The TaSnRK1-TabHLH489 module integrates brassinosteroid and sugar signalling to regulate the grain length in bread wheat.

Regulation of grain size is a crucial strategy for improving the crop yield and is also a fundamental aspect of developmental biology. However, the underlying molecular mechanisms governing grain development in wheat remain largely unknown. In this study, we identified a wheat atypical basic helix-loop-helix (bHLH) transcription factor, TabHLH489, which is tightly associated with grain length through genome-wide association study and map-based cloning. Knockout of TabHLH489 and its homologous genes resulted in increased grain length and weight, whereas the overexpression led to decreased grain length and weight. TaSnRK1α1, the α-catalytic subunit of plant energy sensor SnRK1, interacted with and phosphorylated TabHLH489 to induce its degradation, thereby promoting wheat grain development. Sugar treatment induced TaSnRK1α1 protein accumulation while reducing TabHLH489 protein levels. Moreover, brassinosteroid (BR) promotes grain development by decreasing TabHLH489 expression through the transcription factor BRASSINAZOLE RESISTANT1 (BZR1). Importantly, natural variations in the promoter region of TabHLH489 affect the TaBZR1 binding ability, thereby influencing TabHLH489 expression. Taken together, our findings reveal that the TaSnRK1α1-TabHLH489 regulatory module integrates BR and sugar signalling to regulate grain length, presenting potential targets for enhancing grain size in wheat.

Hybridization affects the structure and function of root microbiome by altering gene expression in roots of wheat introgression line under saline-alkali stress.

The mutually beneficial relationship between plants and their root microbiota is essential for plants to adapt to unfavorable environments. However, the molecular mechanism of wheat regulating the structure of root microbiome and the influence of distant hybridization on this process are poorly understood. In this study, we systematically compared the root transcriptome and microbiome between a saline-alkali tolerant wheat introgression line SR4 (derived from somatic hybridization between wheat and tall wheatgrass) and its parent wheat variety JN177. The results indicated that root microorganisms were key factor maintaining better homeostasis of the sodium and potassium ion contents in SR4 than in JN177 under saline-alkali stress. Through systematic comparisons, we identified SR4-specific root bacterial and fungal taxa under saline-alkali stress. Through a weighted gene correlation network analysis (WGCNA) combining microbiome and transcriptome data, key functional genes and pathways, which were strongly related to root bacteria and fungi with differential abundance between JN177 and SR4, were identified. These results suggest that somatic hybridization has altered the key genes regulating root microbiome in wheat, further improving the saline-alkali tolerance of wheat introgression line. These findings provide the key bacterial and fungal taxa and functional target genes for wheat root microbiome engineering under saline-alkali stress.

Transcriptome profiling reveals the genetic basis of alkalinity tolerance in wheat.

BACKGROUND: Soil alkalinity shows significant constraints to crop productivity; however, much less attention has been paid to analyze the effect of soil alkalinity on plant growth and development. Shanrong No. 4 (SR4) is an alkalinity tolerant bread wheat cultivar selected from an asymmetric somatic hybridization between the bread wheat cultivar Jinan 177 (JN177) and tall wheatgrass (Thinopyrum ponticum), which is a suitable material for studying alkalinity tolerant associate genes. RESULTS: The growth of SR4 plant seedlings was less inhibited than that of JN177 when exposed to alkalinity stress conditions. The root cytosolic Na+/K+ ratio in alkalinity stressed SR4 was lower than in JN177, while alkalinity stressed SR4 contained higher level of nutrient elements than in JN177. SR4 plant seedlings accumulated less malondialdehyde (MDA) and reactive oxygen species (ROS), it also showed higher activity of ROS scavenging enzymes than JN177 under alkalinity stress. The root intracellular pH decreased in both alkalinity stressed JN177 and SR4, however, it was much lower in SR4 than in JN177 under alkalinity stress. The transcriptomes of SR4 and JN177 seedlings exposed to alkalinity stress were analyzed by digital gene expression tag profiling method. Alkalinity stress conditions up- and down-regulated a large number of genes in the seedling roots that play the functions in the categories of transcription regulation, signal transduction and protein modification. CONCLUSIONS: SR4 expresses a superior tolerance to alkaline stress conditions which is due to its strong absorbing ability for nutrient ions, a strong regulating ability for intracellular and rhizosphere pH and a more active ROS scavenging ability.

Isolation and functional characterisation of CDPKs gene from Arachis hypogaea under salt stress.

One of salt-induced calcium-dependent protein kinases (CDPKs) gene was isolated from Arachis hypogeae L. by RACE method. The cDNA full length was 2241bp deposited in GenBank (number KF437909), designated as AhCDPK. The coding region sequence of AhCDPK was 1629bp and encoded a protein of 542 amino acids. The molecular weight and the theoretical isoelectric point of AhCDPK was 60.96kDa and 5.61 respectively. Amino acid sequence analysis indicated that AhCDPK has highest similarity and homology with Glycine max L. In addition, the AhCDPK amino acids were predicted to encode a hydrophilic protein which localised in the endoplasmic reticulum. AhCDPK seemed to transcript in all peanut organs, and had the highest expression in seeds. The expression of AhCDPK could be strongly induced by both Ca2+ and NaCl. When exposed to salt stress, overexpressing AhCDPK in tobacco could alleviate PSII photoinhibition by improving physiological states, such as reducing the accumulation of reactive oxygen species (ROS), improving the activity of antioxidant defence system enzymes and improving the accumulation of osmotic regulation substance. These results showed that AhCDPK has the same functions as that of G. max, and it could play an important role for peanut to resist salt stress.

Isolation and characterization of novel Glu-St1 alleles from Pseudoroegneria spicata and Pd. strigosa.

Pseudoroegneria is a small genus of the Triticeae tribe; its St genome is present in over half of allopolyploid Triticeae species. The high molecular weight (HMW) subunits of glutenin (GS) encoded by the St genome are not well described. In this paper, we report the characterization of fourteen alleles of HMW-GS genes from the two species Pd. spicata and Pd. strigosa. Analysis shows that all fourteen sequences possess a typical primary structure shared by other known HMW-GS, but with some unique modifications. All fourteen Glu-St1 alleles are significantly smaller than normal Glu-1 genes due to fewer repeat motifs in a repetitive region with no indication of large deletion in other conserved regions. Thus, the small size is a common feature of HMW-GS encoded by Glu-St1 loci of Pseudoroegneria species. Sequence analysis indicated that all fourteen Glu-St1 alleles were intermediate type between x- and y-type, which represent an intermediate stage in the evolutionary divergence of x- and y-type subunits.

High frequency of HMW-GS sequence variation through somatic hybridization between Agropyron elongatum and common wheat.

A symmetric somatic hybridization was performed to combine the protoplasts of tall wheatgrass (Agropyron elongatum) and bread wheat (Triticum aestivum). Fertile regenerants were obtained which were morphologically similar to tall wheatgrass, but which contained some introgression segments from wheat. An SDS-PAGE analysis showed that a number of non-parental high-molecular weight glutenin subunits (HMW-GS) were present in the symmetric somatic hybridization derivatives. These sequences were amplified, cloned and sequenced, to deliver 14 distinct HMW-GS coding sequences, eight of which were of the y-type (Hy1-Hy8) and six x-type (Hx1-Hx6). Five of the cloned HMW-GS sequences were successfully expressed in E. coli. The analysis of their deduced peptide sequences showed that they all possessed the typical HMW-GS primary structure. Sequence alignments indicated that Hx5 and Hy1 were probably derived from the tall wheatgrass genes Aex5 and Aey6, while Hy2, Hy3, Hx1 and Hy6 may have resulted from slippage in the replication of a related biparental gene. We found that both symmetric and asymmetric somatic hybridization could promote the emergence of novel alleles. We discussed the origination of allelic variation of HMW-GS genes in somatic hybridization, which might be the result from the response to genomic shock triggered by the merger and interaction of biparent genomes.

Proteomic analysis on a high salt tolerance introgression strain of Triticum aestivum/Thinopyrum ponticum.

Soil salinity is a major abiotic constraint to agricultural productivity. We successfully bred a new common wheat (Triticum aestivum L.) introgression variety (Shanrong No. 3) with high salt-tolerance via asymmetric somatic hybridization between common wheat cultivar (Jinan 177) and UV-irradiated Agropyron elongatum (Thinopyrum ponticum Podp). We report here a comparative proteomic analysis to investigate variety-specific and salt-responsive proteins between seedling-roots of Shanrong No. 3 and Jinan 177. In total, 114 spots reproducibly presented differential expression patterns on 2-DE maps. Of them, 34 were variety-specific and 49 were salt-responsive. We identified 110 spots by MALDI-TOF MS and partially confirmed by MALDI-TOF-TOF MS, and functionally classified them into signal transduction, transcription and translation, transporting, chaperones, proteolysis and detoxification, etc. Meanwhile, we also found the alteration of protein expression of Shanrong No. 3 through inhibition of old proteins and production of novel ones, change in abundance and sensitivity of some nonsalt-responsive and salt-responsive proteins, as well as PTMs. Furthermore, comparison between proteome and transcripteome using cDNA microarray showed that there were only 20 proteins with abundances correlative to signal densities of corresponding EST probes. This study gives us a global insight into proteomic difference between Shanrong No. 3 and Jinan 177 in constitute and to salt-response.

Characterization of the genes coding for the high molecular weight glutenin subunits in Lophopyrum elongatum.

In order to reveal the evolution of Glu-E1 loci of Lophopyrum elongatum (Host) A. Löve and find novel alleles for wheat quality breeding, four Glu-1 alleles were isolated and characterized via genomic PCR, from this wheat-related species. Of them, 1Ex2 and 1Ey2 were novel alleles, which differ from all the previously known HMW-GS alleles of L. elongatum. Two alleles 1Ex1 and 1Ey2, which contain intact open reading frames, have been successfully expressed in E. coli. The expressed proteins showed similar electrophoresis mobility with the candidate high molecular weight glutenin subunit bands found in seeds. Sequence alignment indicated that proteins encoded by the novel alleles showed similar primary structure with those of wheat and other wheat-related grasses, however, they possess some unique modifications in their own structure. For example, the number of residues in the N-terminal domain is different from those of wheat, an irregular tripeptide present between two nonapeptide motifs and a unique cysteine position in the repetitive region. Phylogenetic analyses using N-terminal conserved sequences showed that 1Ex2 was homologous to those from the D genome; but 1Ey2 was homologous to a y-type allele 1Ky from the K genome. The evolution relationship of Glu-E1 alleles and the possible utilization of the alleles in wheat breeding are discussed.

Transgenic wheat progeny resistant to powdery mildew generated by Agrobacterium inoculum to the basal portion of wheat seedling.

To improve the transformation efficiency of wheat (Triticum aestivum L.) mediated by Agrobacterium tumefaciens, we explored the possibility of employing the basal portion of wheat seedling (shoot apical meristem) as the explants. Three genotypes of wheat were transformed by A. tumefaciens carrying beta-1, 3-glucanase gene. After vernalization, the seeds to be transformed were germinated. When these seedlings grew up to 2 approximately 5 cm, their coleoptile and half of the cotyledon were cut out, and the basal portions were infected by A. tumefaciens. A total 27 T(0) transgenic plants were obtained, and the average transformation efficiency was as high as 9.82%. Evident segregation occurred in some of the T(1) plants, as was indicated by PCR and Southern blotting analysis. Investigation of the T(2) plants revealed that some transformed plants had higher resistance to powdery mildew than the controls. Northern blotting revealed that beta-1, 3-glucanase gene was normally expressed in the T(2) plants, which showed an increased resistance to powdery mildew. The results above indicate that the exogenous gene has been successfully integrated into the genome of wheat, transmitted and expressed in the transgenic progeny. From all the results above, it can be concluded that Agrobacterium inoculum to the basal portion of wheat seedling is a highly efficient and dependable transformation method. It can be developed into a practicable method for transfer of target gene into wheat.

[Isolation and sequence analysis of a omega-gliadin homologous gene from wheat].

The DNA sequence of a full-length Triticum astivum CV. Jinan 177omega-gliadin homologous gene (omega1236) containing partial 5' and 3' flanking sequences with no intron was cloned by genomic PCR-based technology. The omega1236 sequence possibly encode a putative 47.2 kDa protein except for eight stop codons at amino acid residue positions 87, 117, 125, 157, 198, 313, 357 and 365 respectively. All the eight stop codons were caused by base transition. Sequence analysis revealed that omega1236 had 98% homology to a omega-gliadin gene of wheat (AB059812). Like all other gliadin gene families characterized in cereals, this gene possessed all the features in other plant reported previously. Phylogenetic analysis of the completely sequenced gene as well as those omega-genes in wheat, omega-secalin and C-horden genes in rye and barley, and alpha-, beta- and gamma-gliadin genes in wheat indicated that the omega1236 was more closely related to omega-gliadin gene family, much less homology to alpha- , beta- and gamma-gliadin gene families. Short peptide was produced in the culture of transformed E. coli induced by IPTG in early 2 h. It indicated that stop codon would be in omega1236. The result is consistent with that of the sequenced gene. The present paper could accumulate data useful for both omega-gliadin gene cloning by PCR and the study on structures and functions of these genes.

[Research on Festuca arundinacea transformation mediated by Agrobacterium tumefaciens].

The embryo-derived calli from four types of tall fescues (Festuca arundinacea Schreb) were transformed with two Agrobactrium tumefaciens strains AGL1 and GV3101. AGL1 harbors an intron-AtNHX1 expression vector pROK2U containing ubiqutin promoter and npt II marker gene. GV3101 harbors an intron-AtNHX1 expression vector pROK2 containing 35S promoter and npt II gene. After infection and co-culture with AGL1 or GV3101, the embyogenic calli were selected with 50-150 mg/L paromomycine and 1126 resistant plants regenerated from the resistant calli. All plants were selected further with 10-20 mg/L Kanamycin and 525 of them remained green. Genome DNA of the resistant plants was checked with specific primers and probe from AtNHX1 gene. The results of PCR assay and Southern blot analysis indicted that exogenous target gene (AtNHX1 gene) had been transferred into different cultivars of Festuca arundinacea. Different transformation frequencies among the four cultivars were obtained.

[The characteristics of nuclear/cytoplasmic genomes of asymmetric somatic hybrid of Daucus carota var. sativa and Swertia mussotii].

Protoplasts of Swertia mussotii irradiated by ultra-violet light (UV) 260 microW/cm(2) were fused with protoplasts of Daucus carota var. sativa using the PEG method. Analysis of 5S rDNA spacer sequences and random amplified polymorphic DNA (RAPD) showed the presence and combination of nuclear DNA from both parents. All hybrids mainly contained genomes of D. carota (receptor) RAPD bands, and a few donor RAPD bands of S. mussotii. There is no obvious relationship between the nuclear DNA composition was not affected by UV-dosage. SSR analysis of chloroplast DNA of the hybrid clones further revealed that cpDNAs of the parents were integrated and recombined in the hybrids randomly.

[Research advances on the mechanism of RNA silencing in plants].

RNA silencing is a supervising mechanism in eukaryote which can prevent virus duplication, repress transposition of transposon and regulate gene expression. Compared with that in animals, RNA silencing in plants shows some differences in the function of RdRP, in bi-directional transitive RNAi, and in systemic propagation, etc. Meanwhile,the endogenous small RNAs in plants are more diverse than those in animals. In this paper, it is reviewed of the mechanism of RNA silencing in plants and the possibility of its use in plant functional Genomics.

Intermediate fertile Triticum aestivum (+) Agropyron elongatum somatic hybrids are generated by low doses of UV irradiation.

We report the production and characterization of somatic hybrids between Triticum aestivum L. and Agropyron elongatum (Host) Nevishi (the synonym is Thinopyrum ponticum). Asymmetric protoplast fusion was performed between Agropyron elongatum protoplasts irradiated with a low UV dose and protoplasts of wheat taken from nonregenerable suspension cultures. More than 40 green plantlets were obtained from 15 regenerated clones and one of them produced seeds. The phenotypes of the hybrid plants and seeds were intermediate between wheat and Agropyron elongatum. All of the regenerated calli and plants were verified as intergeneric hybrids on the basis of morphological observation and analysis of isozyme, cytological, 5SrDNA spacer sequences and random amplified polymorphic DNA (RAPD). RFLP analysis of the mitochondrial genome revealed evidence of random segregation and recombination of mtDNA.

[Asymmetric somatic hybridization between mixed wheat and Psathyrostachys juncea].

Psathyrostachys juncea is a potential source of useful genes, such as the barley yellow dwarf virus resistance, salt tolerance and drought tolerance, for wheat improvement. Conventional sexual hybridization between wheat and Psathyrostachys juncea is very difficult to occur as the two are sexual incompatible. Somatic hybridization is a promising technique for creating hybrids across the sexual border. Here we report a fusion system for somatic hybridization of wheat using PEG method. Mixed protoplasts of two wheat (Triticum aestivum L. cv. Jinan 177) culture cells (cha9 and 176) were used as the recipients to fuse with the donors, the protoplasts of Psathyrostachys juncea (Fisch.) Nevski irradiated with ultraviolet light (UV) at an intensity of 380 microW/cm2 for 1 min or 2 min. Sixteen clones were generated in the combination I, (wheat 176 + wheat cha9 + P. juncea 1 min UV treatment) and five of the hybrid clones could differentiate to green plants. All the regenerated clones were confirmed as somatic hybrids by cytological, isozyme, chromosome and random amplified polymorphic DNA (RAPD) analysis. Chloroplast genome of the hybrids was analyzed using 7 pairs of wheat-specific chloroplast microsatellite (SSR) primers. Three clones were obtained from the combination II (wheat 176 + wheat cha9 + P. juncea 2 min UV treatment), and all browned slowly and died in 3 months. This result indicated that the mixed wheat cells was helpful to the formation and regeneration of hybrid callus and the dosage of the UV had significant effect on the development of the fusion products.

[Nuclear and cytoplasmic genome analysis of somatic hybrid of Triticum aestivum L. and Leymus chinensis (Trin.) Tzvel].

Intergeneric somatic hybrids were obtained by fusion between protoplasts of Triticum aestivum L. cv. Jinan 177 and Leymus chinensis (Trin.) Tzvel. Protoplasts of L. chinensis were exposed to UV (300 microW/cm(2)) for 30 s, 45 s and 1 min before fusion. The results of morphological and chromosomal observation, isozyme pattern as well as RAPD analysis and the 5S rDNA space sequence analysis showed the hybrid nature of the regenerated colonies of fusion combination T (+) L (UV 30 s). Restriction Fragment Length Po1ymorphism (RFLP) analysis using mitochondrion-specific atp6 and chloroplast-specific rbcL probes showed that most of the organelle genomes of the hybrids originated from L. chinensis and some recombination happened between wheat and L. chinensis. The effects of elimination of the nuclear genome of receptor protoplast on hybrid regeneration and receptor organelle genome elimination are discussed.

[Expression vector used in chloroplast genetic transformation].

Chloroplast genetic transformation is a new way of plant genetic engineering. This paper reviews the construction methods of expression vector used in chloroplast genetic transformation. It contains the homologous recombinant fragments, the chloroplast specific promoter and terminater, the selectable marker genes and the interest genes whose expression in chloroplast have been achieved.

[Introduction of a simple and effective method for plant total RNA isolation].

Wheat leaf and seeds at different development stages had been squashed in liquid nitrogen, then lysed by urea buffer which contains 0.1% SDS and 0.1% LDS, denatured protein had been removed by NaAc and chloroform precipitation, total RNA was further purified by LiCl. The RNA we obtained had sharp bands of 28S and 18S after agarose gel electrophoresis, 23S and 16S RNA bands can also be seen clearly in leaf RNA extract, the value of OD260/OD280 of RNA was 2.05-2.105 mg RNA can been isolated from 10 g leaf of wheat. This method can also been used in high molecular weight DNA isolation but the concentration of SDS and LDS must be increased to 1%.

[Somatic hybridization between carrot and Bupleurum scorzonerifolium and esteras isoenzyme analysis].

OBJECTIVE: To transfer the effective elements of Bupleurum scorzonerifolium into carrot, and provide theoretical data for the exploitation, improvement and selection of the germplasm of Chinese medicinal plants. METHOD: The protoplasta of Bupleurum scorzonerifolium irradiated by ultraviolet light (UV) at an intensity of 300 microW.(cm2)-1 for 0, 1, 2 min respectively were fused with those of carrot Fisch by PEG method. The regenerated clones, derived form a single fused cell, were examined for their hybrid nature by phenotype and Esterase isoenzyme analysis. RESULT: Nine clones were identified as the somatic hybrids between B. scorzonerifolium and carrot. CONCLUSION: This provides a firm foundation for the further analysis of the main active components saikosaponin of somatic hybrids and the screening out of high-medicine-content hybrid cell lines.