Identification of pathogen-specific novel sources of genetic resistance against ascochyta blight and their underlying genetic control.

Global chickpea production is restricted by ascochyta blight caused by the necrotrophic fungi ascochyta rabiei. Developing locally adapted disease-resistant cultivars is an economically and environmentally sustainable approach to combat this disease. However, the lack of genetic variability in cultivated chickpeas and breeder-friendly markers poses a significant challenge to ascochyta blight-resistant breeding efforts in chickpeas. In this study, we screened the mini-core germplasm of Cicer reticulatum against a local pathotype of ascochyta rabiei. A modified mini-dome screening approach resulted in the identification of five accessions showing a high level of resistance. The mean disease score of resistant accessions ranged between 1.75±0.3 and 2.88±0.4 compared to susceptible accessions, where the mean disease score ranged between 3.59±0.62 and 8.86±0.14. Genome-wide association analysis revealed a strong association on chromosome 5, explaining ~58% of the phenotypic variance. The underlying region contained two candidate genes (Cr_14190.1_v2 and Cr_14189.1_v2), characterization of which showed the presence of a DNA binding domain (cl28899 & cd18793) in Cr_14190.1_v2 and its orthologs in C. arietinum, whereas Cr_14190.1_v2 carried an additional N-terminal domain (cl31759). qPCR expression analysis in resistant and susceptible accessions revealed ~3 and ~110-fold higher transcript abundance for Cr_14189.1 and Cr_14190.1, respectively.

Capturing Wheat Phenotypes at the Genome Level.

Recent technological advances in next-generation sequencing (NGS) technologies have dramatically reduced the cost of DNA sequencing, allowing species with large and complex genomes to be sequenced. Although bread wheat (Triticum aestivum L.) is one of the world's most important food crops, efficient exploitation of molecular marker-assisted breeding approaches has lagged behind that achieved in other crop species, due to its large polyploid genome. However, an international public-private effort spanning 9 years reported over 65% draft genome of bread wheat in 2014, and finally, after more than a decade culminated in the release of a gold-standard, fully annotated reference wheat-genome assembly in 2018. Shortly thereafter, in 2020, the genome of assemblies of additional 15 global wheat accessions was released. As a result, wheat has now entered into the pan-genomic era, where basic resources can be efficiently exploited. Wheat genotyping with a few hundred markers has been replaced by genotyping arrays, capable of characterizing hundreds of wheat lines, using thousands of markers, providing fast, relatively inexpensive, and reliable data for exploitation in wheat breeding. These advances have opened up new opportunities for marker-assisted selection (MAS) and genomic selection (GS) in wheat. Herein, we review the advances and perspectives in wheat genetics and genomics, with a focus on key traits, including grain yield, yield-related traits, end-use quality, and resistance to biotic and abiotic stresses. We also focus on reported candidate genes cloned and linked to traits of interest. Furthermore, we report on the improvement in the aforementioned quantitative traits, through the use of (i) clustered regularly interspaced short-palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated gene-editing and (ii) positional cloning methods, and of genomic selection. Finally, we examine the utilization of genomics for the next-generation wheat breeding, providing a practical example of using in silico bioinformatics tools that are based on the wheat reference-genome sequence.

Characterizing reduced height wheat mutants for traits affecting abiotic stress and photosynthesis during seedling growth.

Most high-yielding, semidwarf wheat (Triticum aestivum L.) grown around the world contains either Rht1 or Rht2 genes. The success of these high-yielding cultivars is greatest in the most productive farming environments but provide marginal benefits in less favorable growing conditions such as shallow soils and low-precipitation dryland farming. Further, growing evidence suggests semidwarf genes not only affect early seedling growth but limit grain yield, especially under abiotic stress conditions. There are 23 other reduced-height mutants reported in wheat, most of which have not been functionally characterized. We evaluated these mutants along with their parents for several traits affecting seedling emergence, early seedling growth, and photosynthetic efficiency. Two- to seven-fold differences in coleoptile length, first leaf length, root length, and root angle were observed among the genotypes. Most of the mutations had a positive effect on root length, while the root angle narrowed. Coleoptile and first leaf lengths were strongly correlated with emergence. A specialized deep planting experiment identified Rht5, Rht6, Rht8, and Rht13 with significantly improved seedling emergence compared to the parent. Among the mutants, Rht4, Rht19, and Rht12 ranked highest for photosynthetic traits while Rht9, Rht16, and Rht15 performed best for early seedling growth parameters. Considering all traits collectively, Rht15 showed the most promise for utilization in marginal environments followed by Rht19 and Rht16. These wheat mutants may be useful for deciphering the underlying molecular mechanisms of understudied traits and breeding programs in arid and semiarid regions where deep planting is practiced.

Enhanced N-metabolites, ABA and IAA-conjugate in anthers instigate heat sensitivity in spring wheat.

Unraveling the metabolic and phytohormonal changes in anthers exposed to heat stress would help identify mechanisms regulating heat stress tolerance during the sensitive reproductive stage. Two spring wheat genotypes contrasting for heat tolerance were exposed to heat stress during heading in controlled environment chambers. Anthers were collected from main and primary spikes for metabolic and phytohormonal profiling. A significant reduction in seed set (38%), grain number (54%) and grain weight (52%) per plant was recorded in the sensitive (KSG1177) but not in the tolerant (KSG1214) genotype under heat stress compared to control. Anther metabolite accumulation did not vary quantitatively between main and primary spikes. Hierarchical clustering of the genotypes and treatments using metabolites and phytohormones revealed a distinct cluster for KSG1177 under heat stress from that of control and KSG1214. A significant increase in N-based amino acids, ABA, IAA-conjugate and a decrease in polyamines and organic acids were observed in wheat anthers exposed to heat stress. Unlike KSG1214, a significantly higher accumulation of amino acids, ABA and IAA-conjugate in anthers of the sensitive KSG1177 was recorded under heat stress. These findings provide the rationale and direction towards developing molecular markers for enhancing heat stress tolerance in wheat.

Genome-wide analysis of the HSP101/CLPB gene family for heat tolerance in hexaploid wheat.

Heat Shock Protein 101 (HSP101), the homolog of Caseinolytic Protease B (CLPB) proteins, has functional conservation across species to play roles in heat acclimation and plant development. In wheat, several TaHSP101/CLPB genes were identified, but have not been comprehensively characterized. Given the complexity of a polyploid genome with its phenomena of homoeologous expression bias, detailed analysis on the whole TaCLPB family members is important to understand the genetic basis of heat tolerance in hexaploid wheat. In this study, a genome-wide analysis revealed thirteen members of TaCLPB gene family and their expression patterns in various tissues, developmental stages, and stress conditions. Detailed characterization of TaCLPB gene and protein structures suggested potential variations of the sub-cellular localization and their functional regulations. We revealed homoeologous specific variations among TaCLPB gene copies that have not been reported earlier. A study of the Chromosome 1 TaCLPB in four wheat genotypes demonstrated unique patterns of the homoeologous gene expression under moderate and extreme heat treatments. The results give insight into the strategies to improve heat tolerance by targeting one or some of the TaCLPB genes in wheat.

Molecular Characterization of Auxin Efflux Carrier- ABCB1 in hexaploid wheat.

Auxin is an important phytohormone that regulates response, differentiation, and development of plant cell, tissue, and organs. Along with its local production, long-distance transport coordinated by the efflux/influx membrane transporters is instrumental in plant development and architecture. In the present study, we cloned and characterized a wheat (Triticum aestivum) auxin efflux carrier ABCB1. The TaABCB1 was physically localized to the proximal 15% of the short arm of wheat homoeologous group 7 chromosomes. Size of the Chinese spring (CS) homoeologs genomic copies ranged from 5.3-6.2 kb with the 7A copy being the largest due to novel insertions in its third intron. The three homoeologous copies share 95-97% sequence similarity at the nucleotide, 98-99% amino acid, and overall Q-score of 0.98 at 3-D structure level. Though detected in all analyzed tissues, TaABCB1 predominantly expressed in the meristematic tissues likely due to the presence of meristem-specific activation regulatory element identified in the promoter region. RNAi plants of TaABCB1 gene resulted in reduced plant height and increased seed width. Promoter analysis revealed several responsive elements detected in the promoter region including that for different hormones as auxin, gibberellic acid, jasmonic acid and abscisic acid, light, and circadian regulated elements.

The novel function of the Ph1 gene to differentiate homologs from homoeologs evolved in Triticum turgidum ssp. dicoccoides via a dramatic meiosis-specific increase in the expression of the 5B copy of the C-Ph1 gene.

The Ph1 gene is the principal regulator of homoeologous chromosome pairing control (HECP) that ensures the diploid-like meiotic chromosome pairing behavior of polyploid wheat. The HECP control was speculated to have evolved after the first event of polyploidization. With the objective to accurately understand the evolution of the HECP control, wild emmer wheat accessions previously known to differ for HECP control were characterized for the structure and expression of the candidate Ph1 gene, C-Ph1. The C-TdPh1-5A and 5B gene copies of emmer wheat showed 98 and 99% DNA sequence similarity respectively with the corresponding hexaploid wheat copies. Further, the C-TdPh1-5B carried the C-Ph1-5B specific structural changes and transcribed three splice variants as observed in the hexaploid wheat. Further, single nucleotide changes differentiating accessions varying for HECP control were identified. Analyzed by quantitative expression analysis, the wild emmer accessions with HECP control showed ~ 10,000-fold higher transcript abundance of the C-TdPh1-5B copy during prophase-I compared to accessions lacking the control. Differential transcriptional regulation of C-TdPh1-5B splice variants further revealed that C-Ph1-5Balt1 variant is mainly responsible for differential accumulation of C-Ph1-5B copy in accessions with HECP control. Taken together, these results showed that the HECP control evolved via transcriptional regulation of splice variants during meiosis.

Inheritance and Genetic Mapping of the Reduced Height (Rht18) Gene in Wheat.

Short-statured plants revolutionized agriculture during the 1960s due to their ability to resist lodging, increased their response to fertilizers, and improved partitioning of assimilates which led to yield gains. Of more than 21 reduced-height (Rht) genes reported in wheat, only three-Rht-B1b, Rht-D1b, and Rht8-were extensively used in wheat breeding programs. The remaining reduced height mutants have not been utilized in breeding programs due to the lack of characterization. In the present study, we determined the inheritance of Rht18 and developed a genetic linkage map of the region containing Rht18. The height distribution of the F2 population was skewed towards the mutant parent, indicating that the dwarf allele (Rht18) is semi-dominant over the tall allele (rht18). Rht18 was mapped on chromosome 6A between markers barc146 and cfd190 with a genetic distance of 26.2 and 17.3 cM, respectively. In addition to plant height, agronomically important traits, like awns and tiller numbers, were also studied in the bi-parental population. Although the average tiller number was very similar in both parents, the F2 population displayed a normal distribution for tiller number with the majority of plants having phenotype similar to the parents. Transgressive segregation was observed for plant height and tiller number in F2 population. This study enabled us to select a semi-dwarf line with superior agronomic characteristics that could be utilized in a breeding program. The identification of SSRs associated with Rht18 may improve breeders' effectiveness in selecting desired semi-dwarf lines for developing new wheat cultivars.

Evolution of Rubisco activase gene in plants.

KEY MESSAGE: Rubisco activase of plants evolved in a stepwise manner without losing its function to adapt to the major evolutionary events including endosymbiosis and land colonization. Rubisco activase is an essential enzyme for photosynthesis, which removes inhibitory sugar phosphates from the active sites of Rubisco, a process necessary for Rubisco activation and carbon fixation. The gene probably evolved in cyanobacteria as different species differ for its presence. However, the gene is present in all other plant species. At least a single gene copy was maintained throughout plant evolution; but various genome and gene duplication events, which occurred during plant evolution, increased its copy number in some species. The exons and exon-intron junctions of present day higher plant's Rca, which is conserved in most species seem to have evolved in charophytes. A unique tandem duplication of Rca gene occurred in a common grass ancestor, and the two genes evolved differently for gene structure, sequence, and expression pattern. At the protein level, starting with a primitive form in cyanobacteria, RCA of chlorophytes evolved by integrating chloroplast transit peptide (cTP), and N-terminal domains to the ATPase, Rubisco recognition and C-terminal domains. The redox regulated C-terminal extension (CTE) and the associated alternate splicing mechanism, which splices the RCA-α and RCA-ß isoforms were probably gained from another gene in charophytes, conserved in most species except the members of Solanaceae family.

Conservation and divergence of Starch Synthase III genes of monocots and dicots.

Starch Synthase (SS) plays an important role in extending the α-1,4 glucan chains during starch biosynthesis by catalyzing the transfer of the glucosyl moiety from ADP-glucose to the non-reducing end of a pre-existing glucan chain. SS has five distinct isoforms of which SSIII is involved in the formation of longer glucan chain length. Here we report identification and detailed characterization of 'true' orthologs of the well-characterized maize SSIII (ZmSSIII), among six monocots and two dicot species. ZmSSIII orthologs have nucleotide sequence similarity ranging from 56-81%. Variation in gene size among various orthologs ranged from 5.49 kb in Arabidopsis to 11.62 kb in Brachypodium and the variation was mainly due to intron size and indels present in the exons 1 and 3. Number of exons and introns were highly conserved among all orthologs however. While the intron number was conserved, intron phase showed variation at group, genera and species level except for intron 1 and 5. Several species, genera, and class specific cis-acting regulatory elements were identified in the promoter region. The predicted protein size of the SSIII orthologs ranged from 1094 amino acid (aa) in Arabidopsis to 1688 aa in Brachypodium with sequence identity ranging from 60%-89%. The N-terminal region of the protein was highly variable whereas the C-terminal region containing the Glycosyltransferase domain was conserved with >80% sequence similarity among the orthologs. In addition to confirming the known motifs, eleven novel motifs possibly providing species, genera and group specific functions, were identified in the three carbohydrate binding domains. Despite of significant sequence variation among orthologs, most of the motifs and their relative distances are highly conserved among the orthologs. The 3-D structure of catalytic region of SSIII orthologs superimposed with higher confidence confirming the presence of similar binding sites with five unidentified conserved regions in the catalytic (glycosyltransferase) domain including the pockets involved in catalysis and binding of ligands. Homeologs of wheat SSIII gene showed tissue and developmental stage specific expression pattern with the highest expression recorded in developing grains.

Genome-Wide Association Study Reveals Novel Genes Associated with Culm Cellulose Content in Bread Wheat (Triticum aestivum, L.).

Plant cell wall formation is a complex, coordinated and developmentally regulated process. Cellulose is the most dominant constituent of plant cell walls. Because of its paracrystalline structure, cellulose is the main determinant of mechanical strength of plant tissues. As the most abundant polysaccharide on earth, it is also the focus of cellulosic biofuel industry. To reduce culm lodging in wheat and for improved ethanol production, delineation of the variation for stem cellulose content could prove useful. We present results on the analysis of the stem cellulose content of 288 diverse wheat accessions and its genome-wide association study (GWAS). Cellulose concentration ranged from 35 to 52% (w/w). Cellulose content was normally distributed in the accessions around a mean and median of 45% (w/w). Genome-wide marker-trait association study using 21,073 SNPs helped identify nine SNPs that were associated (p < 1E-05) with cellulose content. Four strongly associated (p < 8.17E-05) SNP markers were linked to wheat unigenes, which included ß-tubulin, Auxin-induced protein 5NG4, and a putative transmembrane protein of unknown function. These genes may be directly or indirectly involved in the formation of cellulose in wheat culms. GWAS results from this study have the potential for genetic manipulation of cellulose content in bread wheat and other small grain cereals to enhance culm strength and improve biofuel production.

Evolution of Gene Expression Balance Among Homeologs of Natural Polyploids.

Polyploidy is a major evolutionary process in eukaryotes, yet the expression balance of homeologs in natural polyploids is largely unknown. To study this expression balance, the expression patterns of 2180 structurally well-characterized genes of wheat were studied, of which 813 had the expected three copies and 375 had less than three. Copy numbers of the remaining 992 ranged from 4 to 14, including homeologs, orthologs, and paralogs. Of the genes with three structural copies corresponding to homeologs, 55% expressed from all three, 38% from two, and the remaining 7% expressed from only one of the three copies. Homeologs of 76-87% of the genes showed differential expression patterns in different tissues, thus have evolved different gene expression controls, possibly resulting in novel functions. Homeologs of 55% of the genes showed tissue-specific expression, with the largest percentage (14%) in the anthers and the smallest (7%) in the pistils. The highest number (1.72/3) of homeologs/gene expression was in the roots and the lowest (1.03/3) in the anthers. As the expression of homeologs changed with changes in structural copy number, about 30% of the genes showed dosage dependence. Chromosomal location also impacted expression pattern as a significantly higher proportion of genes in the proximal regions showed expression from all three copies compared to that present in the distal regions.

Comparative Analysis of AGPase Genes and Encoded Proteins in Eight Monocots and Three Dicots with Emphasis on Wheat.

ADP-glucose pyrophosphorylase (AGPase) is a heterotetrameric enzyme with two large subunits (LS) and two small subunits (SS). It plays a critical role in starch biosynthesis. We are reporting here detailed structure, function and evolution of the genes encoding the LS and the SS among monocots and dicots. "True" orthologs of maize Sh2 (AGPase LS) and Bt2 (AGPase SS) were identified in seven other monocots and three dicots; structure of the enzyme at protein level was also studied. Novel findings of the current study include the following: (i) at the DNA level, the genes controlling the SS are more conserved than those controlling the LS; the variation in both is mainly due to intron number, intron length and intron phase distribution; (ii) at protein level, the SS genes are more conserved relative to those for LS; (iii) "QTCL" motif present in SS showed evolutionary differences in AGPase belonging to wheat 7BS, T. urartu, rice and sorghum, while "LGGG" motif in LS was present in all species except T. urartu and chickpea; SS provides thermostability to AGPase, while LS is involved in regulation of AGPase activity; (iv) heterotetrameric structure of AGPase was predicted and analyzed in real time environment through molecular dynamics simulation for all the species; (v) several cis-acting regulatory elements were identified in the AGPase promoters with their possible role in regulating spatial and temporal expression (endosperm and leaf tissue) and also the expression, in response to abiotic stresses; and (vi) expression analysis revealed downregulation of both subunits under conditions of heat and drought stress. The results of the present study have allowed better understanding of structure and evolution of the genes and the encoded proteins and provided clues for exploitation of variability in these genes for engineering thermostable AGPase.

Comparative and evolutionary analysis of α-amylase gene across monocots and dicots.

α-amylase is an important enzyme involved in starch degradation to provide energy to the germinating seedling. The present study was conducted to reveal structural and functional evolution of this gene among higher plants. Discounting polyploidy, most plant species showed only a single copy of the gene making multiple isoforms in different tissues and developmental stages. Genomic length of the gene ranged from 1472 bp in wheat to 2369 bp in soybean, and the size variation was mainly due to differences in the number and size of introns. In spite of this variation, the intron phase distribution and insertion sites were mostly conserved. The predicted protein size ranged from 414 amino acid (aa) in soybean to 449aa in Brachypodium. Overall, the protein sequence similarity among orthologs ranged from 56.4 to 97.4 %. Key motifs and domains along with their relative distances were conserved among plants although several species, genera, and class specific motifs were identified. The glycosyl hydrolase superfamily domain length varied from 342aa in soybean to 384aa in maize and sorghum while length of the C-terminal ß-sheet domain was highly conserved with 61aa in all monocots and Arabidopsis but was 59aa in soybean and Medicago. Compared to rice, 3D structure of the proteins showed 89.8 to 91.3 % similarity among the monocots and 72.7 to 75.8 % among the dicots. Sequence and relative location of the five key aa required for the ligand binding were highly conserved in all species except rice.

Developmental program impacts phenological plasticity of spring wheat under drought.

BACKGROUND: Developing drought-tolerant crops critically depends on the efficient response of a genotype to the limited water availability, a trait known as phenological plasticity. Our understanding of the phenological plasticity remains limited, in particular, about its relationships with plant developmental program. Here, we examined the plastic response of spring wheat at tillering, booting, heading, and anthesis stages to constant or periodic drought stress. The response was assessed by morphological and physiological parameters including symptoms. RESULTS: The dynamics of morphological symptoms were indicators of the plasticity identification of drought. We found that spring wheat exhibits higher phenological plasticity during tillering stage followed by the heading stage, while booting and anthesis stages are the most sensitive. Also, the adaptive response is thought to be influenced with the plant height genes. Furthermore, periodic stress caused more pronounced inhibition of yield than the constant stress, with limited resistance resolution under long period. CONCLUSIONS: Our study shows the importance of considering the phenological plasticity in designing screens for drought tolerance in spring wheat and proposes tillering as the most informative stage for capturing genotypes with tolerance to limit water availability.

An Ethylmethane Sulfonate Mutant Resource in Pre-Green Revolution Hexaploid Wheat.

Mutagenesis is a powerful tool used for studying gene function as well as for crop improvement. It is regaining popularity because of the development of effective and cost efficient methods for high-throughput mutation detection. Selection for semi-dwarf phenotype during green revolution has reduced genetic diversity including that for agronomically desirable traits. Most of the available mutant populations in wheat (Triticum aestivum L.) were developed in post-green revolution cultivars. Besides the identification and isolation of agronomically important alleles in the mutant population of pre-green revolution cultivar, this population can be a vital resource for expanding the genetic diversity for wheat breeding. Here we report an Ethylmethane Sulfonate (EMS) generated mutant population consisting of 4,180 unique mutant plants in a pre-green revolution spring wheat cultivar 'Indian'. Released in early 1900s, 'Indian' is devoid of any known height-reducing mutations. Unique mutations were captured by proceeding with single M2 seed from each of the 4,180 M1 plants. Mutants for various phenotypic traits were identified by detailed phenotyping for altered morphological and agronomic traits on M2 plants in the greenhouse and M3 plants in the field. Of the 86 identified mutants, 75 (87%) were phenotypically stable at the M4 generation. Among the observed phenotypes, variation in plant height was the most frequent followed by the leaf morphology. Several mutant phenotypes including looped peduncle, crooked plant morphology, 'gritty' coleoptiles, looped lower internodes, and burnt leaf tips are not reported in other plant species. Considering the extent and diversity of the observed mutant phenotypes, this population appears to be a useful resource for the forward and reverse genetic studies. This resource is available to the scientific community.

Sequencing-based high throughput mutation detection in bread wheat.

BACKGROUND: Forward genetic approaches have limited use for agronomic traits that can't be reliably scored on a single plant basis. Thus, mutants in wheat and other crops are more useful for gene function studies by reverse genetic approach. With a long-term goal to develop a sequence-based mutation detection resource in hexaploid wheat, we conducted a feasibility study to accurately differentiate induced mutations from the homoeologs' sequence variations present among the three wheat genomes. RESULTS: A reduced representation ApeKI library consisting of 21 Ethylmethane Sulfonate (EMS) induced mutants and two wild type cv. Indian plants was developed using individual barcode adapters and sequenced. A novel bioinformatics pipeline was developed to identify sequence variants using 178,464 wheat unigenes as a reference wheat transcriptome. In total, 14,130 mutational changes [Single Nucleotide Polymorphisms (SNPs) and Insertions/Deletions (INDELs)] and 150,511 homoeologous sequence changes were detected. On an average, 662 SNPs (ranging from 46 to 1,330) and 10 small INDELs (ranging from 0 to 23) were identified for each of the mutants. A mutation frequency of one per 5 Kb was observed with 70 % being transitions and 30 % transversions. The pipeline was tested using the known sequence changes in the three wheat genes. Genes present in the distal regions of the chromosomes were found to be more prone to EMS compared to genes present in the proximal regions. Redefined parameters identified a total of 28,348 mutational changes (1,349/plant). CONCLUSIONS: We conclude that sequencing based mutation detection is a valuable method to identify induced mutations at large.

Evolutionary, Comparative and Functional Analyses of the Brassinosteroid Receptor Gene, BRI1, in Wheat and Its Relation to Other Plant Genomes.

Brassinosteroids (BRs) are plant hormones, fundamental for the growth and development of plants. A trans-membrane protein receptor kinase, Brassinosteroid-Insensitive 1 (BRI1), is known to interact with BRs and be directly involved in plant development. This study investigates the structural organization of BRI1 orthologs in several taxa, with a specific interest in Triticum aestivum. True orthologs of Arabidopsis thaliana BRI1 (AtBRI1) from seven-plant species showed sequence identity ranging from 54% to 95% at the protein level. All gene sequences lacked introns, leading to speculation that post-transcriptional processing in TaBRI1 is similar to AtBRI1. Based on in silico analysis, a single copy of BRI1 was present in each of the three wheat genomes on the long arm of chromosome 3. Domain structure of BRI1 orthologs among different taxa showed multiple leucine rich repeats (LRRs), an island domain (ID), a juxtamembrane/transmembrane domain (JTMD), a catalytic kinase domain (KD), C and N-Terminal domains. The KD showed the highest level of conservation while the LRRs and JTMD were most variable. Phosphorylation of residues in the juxtamembrane domain, known to be involved in the activation of the KD, is conserved in TaBRI1. While TaBRI1 has well-defined differences in the ID and LRR domains, many residues involved in ligand binding are conserved. The activation loop present in the KD showed 100% conservation in all taxa. Despite residue differences, hydrophobicity was conserved in the BR binding pocket across taxa, suggesting that function may not differ as drastically as residue identity may suggest. Predicted 3D structure of AtBRI1 and TaBRI1 showed a conserved super helical assembly, a feature essential in protein-protein interactions. An unrooted phylogram showed TaBRI1 in the monocot clade to be distinct from that of dicots. New insight in the structure and functions of BRI1 may help in targeting BR pathway for crop improvement.

Comparative analysis of ABCB1 reveals novel structural and functional conservation between monocots and dicots.

Phytohormone auxin plays a critical role in modulating plant architecture by creating a gradient regulated via its transporters such as ATP-binding cassette (ABC) B1. Except for Arabidopsis and maize, where it was shown to interrupt auxin transport, ABCB1's presence, structure and function in crop species is not known. Here we describe the structural and putative functional organization of ABCB1 among monocots relative to that of dicots. Identified from various plant species following specific and stringent criteria, ZmABCB1's "true" orthologs sequence identity ranged from 56-90% at the DNA and 75-91% at the predicted amino acid (aa) level. Relative to ZmABCB1, the size of genomic copies ranged from -27 to +1.5% and aa from -7.7 to +0.6%. With the average gene size being similar (5.8 kb in monocots and 5.7 kb in dicots), dicots have about triple the number of introns with an average size of 194 bp (total 1743 bp) compared to 556 bp (total 1667 bp) in monocots. The intron-exon junctions across species were however conserved. N-termini of the predicted proteins were highly variable: in monocots due to mismatches and small deletions of 1-13 aa compared to large, species-specific deletions of up to 77 aa in dicots. The species-, family- and group- specific conserved motifs were identified in the N-terminus and linker region of protein, possibly responsible for the specific functions. The near-identical conserved motifs of Nucleotide Binding Domains (NBDs) in two halves of the protein showed subtle aa changes possibly favoring ATP binding to the N-terminus. Predicted 3-D protein structures showed remarkable similarity with each other and for the residues involved in auxin binding.

Silencing of a metaphase I-specific gene results in a phenotype similar to that of the Pairing homeologous 1 (Ph1) gene mutations.

Although studied extensively since 1958, the molecular mode of action of the Pairing homeologous 1 (Ph1) gene is still unknown. In polyploid wheat, the diploid-like chromosome pairing is principally controlled by the Ph1 gene via preventing homeologous chromosome pairing (HECP). Here, we report a candidate Ph1 gene (C-Ph1) present in the Ph1 locus, transient as well as stable silencing of which resulted in a phenotype characteristic of the Ph1 gene mutants, including HECP, multivalent formation, and disrupted chromosome alignment on the metaphase I (MI) plate. Despite a highly conserved DNA sequence, the C-Ph1 gene homeologues showed a dramatically different structure and expression pattern, with only the 5B copy showing MI-specific expression, further supporting our claim for the Ph1 gene. In agreement with the previous reports about the Ph1 gene, the predicted protein of the 5A copy of the C-Ph1 gene is truncated, and thus perhaps less effective. The 5D copy is expressed around the onset of meiosis; thus, it may function during the earlier stages of chromosome pairing. Along with alternate splicing, the predicted protein of the 5B copy is different from the protein of the other two copies because of an insertion. These structural and expression differences among the homeologues concurred with the previous observations about Ph1 gene function. Stable RNAi silencing of the wheat gene in Arabidopsis showed multivalents and centromere clustering during meiosis I.