Wheat avenin-like protein and its significant Fusarium Head Blight resistant functions

Abstract

Wheat Avenin-like proteins (TaALP) are atypical storage proteins belonging to the Prolamin superfamily. Previous studies on ALPs have focused on the proteins' positive effects on dough strength, whilst no correlation has been made between TaALPs and the plant immune system. Here, we performed genome-wide characterization of ALP encoding genes in bread wheat. In silico analyses indicated the presence of critical peptides in TaALPs that are active in the plant immune system. Pathogenesis-related nucleotide motifs were also identified in the putative promoter regions of TaALP encoding genes. RT-PCR was performed on TaALP and previously characterised pathogenesis resistance genes in developing wheat caryopses under control and Fusarium graminearum infection conditions, respectively. The results showed 2 that TaALP and NMT genes were upregulated upon F. graminearum inoculation. mRNA insitu hybridization showed that TaALP genes were expressed in the embryo, aleurone and sub-aleurone layer cells. Seven TaALP genes were cloned for the expression of recombinant proteins in Escherichia coli, which displayed significant inhibitory function on F. graminearum under anti-fungal tests. In addition, FHB index association analyses showed that allelic variations of two ALP genes on chromosome 7A were significantly correlated with FHB symptoms. Over-expression of an ALP gene on chromosome 7A showed an enhanced resistance to FHB. Yeast two Hybridization results revealed that ALPs have potential proteases inhibiting effect on metacaspases and beta-glucosidases. A vital infection process related pathogen protein, F. graminearum Beta-glucosidase was found to interact with ALPs.Our study is the first to report a novel function for wheat storage protein in fungal resistance, which greatly advances our understanding of the biological roles of this protein class. The findings in this study is of great significance for future wheat breeding and production. Preparation of Albumin and Globulin Protein extractsAustralia spring bread wheat varieties Mace and Spitfire were used for albumin and globulin protein extraction. The albumin/globulin proteins were extracted from 100 mg of flour according to the procedure of Dupont et al. (48). Briefly, 100 mg of flour was extracted with 1 mL of 0.3 M NaI, 7.5% 1-propanol (NaI-propanol), and centrifuged at 4500 g for 10 min, After two extractions, the supernatant fractions were pooled in 15 ml tubes, precipitated with four volumes of ice-cold (-20°C) NH 4 Ac-MeOH (0.1 M ammonium acetate in 100% methanol), stored at -20 °C for at least 48 h, and centrifuged as above. The supernatant fluid was transferred into 50 ml tubes and precipitated with four volumes of ice-cold acetone and incubated at -20 °C overnight. Following incubation, the fluid was centrifuged as above to yield albumin/globulin fraction pellets. Determination of Individual ALPs of wheat cv. Mace and Spitfire via RP-HPLC Analysis coupled with SDS-PAGE, MALDI-TOF, and LC/MS RP-HPLC Freeze-dried protein pellets were dissolved in 500 µL 6 M guani...

Wheat gluten protein and its impacts on wheat processing quality

“…Mutations or silencing of such genes are not lethal for the plant, so the evolutionary selection pressure on these genes is much lower than for functional genes [6] . As a result, these genes can accumulate more mutations, making them ideal model molecules for studying a range of biological fundamental processes [7][8][9][10] . It is worth noting that past work has been primarily focused on the applied aspects of using these proteins to increase wheat end product quality.…”

“…Most alleles in modern hexaploid wheat cultivars emerged before hexaploid wheat was formed, only By18 subunit has been confirmed to have emerged after hexaploid wheat formation [48] . Many gluten alleles did not enter into the hexaploid wheat during evolution or have been discarded through modern breeding [7,35] . Although six genes exist for HMW glutenin subunits, due to gene silencing, most hexaploid wheat cultivars possess three to five HMW glutenin subunits [33] .…”

Characterising avenin-like proteins (ALPs) from albumin/globulin fraction of wheat grains by RP-HPLC, SDS-PAGE, and MS/MS peptides sequencing

“…a The MALDI-TOF profile of C-terminal TaALP-by-4AL/ 7DS and TaALP-by-4AL/7AS/7DS in peak 8. b The MALDI-TOF profile of TaALP-ay-7AS in peak 11. c The MALDI-TOF profile of TaALP-ay-4AL, TaALP-by-4AL/7AS/7DS and TaALP-bx-4AL/7DS in peak 17. d The MALDI-TOF profile of TaALP-ax-4AL in peak 20. e The MALDI-TOF profile of TaALP-ax-7AS in peak 26. f The MALDI-TOF profile of TaALP-ax-7DS in peak 29. Those peaks not identified as ALP and its derivatives in the MALDI-TOF profile were not labelled based on a yeast two hybrid assay [59]. Further, the differences between the calculated MWs and the MALDI-TOF analysed results further indicated the occurrence of more than one PTMs, such as the acetylation, formylation, methionine oxidation, phosphorylation, ubiquitination and glycosylation, that are likely to happen to the ALPs (Table 2, Figs.…”

“…Further, a detailed phylogenetic analysis was performed on the genome-wide TaALPs genes and its close relatives to wheat and other monocots species [47], suggesting that ALPs might have the protease inhibition activity like α-amylase inhibitors (AAIs), yet the substrates of ALPs can be further identified. Zhang et al have studied the ALPs and its potential Fusarium head blight resistant functions [59], further illustrated their antifungal properties. Other research suggest that ALP type b are minor storage proteins which are important to protect endosperm starch reserves from degradation [60].…”

Synthesis and accumulation of amylase-trypsin inhibitors and FODMAPs in bread wheat (Triticum aestivum L.) during grain development

“…Additionally, ATIs have the potential to inhibit the activity of two important digestive enzymes in the gastrointestinal system, amylase and trypsin, causing a signi cant impairment of digestion [6]. Due to this inhibitory feature, ATIs and other proteins from the prolamine superfamily are considered crucial for the natural defence mechanism of the plant itself [7]. They are located in the endosperm of cereal seeds, where they defend starch and protein reserves by blocking amylase and trypsin activities of invading parasites and insects [8,9].…”