irradiation in a dose-dependent manner in the seedlings of wheat. These results suggest that TaKu70 and TaKu80 form a functional heterodimer and are associated with the repair of the induced DSBs in wheat.

Novel mutant alleles of the starch synthesis gene TaSSIVb - D result in the reduction of starch granule number per chloroplast in wheat

BackgroundTransient starch provides carbon and energy for plant growth, and its synthesis is regulated by the joint action of a series of enzymes. Starch synthesis IV (SSIV) is one of the important starch synthase isoforms, but its impact on wheat starch synthesis has not yet been reported due to the lack of mutant lines.ResultsUsing the TILLING approach, we identified 54 mutations in the wheat gene TaSSIVb-D, with a mutation density of 1/165 Kb. Among these, three missense mutations and one nonsense mutation were predicted to have severe impacts on protein function. In the mutants, TaSSIVb-D was significantly down-regulated without compensatory increases in the homoeologous genes TaSSIVb-A and TaSSIVb-B. Altered expression of TaSSIVb-D affected granule number per chloroplast; compared with wild type, the number of chloroplasts containing 0–2 granules was significantly increased, while the number containing 3–4 granules was decreased. Photosynthesis was affected accordingly; the maximum quantum yield and yield of PSII were significantly reduced in the nonsense mutant at the heading stage.ConclusionsThese results indicate that TaSSIVb-D plays an important role in the formation of transient starch granules in wheat, which in turn impact the efficiency of photosynthesis. The mutagenized population created in this study allows the efficient identification of novel alleles of target genes and could be used as a resource for wheat functional genomics.

Transcriptome and proteomic analyses reveal multiple differences associated with chloroplast development in the spaceflight-induced wheat albino mutant mta

Chloroplast development is an integral part of plant survival and growth, and occurs in parallel with chlorophyll biosynthesis. However, little is known about the mechanisms underlying chloroplast development in hexaploid wheat. Here, we obtained a spaceflight-induced wheat albino mutant mta. Chloroplast ultra-structural observation showed that chloroplasts of mta exhibit abnormal morphology and distribution compared to wild type. Photosynthetic pigments content was also significantly decreased in mta. Transcriptome and chloroplast proteome profiling of mta and wild type were done to identify differentially expressed genes (DEGs) and proteins (DEPs), respectively. In total 4,588 DEGs including 1,980 up- and 2,608 down-regulated, and 48 chloroplast DEPs including 15 up- and 33 down-regulated were identified in mta. Classification of DEGs revealed that most were involved in chloroplast development, chlorophyll biosynthesis, or photosynthesis. Besides, transcription factors such as PIF3, GLK and MYB which might participate in those pathways were also identified. The correlation analysis between DEGs and DEPs revealed that the transcript-to-protein in abundance was functioned into photosynthesis and chloroplast relevant groups. Real time qPCR analysis validated that the expression level of genes encoding photosynthetic proteins was significantly decreased in mta. Together, our results suggest that the molecular mechanism for albino leaf color formation in mta is a thoroughly regulated and complicated process. The combined analysis of transcriptome and proteome afford comprehensive information for further research on chloroplast development mechanism in wheat. And spaceflight provides a potential means for mutagenesis in crop breeding.

Gibberellins regulate the stem elongation rate without affecting the mature plant height of a quick development mutant of winter wheat (Triticum aestivum L.).

Gibberellin (GA) is essential for determining plant height. Alteration of GA content or GA signaling results in a dwarf or slender phenotype. Here, we characterized a novel wheat mutant, quick development (qd), in which GA regulates stem elongation but does not affect mature plant height. qd and wild-type plants did not exhibit phenotypic differences at the seedling stage. From jointing to heading stage, qd plants were taller than wild-type plants due to elongated cells. However, wild-type and qd plants were the same height at heading. Unlike wild-type plants, qd plants were sensitive to exogenous GA due to mutation of Rht-B1. With continuous GA stimulation, qd seedlings and adult plants were taller than wild-type. Thus, the GA content of qd plants might differ from that of wild-type during the growth process. Analysis of GA biosynthetic gene expression verified this hypothesis and showed that TaKAO, which is involved in catalyzing the early steps of GA biosynthesis, was differentially expressed in qd plants compared with wild-type. The bioactive GA associated gene TaGA20ox was downregulated in qd plants during the late growth stages. Measurements of endogenous GA content were consistent with the gene-expression analysis results. Consistent with the GA content variation, the first three basal internodes were longer and the last two internodes were shorter in qd than in wild-type plants. The qd mutant might be useful in dissecting the mechanism by which GA regulates stem-growing process, and it may be serve as a GA responsive semi-dwarf germplasm in breeding programs.

Generation of peanut mutants by fast neutron irradiation combined with in vitro culture

Induced mutations have played an important role in the development of new plant varieties. In this study, we investigated the effects of fast neutron irradiation on somatic embryogenesis combined with plant regeneration in embryonic leaflet culture to develop new peanut (Arachis hypogaea L.) germplasm for breeding. The dry seeds of the elite cultivar Luhua 11 were irradiated with fast neutrons at dosages of 9.7, 14.0 and 18.0 Gy. The embryonic leaflets were separated and incubated in a medium with 10.0-mg/l 2,4-D to induce somatic embryogenesis. Next, they were incubated in a medium with 4.0-mg/l BAP for plant regeneration. As the irradiation dosage increased, the frequency of both somatic embryo formation and plantlet regeneration decreased. The regenerated plantlets were grafted onto rootstocks and were transplanted into the field. Later, the mature seeds of the regenerated plants were harvested. The M2 generation plants from most of the regenerated cultivars exhibited variations and segregation in vigor, plant height, branch and pod number, pod size, and pod shape. To determine whether the phenotypes were associated with genomic modification, we compared the DNA polymorphisms between the wild-type plants and 19 M3-generation individuals from different regenerated plants. We used 20 pairs of simple sequence repeat (SSR) primers and detected polymorphisms between most of the mutants and the wild-type plants (Luhua 11). Our results indicate that using a combination of fast neutron irradiation and tissue culture is an effective approach for creating new peanut germplasm.

Characterization of a Novel Chlorophyll-Deficient Mutant Mt6172 in Wheat

Abstract Identification of new chlorophyll-deficient mutants will provide materials for studying signaling components and pathways between plastid and nucleus. A novel chlorophyll-deficient mutant, named Mt6172, was obtained by spaceflight environment induction. Genetic analysis showed that its inheritance was controlled by nuclear and cytoplamic genes. Leaf color of its self-fertilized progenies was albino, narrow-white striped, or green. Only a few cells with abnormal chloroplasts were observed in albino plants and white section of narrow-white striped plants. These chloroplasts had obvious flaws in inner structure, and granum lamellae was extremely disordered. The narrow-white striped plants were characterized with green-and-narrow-white striped leaves, and the width of stripes between different plants was even, their plant height, number of productive tillers, and 1000-grain weight were lower than those of the wild type. The narrow-white striped plants and the wild type had significant difference in the value of potential activity of photosystem II at all tested stages. At elongation stage, which was impacted the most seriously, effective quantum yield significantly decreased, whereas the energy for photoprotection and photodamage significantly increased. Under different photosynthetic active radiation conditions, changes of electron transport rate, photochemical dissipation, and effective quantum yield were different, electron transport rate was more impacted than other parameters. Therefore, the leaf morphology and inheritance of mutant Mt6172 was different from the other reported mutants in wheat, and it was a novel mutant of chlorophyll deficiency.

Agronomic Traits and Photosynthetic Characteristics of Chlorophyll-Deficient Wheat Mutant Induced by Spaceflight Environment

Abstract Chlorophyll deficient mutants are ideal materials to study photosynthetic mechanism, pathways of chlorophyll biosynthesis and degradation, and the genes related to photosynthesis. A chlorophyll-deficient mutant of wheat (Triticum aestivum L.), Mt135, was obtained by space mutagenesis. The main agronomic traits and photosynthetic characteristics of this mutant were compared with its wild type in field experiment. The leaf color of Mt135 showed albino, striped, and green phenotypes. The albino plants with entire albino leaves died at seedling stage. The plants with green-and-white striped leaves usually matured 5–7 d later than the wild type, and had lower plant height, shorter spike length, fewer grain number, and smaller grain weight per plant and 1000-grain weight. In contrast, the green plants of the mutant had similar agronomic traits to the wild type. Preliminary genetic analysis showed that the inheritance of leaf color in Mt135 was controlled by nucleocytoplasmic interaction. When exposed to photosynthetic active radiation (PAR) at 110 μmol m−2 s−1, the green tissue of striped plant (S-G) had a similar value of maximum photosystem II quantum yield to the wide plant, but a significantly lower potential activity of photosystem II; the photochemical quenching, nonphotochemical quenching, effective quantum yield, regulated nonphotochemical energy dissipation, and nonregulated energy dissipation varied differently at seeding, elongation, and heading stages. In addition, for electron transport rate, photochemical quenching, and effective quantum yield, the differences between S-G plant and the wild plant varied under different PAR conditions. Nevertheless, the other 2 types of the mutant, white tissue of striped plant and albino plant, showed complete losses of photosynthetic function. As a result, the photosynthesis of Mt135 was greatly restricted. High photosynthetic active radiation had tremendous impact on the mutant at elongation stage and relatively weak impact at heading stage. The changes of photosynthetic function of striped plants are in accordance with the reduction of their plant height, spike length, and yield related traits.

Development of a High-Efficient Mutation Resource with Phenotypic Variation in Hexaploid Winter Wheat and Identification of Novel Alleles in the TaAGP.L-B1 Gene

Mutated genetic resources play an important role in gene/allele characterization. Currently, there are few hexaploid winter wheat mutated resources available. Here, we developed a hexaploid winter wheat resource by inducing mutations via EMS treatment by the single seed descent method. A broad mutation spectrum with high mutation frequency (∼19%) on phenotypic variations was identified. These mutations included spike, leaf and seed morphology, plant architecture, and heading date variations. To evaluate the efficiency of the resource for reverse genetic analysis, allelic variations in the TaAGP.L-B1 gene, encoding the AGPase large subunit, were screened by the TILLING approach. Four missense mutations were identified and one allele in line E3-1-3, resulted in an amino acid change predicated to have severe effects on gene function. The other three mutations were predicted to have no effect. Results of gene expression patterns and grain starch content demonstrated that the novel allele in E3-1-3 altered the function of TaAGP.L-B1. Our results indicated that this mutated genetic wheat resource contained broad spectrum phenotypic and genotypic variations, that may be useful for wheat improvement, gene discovery, and functional genomics.

Development of a new wheat germplasm with high anther culture ability by using a combination of gamma-ray irradiation and anther culture

BACKGROUND Wheat with high anther culture ability would be beneficial for breeding. We aimed to screen a wheat germplasm to with high anther culture ability as well as good agronomic characteristics. RESULTS The F1 young spikes of winter wheat cross combination Yanfu188/Jimai37 were irradiated with gamma rays at a dose of 1.5 Gy to develop a new germplasm H307 with high anther culture ability. The proportion of green plantlets per 100 anthers (GP/100A) of H307 was 14.50% which was higher than other H2 lines (P < 0.05). Analysis over three successive years (2006-2008) revealed that the green plantlet regeneration ability of H307 remained high in all 3 years. Reciprocal crosses between H307 and Nongda3308 showed no significant differences in their values for calli per 100 anthers (CA/100A), green plantlets per 100 calli (GP/100C) and GP/100A (P > 0.05). Five main wheat varieties used in production, namely Yumai68, Yanzhan4110, Bainongaikang58, Zhoumai18 and Xinmai18, were selected to cross with the new H307. CA/100A, GP/100C and GP/100A were used to assess the anther culture ability of F1 hybrids, demonstrating that the anther culture ability of H307 was heritable. CONCLUSION H307 possessed high anther culture ability that was heritable, which would be potential germplasm for improving wheat anther breeding ability.