We focused on unravelling gene networks, mechanisms and pathways associated with metribuzin tolerance in hexaploid wheat using a unique top-to-bottom three tiered strategy. In the first tier, metribuzin effects were investigated in 946 wheat germplasms (Australian winter wheat collection) from different regions of the world [7]. Our metribuzin tolerance screening identified promising contrasting genotypes. Identification of the most contrasting genotypes is a pre-requisite for better resolution and deeper insight into genes and mechanisms involved in herbicide tolerance. Therefore, in the second tier, a detailed dose-response experiment and field screening were conducted using potential contrasting genotypes to identify the most contrasting genotypes. Chuan Mai 25 and Ritchie were the most contrasting genotypes for metribuzin tolerance when compared with the present known sources. Discovery of the most contrasting genotypes lays a strong foundation for genetic and genomic studies to assist in the development of herbicide-tolerant cultivars with a wide safety margin (Fig. 5). The third tier focused on transcriptome sequencing of Chuan Mai 25 and Ritchie using the Illumina NovaSeq6000 platform. The DEGs identified gene networks, pathways/metabolic enzymes and mechanism(s) contributing to metribuzin tolerance in wheat.
Mechanism(s) for metribuzin tolerance in wheat
Metribuzin stress limits CO2 fixation and over‐reduction of the electron transport chain resulting in ROS [8]. Herbicides generate an abiotic stress that produces ROS, such as O2·−, H2O2, 1O2, OH·, which are extremely toxic and trigger membrane lipid peroxidation and rapid destruction of cellular constituents, resulting in oxidative stress and cell injury or death [9]. Metribuzin is a potent PSII inhibitor. It binds the target site D1 protein in PSII and inhibits electron flow between the primary electron acceptor and plastoquinone. This leads to selective and specific cleavage of the D1 protein. The D1 protein turnover cause the breakdown of PSII, reducing photosynthetic electron transport chain, which produce superoxide radicals and singlet oxygen in the chloroplasts [10-12]. This limits the generation of the energy currencies of cells, ATP and NADPH, inhibiting CO2 fixation in the Calvin cycle.
The present study suggests that metribuzin tolerance in wheat is metabolism-based (Fig. 6). Two major metabolic pathways—glycolysis and pentose phosphate pathway—are over-regulated in response to early metribuzin stress in tolerant wheat. The co-ordinated interplay between these metabolic pathways increases—the influx of energy (ATP), reducing powers [reduced nicotinamide adenine dinucleotide (NADH), NADPH and flavin adenine dinucleotide (FADH2)], and intermediates for biosynthetic and metabolic detoxification processes [13, 14] (Additional file 1: Table S7) are essential for supporting the antioxidant system and preventing oxidative damage to DNA, proteins and lipids [15].
Early genes regulated in response to metribuzin stress (24 h HE) (Fig. 6) belong to carbon metabolism, fructose and mannose metabolism, homologous recombination, amino acid biosynthesis, pyrimidine metabolism, galactose metabolism and amino sugar and nucleotide sugar metabolism. Metabolites such as fructose and mannose are synthesised to protect membranes and proteins from oxidative stress by ROS. Genes involved in homologous recombination are significantly enriched to repair harmful breaks in DNA and restore the essential molecular function in cells [16, 17]. Galactose is involved in glucose synthesis, and pyrimidines serves the role of ATP for glucose synthesis (Zrenner et al., 2006)[18], promoting nutrient remobilisation and preventing senescence.
Increased exposure to metribuzin (60 h) caused over-expression of photosynthetic and metabolic enzymes, antenna proteins (Lhc a/b-binding proteins), PSII stability/assembly factor HCF136, and glutathione/ascorbic acid (Fig. 6). Photosynthetic enzymes and antenna proteins are involved in carbon fixation and glucose synthesis catalysed by Rubisco (Additional file 1: Table S6). Glutathione metabolism removes free radicals and prevents oxidative damage to DNA, proteins and lipids. Ascorbic acid (antioxidant) functions as a cofactor for enzymes in photosynthesis, and the synthesis of plant hormones [19] and affects gene expression and transcription, cell division, and growth [20].
Enzymatic and non-enzymatic components for ROS detoxification
The DEG analysis suggested that metribuzin tolerance is wheat is metabolism-based involving over-expression of several ROS‐scavenging enzymes such as superoxide dismutase, catalase, glutathione S-transferase (GSTs), glutathione peroxidase, cytochrome P450 (CYPs), cytochrome reductase, cytochrome peroxidase, oxidoreductase, ABC transporters, glycosyltransferase (GT), UDP-galactosyltransferase and ubiquitin transferase to prevent oxidative stress during herbicide stress in the tolerant wheat genotype, Chuan Mai 25. Some of the herbicide is detoxified before it reaches target site. CYPs add a reactive group such as hydroxyl, carboxyl, or an amino group through oxidation to herbicide molecule, making it a polar molecule (phase I detoxification) and transferases (phase II detoxification enzymes) conjugates the addition of water-soluble group to the reactive site of polar molecule. The identified gene superfamilies or domains are essentially xenobiotic detoxifying enzymes involved in vacuolar sequestration of conjugated herbicide metabolites. The non-enzymatic components/phytohormones such as ascorbic acid and glutathione (GSH) have ROS scavenging function and plays a protective role during metribuzin stress. GSH function with GSTs to detoxify herbicides by tagging electrophilic compounds for removal during oxidative stress [21, 22].
Overexpression of ROS‐responsive regulatory genes (Additional file 1: Table S6, S7), which regulate a large set of genes involved in acclimation mechanisms, is a powerful strategy for enhancing herbicide tolerance in wheat. The ability of wheat genotypes to metabolize herbicides are largely dependent on the genetic expression of these enzymes. Difference in metribuzin tolerance expression is a result of genetic polymorphisms resulting in an altered expression. This is confirmed by SNP discovery in metribuzin-tolerant and –susceptible wheat groups using 90K iSelect SNP genotyping assay. The polymorphic SNP loci between the two groups detected genes on chromosomes (2A, 2D, 3B, 4A, 4B, 7A, 7B, 7D) encoding metabolic detoxification enzymes (cytochrome P450, glutathione S-transferase, glycosyltransferase, ATP-binding cassette transporters and glutathione peroxidase) [23]. We have mapped QTLs for metribuzin tolerance in wheat. The genes underlying the QTL support range on chromosomes-1AS (oxidoreductase), 2DS (glycosyltransferase), 4AL (transferase activity) are involved in metabolic detoxification. The integration of present transcriptomic analyses, previous metribuzin-tolerant QTL mapping [6], and SNP discovery using 90K iSelect SNP genotyping assay in metribuzin-tolerant and -susceptible wheat genotypes [7] suggests that enzymatic components play a significant role in modulating ROS homeostasis and the acclimation response of wheat to metribuzin tolerance.
Over-Expression of Lhc a/b-binding proteins and PSII stability/assembly factor HCF136 confers metribuzin tolerance in wheat
PSII functions as a water-plastoquinone oxidoreductase in oxygenic photosynthesis. The redox components, required for PSII function are localised on the heterodimer of the Dl and D2 proteins of the PSII reaction centre (Fig. 4). Lhc a/b-binding proteins are typically complexed with chlorophyll and xanthophylls and serve as the antenna complex, which regulate the distribution of excitation energy between PSII and PSI [24]. Regulation of Lhc a/b-binding proteins is an important mechanism in plants to modulate chloroplast functions [25, 26]. This study suggests that over-expression of Lhc a/b binding proteins in metribuzin tolerant wheat (Chuan Mai 25), promotes carbon fixation and modulates ROS homeostasis during metribuzin stress.
HCF136—the thylakoid-embedded large pigment-protein complexes of the photosynthetic electron transfer chain—is involved in the assembly of PSII reaction centre complexes, de novo synthesis of the D1 protein and the selective replacement of damaged D1 protein during PSII repair [27]. Lower expression of HCF136 in susceptible Ritchie during metribuzin stress resulted in the accumulation of damaged PSII proteins, which increased oxidative stress. Photosynthesis cease when degradation and PSII repair do not balance under herbicide stress. This implies that in susceptible wheat, a reduction in fundamental processes such as photosynthesis produce oxidative stress in chloroplast, which extends beyond PSII to cause a down-regulation of total carbon gain and imbalance between the rate of photo-damage to PSII and the rate of the repair of damaged PSII, reducing plant yield in susceptible genotypes. 90K iSelect SNP genotyping assay in our previous investigation detected polymorphism between tolerant and susceptible wheat genotypes in the gene encoding PSII assembly factor involved in PSII repair [23]. This suggests that metribuzin-tolerant wheat genotypes have inherently high photosynthetic efficiency.
Transcription factors
Biotic and abiotic stresses trigger a wide range of plant responses, from the alteration of gene expression and cellular metabolism to changes in plant growth and development. The TFs play critical roles in regulating stress responses in plants. The present study suggests that TFs belonging to the MYB, AP2-EREBP, ABI3VP1, bHLH, NAC, FAR1, mTERF, WRKY families are over-expressed to enrich ROS scavenging activity and photosynthetic genes during metribuzin stress in tolerant wheat. The MYB and NAC genes are the largest families of plant-specific TFs that play important roles in the regulation of the transcriptional reprogramming of plant stress responses. Genetic and molecular studies using knockout/knockdown mutants and overexpression in model plants and crop plants have demonstrated that TFs belonging to the MYB, NAC [28-30], AP2/EREBP [31], WRKY [32, 33], and bHLH families play important roles in plant responses to abiotic and biotic stresses [34].
Herbicide-tolerant wheats
The EST-based SSR markers identified in significantly enriched genes relating to photosynthetic and metabolic detoxification enzymes with present-absent variation (PAV), with significant differential expression will be a great resource for metribuzin tolerance breeding. The PAV is a sequence in one genome, but entirely missing in another genome. This is an important source of genetic diversity in plants [35, 36]. We propose the use of functional specific markers for a desired traits which reduces genotype-phenotype gaps in crop plants to maximize genetic gains in breeding. High-throughput identification of PAV on a whole-genome level has become possible with the advent of next-generation sequencing (NGS) technologies, at affordable prices [37]. There is a rapidly rising trends in the application of genome editing based crop improvement using CRISPR/Cas genome engineering system [38, 39]. The improved understanding of genetic and genomic knowledge of herbicide tolerance will open up the utilities for inducing multiple cleavage events, controlling gene expression, and site specific transgene insertion.
In conclusion, the use of improved metribuzin-tolerant wheats will help farmers to (1) minimise the early cohorts of problematic weeds, removing early wheat and weed competition and increasing wheat productivity, and (2) promote crop rotations with other herbicide-tolerant crops, such as narrow-leafed lupin (Lupinus angustifolius L.) and canola (Brassica napus L.) to assist in sustainable farming systems.