Genetic mapping and molecular characterisation of the candidate gene responsible for root aerenchyma formation under waterlogging stress in barley

Cen Tong

Waterlogging is when soil is saturated with water due to heavy rain or inefficient drainage, which decreases the oxygen available to plant roots and significantly inhibits plant growth. Approximately 10-12% of the arable land worldwide is affected by waterlogging, leading to substantial grain yield losses. The impact of waterlogging on agricultural production was predicted to rise significantly in the context of climate change, which causes more frequent extreme weather worldwide. Plants, including important crop species, have developed various mechanisms to tolerate waterlogging stress, such as developing adventitious roots, root aerenchyma formation, energy metabolism alterations, and phytohormone signalling changes. Identification and molecular characterisation of their genetic mechanisms are significant for successfully breeding waterlogging-tolerant crop varieties to reduce crop yield losses. Domesticated barley (Hordeum vulgare L.), the fourth most important cereal crop, is considered moderately susceptible to waterlogging stress compared to other crops. However, significant phenotypic variations in plant biomass, photosynthesis rate, adventitious roots development, and aerenchyma formation under waterlogging stress have been observed across diverse barley genotypes, which offer promising opportunities for the selection and genetic characterisation of waterlogging-tolerant barley varieties. Among the waterlogging-adaptive phenotypic traits, adventitious root growth and aerenchyma formation were identified as the major adaptive traits adopted by barley to address waterlogging stress. This study aimed to identify and functionally characterise a candidate gene controlling aerenchyma formation in adventitious roots under waterlogging conditions so that future research could utilize genetic engineering for waterlogging-tolerant barley cultivars development. An F2 population derived from crossing a Chinese waterlogging-tolerant barley line YYXT and a waterlogging-sensitive Australian variety Franklin was used to pinpoint the genetic locus controlling waterlogging tolerance. The F2 population displayed a clear phenotype segregation in root aerenchyma formation. A customised phenotyping method was developed to assess the root aerenchyma formation. Based on the genotypic data of two molecular markers of 4H598.473 and 4H598.628, a genetic locus associated with aerenchyma formation was finemapped to an interval of 0.15 Mb on chromosome 4H, which contains two annotated genes based on the reference genome (cv. Morex v3). Expression of the candidate genes in the root tissues of YYXT, Franklin, and Yerong, another waterlogging-tolerant line, was measured by RT-qPCR under control and waterlogging treatments. One gene encoding a basic Helix-Loop- Helix (bHLH) transcript factor (HvbHLH_4H) was significantly upregulated in tolerant and susceptible varieties under waterlogging conditions. It displayed relatively higher transcriptional and fold changes in the tolerant genotypes YYXT and Yerong than in the sensitive genotype Franklin. The function of HvbHLH_4H was validated through virusinduced gene silence (VIGS). The inoculated plants formed less aerenchyma than the control. In addition, Sanger sequencing identified a 1-bp deletion located in exon 2 of HvbHLH_4H in the susceptible variety Franklin, resulting in a truncated protein caused by an open reading frame shift. Protein structure modelling predicted by AlphaFold2 of HvbHLH_4H revealed a significant difference between tolerant and susceptible varieties. Thus, gene expression analyses and functional validation indicated that HvbHLH_4H was likely the candidate gene for aerenchyma formation and waterlogging tolerance. RNA sequencing (RNA-seq) was performed using two sister lines (P16A4_FF, P16A4_FY) with different aerenchyma phenotypes. More DEGs between control and waterlogging treatment were identified for the tolerant line (P16A4_FY) than for the susceptible line (P16A4_FF) at three and seven days after the initiation of waterlogging treatment. Gene Ontology (GO) enrichment of DEGs revealed “response to decreased oxygen level” as the main enriched functional term. Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses highlighted that “phenylpropanoid biosynthesis” and “plant hormone signal transduction” were the most enriched terms induced by waterlogging stress. Weighted gene coexpression network analyses (WGCNA) of transcriptional profiles in adventitious root successfully identified the genetic network centred on HvbHLH_4H, covering several genes related to cell wall modification (nuclease S1, polygalacturonase, expansin protein, pectin lyase-like superfamily protein), supporting its potential involvement in waterlogging stress. In addition, the beta-glucan catabolic process and pentose and glucuronate interconversions were also enriched in the HvbHLH_4H-centered network. DNA Affinity Purification sequencing (DAP-seq) analyses revealed HvbHLH_4H-binding peaks in putative promoter regions. Overlapping these genes with the above-identified DEGs of RNA-seq and the co-expressed gene module of WGCNA identified several downstream genes potentially regulated by HvbHLH_4H. GO function analyses highlighted several genes with predicted function in cell wall metabolism and development. Therefore, it can be hypothesised that HvbHLH_4H regulates the aerenchyma formation through cell wall remodelling. Further, a comprehensive bHLH gene family analyses was performed on 20 barley genomes to characterise the evolutionary origin and genetic variation of HvbHLH_4H. Two hundred and one bHLH orthologous gene groups (OGGs) were classified into 141 core/near-core and 60 dispensable bHLH. The dispensable bHLH OGGs displayed a relaxed selection pressure than the core/nearcore OGGs. The expansion of bHLH is biased toward specific OGGs. HvbHLH_4H was identified as core bHLH with no copy number variation. This study is the first to identify a candidate gene HvbHLH_4H controlling root aerenchyma formation in barley. VIGS tentatively supports HvbHLH_4H’s involvement in the waterlogging response. A 1-bp deletion was identified in the sensitive variety, leading to a truncated HvbHLH_4H protein and potentially a variation in aerenchyma formation. Comprehensive transcriptome and DAP-seq analyses indicated that aerenchyma formation may result from cell wall modification through the beta-glucan metabolic process. These data together provided novel genetic and molecular insights into waterlogging tolerance in barley. They lay the foundation for future selection and breeding of waterlogging-tolerant barley varieties in the face of the unprecedented challenge of climate change.

Genetic mapping and molecular characterisation of the candidate gene responsible for root aerenchyma formation under waterlogging stress in barley

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