BackgroundLow-molecular-weight glutenin subunits (LMW-GS), encoded by Glu-3 complex loci in hexaploid wheat, play important roles in the processing quality of wheat flour. To date, the molecular characteristics and effects on dough quality of individual Glu-3 alleles and their encoding proteins have been poorly studied. We used a Glu-A3 deletion line of the Chinese Spring (CS-n) wheat variety to conduct the first comprehensive study on the molecular characteristics and functional properties of the LMW-GS allele Glu-A3a.ResultsThe Glu-A3a allele at the Glu-A3 locus in CS and its deletion in CS-n were identified and characterized by proteome and molecular marker methods. The deletion of Glu-A3a had no significant influence on plant morphological and yield traits, but significantly reduced the dough strength and breadmaking quality compared to CS. The complete sequence of the Glu-A3a allele was cloned and characterized, which was found to encode a B-subunit with longer repetitive domains and an increased number of α-helices. The Glu-A3a-encoded B-subunit showed a higher expression level and accumulation rate during grain development. These characteristics of the Glu-A3a allele could contribute to achieving superior gluten quality and demonstrate its potential application to wheat quality improvement. Furthermore, an allele-specific polymerase chain reaction (AS-PCR) marker for the Glu-A3a allele was developed and validated using different bread wheat cultivars, including near-isogenic lines (NILs) and recombinant inbred lines (RILs), which could be used as an effective molecular marker for gluten quality improvement through marker-assisted selection.ConclusionsThis work demonstrated that the LMW-GS allele Glu-A3a encodes a specific LMW-i type B-subunit that significantly affects wheat dough strength and breadmaking quality. The Glu-A3a-encoded B-subunit has a long repetitive domain and more α-helix structures as well as a higher expression level and accumulation rate during grain development, which could facilitate the formation of wheat with a stronger dough structure and superior breadmaking quality.

BackgroundWheat seed germination directly affects wheat yield and quality. Although transcriptome and proteome analyses during seed germination have been reported in some crop plant species, dynamic transcriptome characterization during wheat seed germination has not been conducted. We performed the first comprehensive dynamic transcriptome analysis during different seed germination stages of elite Chinese bread wheat cultivar Jimai 20 using the Affymetrix Wheat Genome Array.ResultsA total of 61,703 probe sets representing 51,411 transcripts were identified during the five seed germination stages of Jimai 20, of which 2,825 differential expression probe sets corresponding to 2,646 transcripts with different functions were declared by ANOVA and a randomized variance model. The seed germination process included a rapid initial uptake phase (0–12 hours after imbibition [HAI]), a plateau phase (12–24 HAI), and a further water uptake phase (24–48 HAI), corresponding to switches from the degradation of small-molecule sucrose to the metabolism of three major nutrients and to photosynthesis. Hierarchical cluster and MapMan analyses revealed changes in several significant metabolism pathways during seed germination as well as related functional groups. The signal pathway networks constructed with KEGG showed three important genes encoding the phosphofructokinase family protein, with fructose-1, 6-bisphosphatase, and UTP-glucose-1-phosphate uridylyltransferase located at the center, indicating their pivotal roles in the glycolytic pathway, gluconeogenesis, and glycogenesis, respectively. Several significant pathways were selected to establish a metabolic pathway network according to their degree value, which allowed us to find the pathways vital to seed germination. Furthermore, 51 genes involved in transport, signaling pathway, development, lipid metabolism, defense response, nitrogen metabolism, and transcription regulation were analyzed by gene co-expression network with a k-core algorithm to determine which play pivotal roles in germination. Twenty-three meaningful genes were found, and quantitative RT-PCR analysis validated the expression patterns of 12 significant genes.ConclusionsWheat seed germination comprises three distinct phases and includes complicated regulation networks involving a large number of genes. These genes belong to many functional groups, and their co-regulations guarantee regular germination. Our results provide new insight into metabolic changes during seed germination and interactions between some significant genes.

BackgroundThe SR/CAMTA proteins represent a small family of transcription activators that play important roles in plant responses to biotic and abiotic stresses. Seven SlSR/CAMTA genes were identified in tomato as tomato counterparts of SR/CAMTA; however, the involvement of SlSRs/CAMTAs in biotic and abiotic stress responses is not clear. In this study, we performed functional analysis of the SlSR/CAMTA family for their possible functions in defense response against pathogens and tolerance to drought stress.ResultsExpression of SlSRs was induced with distinct patterns by Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000. Virus-induced gene silencing (VIGS)-based knockdown of either SlSR1 or SlSR3L in tomato resulted in enhanced resistance to B. cinerea and Pst DC3000 and led to constitutive accumulation of H2O2, elevated expression of defense genes, marker genes for pathogen-associated molecular pattern-triggered immunity, and regulatory genes involved in the salicylic acid- and ethylene-mediated signaling pathways. Furthermore, the expression of SlSR1L and SlSR2L in detached leaves and whole plants was significantly induced by drought stress. Silencing of SlSR1L led to decreased drought stress tolerance, accelerated water loss in leaves, reduced root biomass and attenuated expression of drought stress responsive genes in tomato. The SlSR1 and SlSR3L proteins were localized in the nucleus of plant cells when transiently expressed in Nicotiana benthamiana and had transcriptional activation activity in yeast.ConclusionsVIGS-based functional analyses demonstrate that both SlSR1 and SlSR3L in the tomato SlSR/CAMTA family are negative regulators of defense response against B. cinerea and Pst DC3000 while SlSR1L is a positive regulator of drought stress tolerance in tomato.

BackgroundThorough understanding of seed starch biosynthesis and accumulation mechanisms is of great importance for agriculture and crop improvement strategies. We conducted the first comprehensive study of the dynamic development of starch granules and the regulation of starch biosynthesis in Brachypodium distachyon and compared the findings with those reported for common wheat (Chinese Spring, CS) and Aegilops peregrina.ResultsOnly B-granules were identified in Brachypodium Bd21, and the shape variation and development of starch granules were similar in the B-granules of CS and Bd21. Phylogenetic analysis showed that most of the Bd21 starch synthesis-related genes were more similar to those in wheat than in rice. Early expression of key genes in Bd21 starch biosynthesis mediate starch synthesis in the pericarp; intermediate-stage expression increases the number and size of starch granules. In contrast, these enzymes in CS and Ae. peregrina were mostly expressed at intermediate stages, driving production of new B-granules and increasing the granule size, respectively. Immunogold labeling showed that granule-bound starch synthase (GBSSI; related to amylose synthesis) was mainly present in starch granules: at lower levels in the B-granules of Bd21 than in CS. Furthermore, GBSSI was phosphorylated at threonine 183 and tyrosine 185 in the starch synthase catalytic domain in CS and Ae. peregrina, but neither site was phosphorylated in Bd21, suggesting GBSSI phosphorylation could improve amylose biosynthesis.ConclusionsBd21 contains only B-granules, and the expression of key genes in the three studied genera is consistent with the dynamic development of starch granules. GBSSI is present in greater amounts in the B-granules of CS than in Bd21; two phosphorylation sites (Thr183 and Tyr185) were found in Triticum and Aegilops; these sites were not phosphorylated in Bd21. GBSSI phosphorylation may reflect its importance in amylose synthesis.

BackgroundMitogen-activated protein kinase (MAPK) cascades are highly conserved signaling modules that mediate the transduction of extracellular stimuli via receptors/sensors into intracellular responses and play key roles in plant immunity against pathogen attack. However, the function of tomato MAPK kinases, SlMKKs, in resistance against Botrytis cinerea remains unclear yet.ResultsA total of five SlMKK genes with one new member, SlMKK5, were identified in tomato. qRT-PCR analyses revealed that expression of SlMKK2 and SlMKK4 was strongly induced by B. cinerea and by jasmonic acid and ethylene precursor 1-amino cyclopropane-1-carboxylic acid. Virus-induced gene silencing (VIGS)-based knockdown of individual SlMKKs and disease assays identified that SlMKK2 and SlMKK4 but not other three SlMKKs (SlMKK1, SlMKK3 and SlMKK5) are involved in resistance against B. cinerea. Silencing of SlMKK2 or SlMKK4 resulted in reduced resistance to B. cinerea, increased accumulation of reactive oxygen species and attenuated expression of defense genes after infection of B. cinerea in tomato plants. Furthermore, transient expression of constitutively active phosphomimicking forms SlMKK2DD and SlMKK4DD in leaves of Nicotiana benthamiana plants led to enhanced resistance to B. cinerea and elevated expression of defense genes.ConclusionsVIGS-based knockdown of SlMKK2 and SlMKK4 expression in tomato and gain-of-function transient expression of constitutively active phosphomimicking forms SlMKK2DD and SlMKK2DD in N. benthamiana demonstrate that both SlMKK2 and SlMKK4 function as positive regulators of defense response against B. cinerea.

BackgroundBrachypodium distachyon L. is a newly emerging model plant system for temperate cereal crop species. However, its grain protein compositions are still not clear. In the current study, we carried out a detailed proteomics and molecular genetics study on grain glutenin proteins in B. distachyon.ResultsSDS-PAGE and RP-HPLC analysis of grain proteins showed that Brachypodium has few gliadins and high molecular weight glutenin subunits. In contrast the electrophoretic patterns for the albumin, globulin and low molecular weight glutenin subunit (LMW-GS) fractions of the grain protein were similar to those in wheat. In particular, the LMW-C type subunits in Brachypodium were more abundant than the equivalent proteins in common wheat. Southern blotting analysis confirmed that Brachypodium has 4–5 copies of LMW-GS genes. A total of 18 LMW-GS genes were cloned from Brachypodium by allele specific PCR. LMW-GS and 4 deduced amino acid sequences were further confirmed by using Western-blotting and MALDI-TOF-MS. Phylogenetic analysis indicated that Brachypodium was closer to Ae. markgrafii and Ae. umbellulata than to T. aestivum.ConclusionsBrachypodium possessed a highly conserved Glu-3 locus that is closely related to Triticum and related species. The presence of LMW-GS in B. distachyon grains indicates that B. distachyon may be used as a model system for studying wheat quality attributes.