## Sub1 Locus and Submergence Tolerance in Rice: Study Notes This document summarizes the key findings regarding the Sub1 locus and its role in conferring submergence tolerance in rice. ### The Sub1 Locus: A Cluster of ERF Genes The Sub1 locus on rice chromosome 9 contains a cluster of two to three genes (Sub1A, Sub1B, and Sub1C). These genes encode proteins with a DNA-binding domain common to ethylene response factors (ERFs), also known as ethylene-responsive element-binding proteins or Apetala2-like proteins. Remember that ERFs are transcription factors that regulate gene expression in response to ethylene. All three Sub1 genes belong to the B-2 subclass of ERF proteins, characterized by a single 58- to 59-residue ERF domain. They interact with GC-rich cis-acting sequences of target genes and regulate ethylene-mediated responses. Cis-acting sequences are DNA sequences that regulate the expression of nearby genes. * Sub1B and Sub1C are found in a wide range of indica and japonica varieties. * Sub1A is limited to a subset of indica varieties. A single amino acid substitution distinguishes the Sub1A-1 allele of tolerant indica varieties (like FR13A) from intolerant varieties. Don't confuse Sub1A, Sub1B, and Sub1C. They are distinct genes within the Sub1 locus. Understanding the different Sub1 alleles is crucial for understanding submergence tolerance. Sub1A alleles in tolerant varieties are highly induced under submergence, unlike those in intolerant varieties. Sub1A is absent from all japonica germplasms studied and some indica germplasms, all of which are submergence-intolerant. Transformation of the intolerant japonica variety Liaogeng with a constitutively expressed Sub1A gene significantly increased submergence tolerance. ### Ethylene's Role in Submergence Response Submergence promotes the biosynthesis of 1-aminocyclopropane-1-carboxylic acid (ACC), which is converted to ethylene in an oxygen-dependent manner. Ethylene plays crucial roles in acclimative responses to submergence and flooding: * Contributes to programmed cell death leading to aerenchyma and adventitious root formation under hypoxia. * Promotes internode and petiole elongation, allowing deepwater rice to extend photosynthetic organs above the water surface. This elongation involves increased accumulation of expansin transcripts, mediating cell wall loosening. In *Rumex palustris*, Expansin A transcripts increased after submergence and ethylene treatment; this induction was suppressed by an ethylene action inhibitor, 1-methylcyclopropene. * Triggers an increase in endogenous gibberellic acid (GA) content and sensitivity, leading to internode and petiole elongation. * Contributes to a decrease in endogenous abscisic acid (ABA) content, a prerequisite for petiole elongation. ### Comparing Submergence Responses in Near-Isogenic Lines The study compared near-isogenic japonica lines differing in Sub1 haplotype. M202 (japonica) lacks Sub1A and possesses Sub1B and Sub1C, while M202(Sub1) contains all three Sub1 genes of indica origin. M202 serves as a control, lacking the Sub1A gene associated with submergence tolerance. M202(Sub1) is genetically modified to include this gene. #### Submergence Stress Studies 14-day-old plants were submerged for up to 14 days. Leaf tissue was collected at various time points for analysis. Semiquantitative RT-PCR confirmed the absence of Sub1A transcript in M202 leaves. Sub1A mRNA increased rapidly in M202(Sub1) leaves after 1 day of submergence and remained elevated. Sub1B transcript was detected in both genotypes and slightly upregulated by stress, with higher levels consistently detected in M202(Sub1). Sub1C transcript increased dramatically in both, but was higher in M202 compared to M202(Sub1). #### Submergence Recovery and Chlorophyll Maintenance M202(Sub1) showed significantly better recovery and chlorophyll maintenance after submergence compared to M202. M202 leaves were more prostrate, spindly, and chlorotic. M202(Sub1) maintained significantly higher chlorophyll levels from day 6 onwards. #### Differential Regulation of Elongation M202 showed significantly greater elongation than M202(Sub1) under submergence. The Sub1 haplotype suppresses elongation during submergence but does not influence plant height under normal conditions. #### Carbohydrate Consumption M202 leaves experienced more rapid and prolonged starch starvation during submergence than M202(Sub1). The Sub1 haplotype controls the regulation of α-amylase and sucrose synthase transcript levels, affecting carbohydrate catabolism. #### Ethanolic Fermentation The Sub1 haplotype influences the magnitude but not the temporal pattern of PDC and ADH activities during submergence. M202(Sub1) showed higher ADH activity induction but not necessarily higher ethanol content. #### Ethylene and GA Responsiveness The Sub1 haplotype influences ethylene and GA responsiveness of genes associated with submergence acclimation. Sub1A mRNA was dramatically increased in M202(Sub1) in response to ethylene. Ethylene treatment did not fully recapitulate the genotype-specific increases in Sus transcript levels observed under submergence. GA treatment induced Sub1C mRNA accumulation, particularly in M202. ### Key Takeaways The Sub1 haplotype plays a crucial role in regulating various acclimative responses to submergence in rice. The presence of Sub1A-1 is associated with enhanced submergence tolerance through the regulation of carbohydrate metabolism, cell elongation, and ethanolic fermentation. This regulation is mediated, at least in part, by the differential responses to ethylene and GA. The Sub1 locus represents a significant genetic resource for improving submergence tolerance in rice cultivation.