Beyond pathways: Accelerated flavonoids candidate identification and novel exploration of enzymatic properties using combined mapping populations of wheat.

Although forward-genetics-metabolomics methods such as mGWAS and mQTL have proven effective in providing myriad loci affecting metabolite contents, they are somehow constrained by their respective constitutional flaws such as the hidden population structure for GWAS and insufficient recombinant rate for QTL. Here, the combination of mGWAS and mQTL was performed, conveying an improved statistical power to investigate the flavonoid pathways in common wheat. A total of 941 and 289 loci were, respectively, generated from mGWAS and mQTL, within which 13 of them were co-mapped using both approaches. Subsequently, the mGWAS or mQTL outputs alone and their combination were, respectively, utilized to delineate the metabolic routes. Using this approach, we identified two MYB transcription factor encoding genes and five structural genes, and the flavonoid pathway in wheat was accordingly updated. Moreover, we have discovered some rare-activity-exhibiting flavonoid glycosyl- and methyl-transferases, which may possess unique biological significance, and harnessing these novel catalytic capabilities provides potentially new breeding directions. Collectively, we propose our survey illustrates that the forward-genetics-metabolomics approaches including multiple populations with high density markers could be more frequently applied for delineating metabolic pathways in common wheat, which will ultimately contribute to metabolomics-assisted wheat crop improvement.

The pathway of melatonin biosynthesis in common wheat (Triticum aestivum).

Melatonin (Mel) is a multifunctional biomolecule found in both animals and plants. In plants, the biosynthesis of Mel from tryptophan (Trp) has been delineated to comprise of four consecutive reactions. However, while the genes encoding these enzymes in rice are well characterized no systematic evaluation of the overall pathway has, as yet, been published for wheat. In the current study, the relative contents of six Mel-pathway-intermediates including Trp, tryptamine (Trm), serotonin (Ser), 5-methoxy tryptamine (5M-Trm), N-acetyl serotonin (NAS) and Mel, were determined in 24 independent tissues spanning the lifetime of wheat. These studies indicated that Trp was the most abundant among the six metabolites, followed by Trm and Ser. Next, the candidate genes expressing key enzymes involved in the Mel pathway were explored by means of metabolite-based genome-wide association study (mGWAS), wherein two TDC genes, a T5H gene and one SNAT gene were identified as being important for the accumulation of Mel pathway metabolites. Moreover, a 463-bp insertion within the T5H gene was discovered that may be responsible for variation in Ser content. Finally, a ASMT gene was found via sequence alignment against its rice homolog. Validations of these candidate genes were performed by in vitro enzymatic reactions using proteins purified following recombinant expression in Escherichia coli, transient gene expression in tobacco, and transgenic approaches in wheat. Our results thus provide the first comprehensive investigation into the Mel pathway metabolites, and a swift candidate gene identification via forward-genetics strategies, in common wheat.

Metabolite-based genome-wide association study enables dissection of the flavonoid decoration pathway of wheat kernels.

The marriage of metabolomic approaches with genetic design has proven a powerful tool in dissecting diversity in the metabolome and has additionally enhanced our understanding of complex traits. That said, such studies have rarely been carried out in wheat. In this study, we detected 805 metabolites from wheat kernels and profiled their relative contents among 182 wheat accessions, conducting a metabolite-based genome-wide association study (mGWAS) utilizing 14 646 previously described polymorphic SNP markers. A total of 1098 mGWAS associations were detected with large effects, within which 26 candidate genes were tentatively designated for 42 loci. Enzymatic assay of two candidates indicated they could catalyse glucosylation and subsequent malonylation of various flavonoids and thereby the major flavonoid decoration pathway of wheat kernel was dissected. Moreover, numerous high-confidence genes associated with metabolite contents have been provided, as well as more subdivided metabolite networks which are yet to be explored within our data. These combined efforts presented the first step towards realizing metabolomics-associated breeding of wheat.

Plants interfere with non-self recognition of a phytopathogenic fungus via proline accumulation to facilitate mycovirus transmission.

Non-self recognition is a fundamental aspect of life, serving as a crucial mechanism for mitigating proliferation of molecular parasites within fungal populations. However, studies investigating the potential interference of plants with fungal non-self recognition mechanisms are limited. Here, we demonstrate a pronounced increase in the efficiency of horizontal mycovirus transmission between vegetatively incompatible Sclerotinia sclerotiorum strains in planta as compared to in vitro. This increased efficiency is associated with elevated proline concentration in plants following S. sclerotiorum infection. This surge in proline levels attenuates the non-self recognition reaction among fungi by inhibition of cell death, thereby facilitating mycovirus transmission. Furthermore, our field experiments reveal that the combined deployment of hypovirulent S. sclerotiorum strains harboring hypovirulence-associated mycoviruses (HAVs) together with exogenous proline confers substantial protection to oilseed rape plants against virulent S. sclerotiorum. This unprecedented discovery illuminates a novel pathway by which plants can counteract S. sclerotiorum infection, leveraging the weakening of fungal non-self recognition and promotion of HAVs spread. These promising insights provide an avenue to explore for developing innovative biological control strategies aimed at mitigating fungal diseases in plants by enhancing the efficacy of horizontal HAV transmission.

Suppressing a phosphohydrolase of cytokinin nucleotide enhances grain yield in rice.

One-step and two-step pathways are proposed to synthesize cytokinin in plants. The one-step pathway is mediated by LONELY GUY (LOG) proteins. However, the enzyme for the two-step pathway remains to be identified. Here, we show that quantitative trait locus GY3 may boost grain yield by more than 20% through manipulating a two-step pathway. Locus GY3 encodes a LOG protein that acts as a 5'-ribonucleotide phosphohydrolase by excessively consuming the cytokinin precursors, which contrasts with the activity of canonical LOG members as phosphoribohydrolases in a one-step pathway. The residue S41 of GY3 is crucial for the dephosphorylation of iPRMP to produce iPR. A solo-LTR insertion within the promoter of GY3 suppressed its expression and resulted in a higher content of active cytokinins in young panicles. Introgression of GY302428 increased grain yield per plot by 7.4% to 16.3% in all investigated indica backgrounds, which demonstrates the great value of GY302428 in indica rice production.

Cloning of wheat keto-acyl thiolase 2B reveals a role of jasmonic acid in grain weight determination.

Grain weight (GW) is one of the component traits of wheat yield. Existing reports have shown that multiple phytohormones are involved in the regulation of GW in different crops. However, the potential role of jasmonic acid (JA) remains unclear. Here, we report that triticale grain weight 1 (tgw1) mutant, with marked reductions in both GW and JA content, is caused by a premature stop mutation in keto-acyl thiolase 2B (KAT-2B) involved in ß-oxidation during JA synthesis. KAT-2B overexpression increases GW in wild type and boosts yield. Additionally, KAT-2B compliments the grain defect in tgw1 and rescues the lethal phenotype of the Arabidopsis kat2 mutant in a sucrose-free medium. Despite the suppression of JA synthesis in tgw1 mutant, ABA synthesis is upregulated, which is accompanied by enhanced expression of SAG3 and reduction of chlorophyll content in leaves. Together, these results demonstrate a role of the JA synthetic gene KAT-2B in controlling GW and its potential application value for wheat improvement.

Breaking the Summer Dormancy of Pinellia ternata by Introducing a Heat Tolerance Receptor-Like Kinase ERECTA Gene.

Pinellia ternata is a perennial traditional Chinese medicinal plant that undergoes different phenological patterns of dormancy depending on where it is growing. Plants grown in central and southern China typically display two growth cycles every year before and after hot summer days, exhibiting a summer dormancy. However, germplasms from these areas do not go into a dormancy phase in northern China where the summer monthly average temperatures range from 29-31°C. The northern China herbal growers prefer plant stocks from central China due to their longer growing quality and better tuber harvests. Here, we introduced a heat responsive receptor-like kinase ERECTA (ER) gene into P. ternata to explore changes in the growth cycle which were aimed at disrupting the summer dormancy. The 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene was also co-transformed with ER to improve the commercial trait. For the thermo-tolerance evaluation, all plants were treated with high temperatures (35°C/40°C) in a growth chamber or grown in natural field temperature in an isolated field before measurement of different agricultural, biochemical and physiological traits. The transgenics showed significantly (P < 0.05) higher heat tolerance, maintaining healthy vegetative growth unlike the empty vector (EV) harboring controls that became chlorotic and necrotic. Better performance in some of the monitored physiological traits was evident for overexpression lines exposed to the heat stress. In open isolated field trials, the transgenic genotypes did not show a summer dormancy but had a survival rate of 84-95%. The tuber biomasses were also significantly (P < 0.05) higher for the transgenic lines as compared to the EV controls, except for line ER118. Metabolites analysis indicated that the HMGR overexpressing lines (HMGR orHMGR + ER) exhibited significantly higher amounts of bioactive compounds including aromadendrene-4, 10-diol and 4, 8, 13-cyclotetradecatriene-1, 3-diol, 1, 5, 9-trimethyl-12-(1-methylethyl). Our findings show that the summer dormancy of P. ternata which is a naturally evolved trait, can be removed by a single heat responsive gene. The study contributes to generating heat tolerant new Pinellia varieties with enhanced commercially valuable chemicals.