Ca2+-dependent TaCCD1 cooperates with TaSAUR215 to enhance plasma membrane H+-ATPase activity and alkali stress tolerance by inhibiting PP2C-mediated dephosphorylation of TaHA2 in wheat.

Alkali stress is a major constraint for crop production in many regions of saline-alkali land. However, little is known about the mechanisms through which wheat responds to alkali stress. In this study, we identified a calcium ion-binding protein from wheat, TaCCD1, which is critical for regulating the plasma membrane (PM) H+-ATPase-mediated alkali stress response. PM H+-ATPase activity is closely related to alkali tolerance in the wheat variety Shanrong 4 (SR4). We found that two D-clade type 2C protein phosphatases, TaPP2C.D1 and TaPP2C.D8 (TaPP2C.D1/8), negatively modulate alkali stress tolerance by dephosphorylating the penultimate threonine residue (Thr926) of TaHA2 and thereby inhibiting PM H+-ATPase activity. Alkali stress induces the expression of TaCCD1 in SR4, and TaCCD1 interacts with TaSAUR215, an early auxin-responsive protein. These responses are both dependent on calcium signaling triggered by alkali stress. TaCCD1 enhances the inhibitory effect of TaSAUR215 on TaPP2C.D1/8 activity, thereby promoting the activity of the PM H+-ATPase TaHA2 and alkali stress tolerance in wheat. Functional and genetic analyses verified the effects of these genes in response to alkali stress, indicating that TaPP2C.D1/8 function downstream of TaSAUR215 and TaCCD1. Collectively, this study uncovers a new signaling pathway that regulates wheat responses to alkali stress, in which Ca2+-dependent TaCCD1 cooperates with TaSAUR215 to enhance PM H+-ATPase activity and alkali stress tolerance by inhibiting TaPP2C.D1/8-mediated dephosphorylation of PM H+-ATPase TaHA2 in wheat.

Alteration of synonymous codon usage bias accompanies polyploidization in wheat.

The diploidization of polyploid genomes is accompanied by genomic variation, including synonymous nucleotide substitutions that may lead to synonymous codon usage bias (SCUB). SCUB can mirror the evolutionary specialization of plants, but its effect on the formation of polyploidies is not well documented. We explored this issue here with hexaploid wheat and its progenitors. Synonymous codons (SCs) ending in either cytosine (NNC) or guanidine (NNG) were more frequent than those ending in either adenosine (NNA) or thymine (NNT), and the preference for NNC/G codons followed the increase in genome ploidy. The ratios between NNC/G and NNA/T codons gradually decreased in genes with more introns, and the difference in these ratios between wheat and its progenitors diminished with increasing ploidy. SCUB frequencies were heterogeneous among exons, and the bias preferred to NNA/T in more internal exons, especially for genes with more exons; while the preference did not appear to associate with ploidy. The SCUB alteration of the progenitors was different during the formation of hexaploid wheat, so that SCUB was the homogeneous among A, B and D subgenomes. DNA methylation-mediated conversion from cytosine to thymine weakened following the increase of genome ploidy, coinciding with the stronger bias for NNC/G SCs in the genome as a function of ploidy, suggesting that SCUB contribute to the epigenetic variation in hexaploid wheat. The patterns in SCUB mirrored the formation of hexaploid wheat, which provides new insight into genome shock-induced genetic variation during polyploidization. SCs representing non-neutral synonymous mutations can be used for genetic dissection and improvement of agricultural traits of wheat and other polyploidies.

Polyploidization is accompanied by synonymous codon usage bias in the chloroplast genomes of both cotton and wheat.

Synonymous codon usage bias (SCUB) of both nuclear and organellar genes can mirror the evolutionary specialization of plants. The polyploidization process exposes the nucleus to genomic shock, a syndrome which promotes, among other genetic variants, SCUB. Its effect on organellar genes has not, however, been widely addressed. The present analysis targeted the chloroplast genomes of two leading polyploid crop species, namely cotton and bread wheat. The frequency of codons in the chloroplast genomes ending in either adenosine (NNA) or thymine (NNT) proved to be higher than those ending in either guanidine or cytosine (NNG or NNC), and this difference was conserved when comparisons were made between polyploid and diploid forms in both the cotton and wheat taxa. Preference for NNA/T codons was heterogeneous among genes with various numbers of introns and was also differential among the exons. SCUB patterns distinguished tetraploid cotton from its diploid progenitor species, as well as bread wheat from its diploid/tetraploid progenitor species, indicating that SCUB in the chloroplast genome partially mirrors the formation of polyploidies.

From Genetic Stock to Genome Editing: Gene Exploitation in Wheat.

Bread wheat (Triticum aestivum) ranks as one of our most important staple crops. However, its hexaploid nature has complicated our understanding of the genetic bases underlying many of its traits. Historically, functional genetic studies in wheat have focused on identifying natural variations and have contributed to assembling and enriching its genetic stock. Recently, mold-breaking advances in whole genome sequencing, exome-capture based mutant libraries, and genome editing have revolutionized strategies for genetic research in wheat. We review new trends in wheat functional genetic studies along with germplasm conservation and innovation, including the relevance of genetic stocks, and the application of sequencing-based mutagenesis and genome editing. We also highlight the potential of multiplex genome editing toolkits in addressing species-specific challenges in wheat.

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.

Characterisation of low molecular weight glutenin subunit genes from Pseudoroegneria spicata and Pd. strigosa.

We report the characterisation of nine novel low molecular weight glutenin subunit (LMW-GS) genes from two Pseudoroegneria species, Pd. spicata and Pd. strigosa. We found that all nine LMW-GS genes possess the same primary structure shared by other published LMW-GS. Five genes encode LMW-i type subunits, three encode LMW-m type subunits and one encodes a peptide similar to B-3 hordeins of Hordeum chilense. No LMW-s type subunit genes were found in Pseudoroegneria. One subunit, PSt24-LMW-2, contains six conserved cysteine residues, and the other eight subunits all contain eight cysteine residues. We show that one cysteine residue is located in the signal peptide of PSt24-LMW-1, suggesting a mature peptide containing only seven cysteine residues. Phylogenetic analysis indicates that the LMW-GS genes from the St genome cluster together and suggests a distant relationship with LMW-GS of the A and B genomes of wheat. Slippage/unequal crossing over and illegitimate recombination are effective mechanisms for enriching variations of seed storage proteins.

Isolation and characterization of a bread wheat salinity responsive ERF transcription factor.

A screen conducted on both a suppression subtractive hybridization and a full length cDNA library made from a salinity tolerant bread wheat cultivar SR3 (Triticum aestivum cv. SR3) resulted in the recognition of TaERF4, a gene including both an AP2/ERF domain and a nuclear localization signal. The 982 bp TaERF4 cDNA comprised a 582 bp open reading frame, encoding a 193 residue polypeptide of molecular weight 20 kDa and calculated pI 8.48. A TaERF4-GFP fusion protein localized preferentially to the nuclei of Arabidopsis thaliana protoplasts. TaERF4 is a member of the B-1 group within the ERF sub-family and was not transactivatable in yeast. The presence of an ERF-associated amphiphilic repression (EAR) motif at its C-terminus suggests that TaERF4 is probably a transcription repressor. TaERF4 was inducible by exposure to salinity and osmotic stresses, but not to exogenously supplied abscisic acid (ABA). The heterologous constitutive expression of TaERF4 in Arabidopsis enhanced the level of sensitivity to salinity stress, possibly via the repression of tonoplast Na(+)/H(+) antiporter activity. There was no phenotype associated with the transgene's presence when plants were subjected to either osmotic stress or ABA treatment. TaERF4 appears to be a transcription repressor acting within the ABA-independent response to salinity stress.

Construction of whole genome radiation hybrid panels and map of chromosome 5A of wheat using asymmetric somatic hybridization.

To explore the feasibility of constructing a whole genome radiation hybrid (WGRH) map in plant species with large genomes, asymmetric somatic hybridization between wheat (Triticum aestivum L.) and Bupleurum scorzonerifolium Willd. was performed. The protoplasts of wheat were irradiated with ultraviolet light (UV) and gamma-ray and rescued by protoplast fusion using B. scorzonerifolium as the recipient. Assessment of SSR markers showed that the radiation hybrids have the average marker retention frequency of 15.5%. Two RH panels (RHPWI and RHPWII) that contained 92 and 184 radiation hybrids, respectively, were developed and used for mapping of 68 SSR markers in chromosome 5A of wheat. A total of 1557 and 2034 breaks were detected in each panel. The RH map of chromosome 5A based on RHPWII was constructed. The distance of the comprehensive map was 2103 cR and the approximate resolution was estimated to be ∼501.6 kb/break. The RH panels evaluated in this study enabled us to order the ESTs in a single deletion bin or in the multiple bins cross the chromosome. These results demonstrated that RH mapping via protoplast fusion is feasible at the whole genome level for mapping purposes in wheat and the potential value of this mapping approach for the plant species with large genomes.

Comparative and evolutionary analysis of new variants of ω-gliadin genes from three A-genome diploid wheats.

A genomic polymerase chain reaction (PCR) cloning strategy was applied to isolate ω-gliadin sequences from three A-genome diploid wheats (Triticum monococcum, T. boeoticum and T. urartu). Amplicon lengths varied from 744 and 1,044 bp, and those of the corresponding deduced mature proteins from 248 to 348 residues. The primary structure of the deduced polypeptides comprised a short N- and C-terminal conserved domain, and a long, variable repetitive domain. A phylogenetic analysis recognised several clades: the first consisted of three T. aestivum sequences; the second and the third two T. boeoticum and six T. monococcum sequences; and the rest four T. urartu and three T. aestivum sequences. Among the functional (non-pseudogene) ARQ/E-type ω-gliadin sequences, two were derived from T. boeoticum and three from T. monococcum; one of the latter sequences appeared to be a chimera originating via illegitimate recombination between the other two T. monococcum sequences. None of the 12 intact ω-gliadin sequences contained any cysteine or methionine residues. We discussed the variation and evolution of A-genome ω-gliadin genes.

From model to crop: functional analysis of a STAY-GREEN gene in the model legume Medicago truncatula and effective use of the gene for alfalfa improvement.

Medicago truncatula has been developed into a model legume. Its close relative alfalfa (Medicago sativa) is the most widely grown forage legume crop in the United States. By screening a large population of M. truncatula mutants tagged with the transposable element of tobacco (Nicotiana tabacum) cell type1 (Tnt1), we identified a mutant line (NF2089) that maintained green leaves and showed green anthers, central carpels, mature pods, and seeds during senescence. Genetic and molecular analyses revealed that the mutation was caused by Tnt1 insertion in a STAY-GREEN (MtSGR) gene. Transcript profiling analysis of the mutant showed that loss of the MtSGR function affected the expression of a large number of genes involved in different biological processes. Further analyses revealed that SGR is implicated in nodule development and senescence. MtSGR expression was detected across all nodule developmental zones and was higher in the senescence zone. The number of young nodules on the mutant roots was higher than in the wild type. Expression levels of several nodule senescence markers were reduced in the sgr mutant. Based on the MtSGR sequence, an alfalfa SGR gene (MsSGR) was cloned, and transgenic alfalfa lines were produced by RNA interference. Silencing of MsSGR led to the production of stay-green transgenic alfalfa. This beneficial trait offers the opportunity to produce premium alfalfa hay with a more greenish appearance. In addition, most of the transgenic alfalfa lines retained more than 50% of chlorophylls during senescence and had increased crude protein content. This study illustrates the effective use of knowledge gained from a model system for the genetic improvement of an important commercial crop.

TaCHP: a wheat zinc finger protein gene down-regulated by abscisic acid and salinity stress plays a positive role in stress tolerance.

The plant response to abiotic stresses involves both abscisic acid (ABA)-dependent and ABA-independent signaling pathways. Here we describe TaCHP, a CHP-rich (for cysteine, histidine, and proline rich) zinc finger protein family gene extracted from bread wheat (Triticum aestivum), is differentially expressed during abiotic stress between the salinity-sensitive cultivar Jinan 177 and its tolerant somatic hybrid introgression cultivar Shanrong No.3. TaCHP expressed in the roots of seedlings at the three-leaf stage, and the transcript localized within the cells of the root tip cortex and meristem. TaCHP transcript abundance was higher in Shanrong No.3 than in Jinan 177, but was reduced by the imposition of salinity or drought stress, as well as by the exogenous supply of ABA. When JN17, a salinity hypersensitive wheat cultivar, was engineered to overexpress TaCHP, its performance in the face of salinity stress was improved, and the ectopic expression of TaCHP in Arabidopsis (Arabidopsis thaliana) also improved the ability of salt tolerance. The expression level of a number of stress reporter genes (AtCBF3, AtDREB2A, AtABI2, and AtABI1) was raised in the transgenic lines in the presence of salinity stress, while that of AtMYB15, AtABA2, and AtAAO3 was reduced in its absence. The presence in the upstream region of the TaCHP open reading frame of the cis-elements ABRE, MYBRS, and MYCRS suggests that it is a component of the ABA-dependent and -independent signaling pathways involved in the plant response to abiotic stress. We suggest that TaCHP enhances stress tolerance via the promotion of CBF3 and DREB2A expression.

The effect of the heterologous expression of Phragmites australis gamma-glutamylcysteine synthetase on the Cd2+ accumulation of Agrostis palustris.

Heavy metal pollution has become one of the most serious environmental problems today. To develop a more efficient plant to clean up heavy metal contaminated soils, a gamma-glutamylcysteine synthetase (GCS) cDNA, named PaGCS, was isolated by PCR from Phragmites australis. The PaGCS sequence was transformed via agroinfection into the heavy metal intolerant grass Agrostis palustris. Five confirmed transgenic A. palustris plants expressing PaGCS were compared with the wild-type line for growth and Cd(2+) accumulation, as well as for the expression of a number of phytochelatin synthesis and stress-responsive enzymes when challenged with Cd(2+) stress. GCS and phytochelatin synthase (PCS) were up-regulated in the transgenic lines. All the transgenic lines accumulated more Cd(2+) and phytochelatins (PCs) than the wild-type line, and three of the five lines grew more effectively than the wild-type after either five or 21 d of Cd(2+) stress. Variation among the transgenics was observed for the distribution of Cd(2+) in the root, shoot and leaf. The malondialdehyde content of all the transgenic lines was lower than that of the wild type under Cd(2+) treatment, while the activity of both superoxide dismutase and peroxidase present in the transgenic lines increased markedly 24 h after Cd(2+) stress, and then rapidly declined.

A novel high molecular weight glutenin subunit from Australopyrum retrofractum.

We describe the sequence of a gene encoding a high molecular weight glutenin subunit (HMW-GS) expressed in the endosperm of the wheat relative Australopyrum retrofractum. Although the subunit has a similar primary structure to that HMW-GS genes present in other Triticeae species, its N-terminal domain is shorter, its central repetitive domain includes a unique dodecameric motif, and its C-terminal domain contain an extra cysteine residue. A phylogenetic analysis showed that the Glu-W1 gene is neither a true x- nor a true y-type subunit, although it is more closely related to the y-type genes present in the K and E genomes than to any other published HMW-GS gene. All these results indicated that this novel subunit may undergo a special evolutionary process different from other Triticeae species. A flour supplementation experiment showed that the Glu-W1 subunit has a negative effect on dough quality, which might be the result of interaction between the two closely placed cysteine residues in the C-terminal region.

Molecular characterisation of the low-molecular weight glutenin subunit genes of tall wheatgrass and functional properties of one clone Ee34.

Wild tall wheatgrass (Lophopyrum elongatum L., 2x = 14) is an important resource for improving bread wheat (Titicum aestivum L.), including HMW-GS and LMW-GS relevant to end-use quality of the wheat flour. A set of 14 distinct sequences were amplified from the genomic DNA of the tall wheatgrass, using degenerate primers targeted at Glu-3, the locus containing the genes encoding the low-molecular weight glutenin subunits (LMW-GS). Three sequences contained an internal stop codon and were classified as pseudogenes. The other 11 all consisted of a single intron-less intact open-reading frame. An alignment of deduced protein sequences showed that the primary structure of all 11 sequences was similar to that of wheat and other wheat-related grass Glu-3 genes. All 11 sequences carried the 14 amino acid residue N-terminal motif MESNIIISFLK/RPWL, and were classified as LMW-m genes, based on the identity of the first amino acid of the mature protein. All but one of the sequences contained seven cysteine residues (the exception had 6). Their repetitive domain differs significantly from that present in Glu-3 genes isolated from the close relative intermediate wheatgrass (Thinopyrum Intermedium, 6x). A phylogenetic analysis showed that the tall wheatgrass sequences were closely related to those of the intermediate wheatgrass, but only distantly so to those from decaploid tall wheatgrass. One of the 11 LMW-GS peptides with a free-cysteine residue was heterologously expressed in E. coli and purified in sufficient scale to perform a flour supplementation test. This showed that the dough strength of bread wheat flour was significantly increased by the presence of the tall wheatgrass LMW-GS.

Natural variation at the DEP1 locus enhances grain yield in rice.

Grain yield is controlled by quantitative trait loci (QTLs) derived from natural variations in many crop plants. Here we report the molecular characterization of a major rice grain yield QTL that acts through the determination of panicle architecture. The dominant allele at the DEP1 locus is a gain-of-function mutation causing truncation of a phosphatidylethanolamine-binding protein-like domain protein. The effect of this allele is to enhance meristematic activity, resulting in a reduced length of the inflorescence internode, an increased number of grains per panicle and a consequent increase in grain yield. This allele is common to many Chinese high-yielding rice varieties and likely represents a relatively recent introduction into the cultivated rice gene pool. We also show that a functionally equivalent allele is present in the temperate cereals and seems to have arisen before the divergence of the wheat and barley lineages.

Generation of high frequency of novel alleles of the high molecular weight glutenin in somatic hybridization between bread wheat and tall wheatgrass.

Somatic hybridization between bread wheat and tall wheatgrass (Agropyron elongatum) has generated fertile introgression progenies with novel combinations of high molecular weight glutenin subunits (HMW-GS). Most of these novel HMW-GS alleles were stably inherited. Sixteen HMW-GS sequences were PCR amplified from three introgression progeny lines and sequenced. The alignment of these sequences indicated that five, probably derived from point mutations of the parental genes, whereas eight likely represent the product of replication slippage. Three Glu-1Ay sequences appear to have lost the transposon presented in the parental gene. Two subunits carry an additional cysteine residue, which may be favorable to the quality of end-use product. We demonstrate that novel HMW-GS alleles can be rapidly generated via asymmetric somatic hybridization.

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.

Characterizing HMW-GS alleles of decaploid Agropyron elongatum in relation to evolution and wheat breeding.

Bread wheat quality is mainly correlated with high molecular weight glutenin subunits (HMW-GS) of endosperm. The number of HMW-GS alleles with good processing quality is limited in bread wheat cultivars, while there are plenty of HMW-GS alleles in wheat-related grasses to exploit. We report here on the cloning and characterization of HMW-GS alleles from the decaploid Agropyron elongatum. Eleven novel HMW-GS alleles were cloned from the grass. Of them, five are x-type and six y-type glutenin subunit genes. Three alleles Aex4, Aey7, and Aey9 showed high similarity with another three alleles from the diploid Lophopyrum elongatum, which provided direct evidence for the Ee genome origination of A. elongatum. It was noted that C-terminal regions of three alleles of the y-type genes Aey8, Aey9, and Aey10 showed more similarity with x-type genes than with other y-type genes. This demonstrates that there is a kind of intermediate state that appeared in the divergence between x- and y-type genes in the HMW-GS evolution. One x-type subunit, Aex4, with an additional cysteine residue, was speculated to be correlated with the good processing quality of wheat introgression lines. Aey4 was deduced to be a chimeric gene from the recombination between another two genes. How the HMW-GS genes of A. elongatum may contribute to the improvement of wheat processing quality are discussed.

Two quality-associated HMW glutenin subunits in a somatic hybrid line between Triticum aestivum and Agropyron elongatum.

High-molecular-weight glutenin subunits (HMW-GSs) from hybrid line II-12 between wheat (Triticum aestivum L.) and Agropyron elongatum (Host) Nivski were characterized with SDS-PAGE. Out of these HMW-GSs, two subunits, h1Bx and h1By, had mobilities similar to the subunits 1Bx13 and 1By16 from common wheat 4072, which was used as control. Polyclonal antibodies (pAbs) of h1Bx and h1By were prepared, and Western blotting showed that the pAbs had strong affinities for h1Bx and h1By, separately. The specificity of h1Bx-pAb was further checked; it preferentially recognized subunits h1Bx and 1Bx13. HMW-GS gene coding sequences were amplified by genomic polymerase chain reaction from hybrid II-12. Two of the five amplicons, marked II2a and II31b, were sequenced. Their coding sequences are clustered to Glu-1Bx7 and Glu-1By9 of common wheat. Three discrepant regions in deduced amino acid sequences of II2a and 31b repeated one time more than Glu-1Bx7 and Glu-1By9. N-terminal sequences of h1Bx and h1By were determined, which were identical to the published sequences of 1Bx13 and 1By16 and in agreement with that deduced from II2a and II31b, respectively. These results indicated that the two novel genes separated from the hybrid wheat derived from the allelic variation of 1Bx7 and 1By9 of the parent wheat. There is an additional cysteine residue positioned at 271st amino acid of the mature peptide of II2a, which may be related to the high quality of the flour.