Flood resilience loci SUBMERGENCE 1 and ANAEROBIC GERMINATION 1 interact in seedlings established underwater

Abstract

Crops with resilience to multiple climatic stresses are essential for increased yield stability. Here, we evaluate the interaction between two loci associated with flooding survival in rice (Oryza sativa L.). ANAEROBIC GERMINATION 1 (AG1), encoding trehalose 6-phosphate phosphatase 7 (TPP7), promotes mobilization of endosperm reserves to enhance the elongation of a hollow coleoptile in seeds that are seeded directly into shallow paddies. SUBMERGENCE 1 (SUB1), encoding the ethylene-responsive transcription factor SUB1A-1, confers tolerance to complete submergence by dampening carbohydrate catabolism, to enhance recovery upon desubmergence. Interactions between AG1/TPP7 and SUB1/SUB1A-1 were investigated under three flooding scenarios using four near-isogenic lines by surveying growth and survival. Pyramiding of the two loci does not negatively affect anaerobic germination or vegetative-stage submergence tolerance. However, the pyramided AG1 SUB1 genotype displays reduced survival when seeds are planted underwater and maintained under submergence for 16 d. To better understand the roles of TPP7 and SUB1A-1 and their interaction, temporal changes in carbohydrates and shoot transcriptomes were monitored in the four genotypes varying at the two loci at four developmental timeponts, from day 2 after seeding through day 14 of complete submergence. TPP7 enhances early coleoptile elongation, whereas SUB1A-1 promotes precocious photoautotrophy and then restricts underwater elongation. By contrast, pyramiding of the AG1 and SUB1 slows elongation growth, the transition to photoautotrophy, and survival. mRNA-sequencing highlights time-dependent and genotype-specific regulation of mRNAs associated with DNA repair, cell cycle, chromatin modification, plastid biogenesis, carbohydrate catabolism and transport, elongation growth, and other processes. These results suggest that interactions between AG1/TPP7 and SUB1/SUB1A-1 could impact seedling establishment if paddy depth is not effectively managed after direct seeding.

Keywords: AG1; Oryza sativa; SUB1; direct seeding; epistasis; pyramiding; submergence tolerance.

FIGURE 1

Pyramiding of AG1 and SUB1 in IR64 maintains anaerobic germination tolerance and vegetative‐stage submergence tolerance under respective submergence conditions. (a) AG1 enhanced plant survival under anaerobic germination in genetic backgrounds with or without SUB1. This assay was performed in the soil by dry seeding under ambient light and temperature in a nethouse. IR64, IR64(AG1), IR64(SUB1), IR64(AG1,SUB1), FR13A, IR42, and KHO were dry seeded in soil and submerged at 10 cm water depth maintained for 21 d. Survival was scored if the shoot emerged above the water surface by day 21. (b–d) A hollow coleoptile develops from seeds grown under water. IR64, IR64(AG1), and IR64(SUB1) seeds were germinated in air or underwater (H₂O) in complete darkness. After 4 d of growth, the length of the dissected coleoptile and plumule (leaf whorl surrounding the shoot apical meristem) were measured, and their was ratio calculated. Lower and upper whiskers indicate the minima and maxima, respectively; the box represents the interquartile range; the line in the box shows the median length from 3 independent biological replicates of 10 dissections. Circles indicate outliers. Dissected coleoptiles were observed with visible light at 5× magnification from underwater‐grown (b) and air‐grown (c) seeds. Plumules are outlined to enhance visualization. White bars indicate 1 mm. (e) SUB1 maintained seedling survival of vegetative‐stage submergence in IR64(AG1,SUB1). 14‐d‐old seedlings of the seven genotypes were completely submerged at 1.3–1.5 m depth and desubmerged after 42 d when the majority of the IR42 plants displayed leaf death. Survival was confirmed as new tiller and leaf growth after 21 d of recovery. The data in panel (a) and (e) represent mean ± SE of 3 biological replicates (n = 30 seedlings of each genotype per replicate). Genotypes, indicated as AG1, SUB1, and AG1 SUB1, are in the IR64 background. Letters indicate significant differences (p < 0.05, ANOVA with Tukey HSD test)

FIGURE 2

Germination, shoot growth, and survival of near‐isogenic lines, continually submerged up to 16 d, indicate AG1 and SUB1 interactions. IR64, IR64(AG1), IR64(SUB1), and IR64(AG1,SUB1) were dry seeded in soil and submerged completely with incremental addition of water for up to 16 d. (a) Percentage of germinated seed (coleoptile emergence ≥ 1 mm) at 4, 6, and 8 d of submergence. (b) Coleoptile/shoot length at 4, 6, 8, 10, 12, and 14 d of submergence. (c) Percentage of plants surviving 16 d of submergence, based on tiller and leaf growth, scored 7 d after desubmergence. Data represent mean ± SE of three biological replicates (n = 25–30 seedlings of each genotype per replicate). Letters represent significant differences (p < 0.05, ANOVA with Tukey HSD test)

FIGURE 3

Transcriptomic changes during germination and seedling establishment of genotypes grown under prolonged submergence. (a) MDS analysis with dimensions 1 and 2 of transcriptomes from four time points and four genotypes determined using mean log₂CPM values. (b) Differentially expressed gene (DEG; log₂ FC > |1|; FDR < 0.05) numbers based on genotypic comparisons of log₂CPM values over four submergence time points. (c) Cluster analyses (n = 10) of DEGs using mean log₂CPM values of genotypes over four submergence time points. Representative Gene Ontology (GO) categories reflect analyses of key clusters for the degree of AG1 and SUB1 loci regulation. Genes and corresponding log₂CPM values and GO terms in each cluster are in Dataset S1d, e

FIGURE 4

Genotypically controlled transcriptome changes during seedling establishment under prolonged submergence. (a) Clustering of DEGs identified in the genotypic comparisons (log₂ FC > |1|; FDR < 0.05) of IR64(AG1), IR64(SUB1), and IR64(AG1,SUB1) with IR64 for all submergence days combined. (b) PAM clustering of DEGs identified by day comparison of IR64(AG1), IR64(SUB1), and IR64(AG1,SUB1) relative to the IR64 2 d transcriptome during specified submergence days (Genotype × Day vs. IR64; GxD). Representative GO enrichment of notable clusters and genes are listed. Genotype interactions diagrammed in Figure S6. A comprehensive list of DEGs, log₂FC values, and GO term association are in Dataset S2

FIGURE 5

Overview of pathways involved in anaerobic germination and vegetative submergence influenced by AG1/TPP7 and SUB1/SUB1A‐1 respectively. The heatmap displays mean log₂CPM values (representing on a scale of 0 to 8) of genes in embryo‐coleoptile‐shoot tissue. Proteins are displayed in black boxes, and genes are italicized. The left panel shows the mobilization of seed reserves that fuel elongation growth during anaerobic germination. The right panel shows the suppression of elongation growth during vegetative submergence. During anaerobic germination, low oxygen and sugar starvation promote the upregulation of CALCINEURIN B‐LIKE INTERACTING PROTEIN KINASE 15 (CIPK15, LOC_Os11g02240) that promotes activation of the catalytic subunit of the energy‐sensing SUCROSE NON‐FERMENTING‐1‐RELATED PROTEIN KINASE 1 (SnRK1A, LOC_Os05g45420). SnRK1A activation enables the sugar starvation‐responsive MYBS1 (LOC_Os01g34060) transcription factor to enhance α‐AMYLASE transcription (i.e., AMY3D, LOC_Os08g36910; AMY3E, LOC_Os08g36900). During germination GA‐regulated MYB (MYBGA, LOC_Os01g59660) acts synergistically with MYBS1 to upregulate AMYs. These AMYs (and possibly BMY2, LOC_Os07g35940) hydrolyze endospermic starch into sugars that can be consumed in anaerobic ATP production within the embryo and developing seedling tissue. Two genes, PYRUVATE DECARBOXYLASE 1 (PDC1, LOC_Os05g39310) and ALCOHOL DEHYDROGENASE 1 (ADH1, LOC_Os11g10480) are vital during anaerobic respiration to produce ATP for growth and survival. This is accompanied by an elevation of EXPANSINs (i.e., EXPA1, LOC_Os04g15840; EXPB11, LOC_Os02g44108; EXPB7, LOC_Os03g01270, etc.). Trehalose‐6‐phosphate (T6P), a sensor of intracellular sucrose status, inhibits SnRK1A activation reducing catabolic metabolism. TPP7 (LOC_Os09g20390) catalyzes T6P conversion to trehalose, reducing the T6P to sucrose ratio to promote SnRK1A expression and coleoptile elongation. Submergence of vegetative‐stage plants entraps ethylene in shoot tissues, promoting the ethylene‐responsive transcription factor SUB1A‐1 to negatively regulate ethylene production. SUB1A‐1 stabilizes two gibberellin (GA) signaling repressors, GRAS domain transcription factors SLENDER RICE 1 (SLR1, LOC_Os03g49990) and SLR1‐LIKE 1 (SLRL1, LOC_Os01g45860) that reduce GA‐responsiveness. Our data indicate that SUB1A‐1 may also influence the upregulation of SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTERs (i.e., SWEET1B, LOC_Os05g35140; SWEET14, LOC_Os11g31190) sugar transporters (i.e., MST2, LOC_Os03g39710; MST4, LOC_Os03g11900) gene family members. Our data further indicate that in seedlings developing underwater SUB1A‐1 directly or indirectly promotes early activation of genes associated with chloroplast development (i.e., type I chlorophyll a/b‐binding protein CAB, LOC_Os01g52240), enzymes involved in chlorophyll biosynthesis (i.e., PORB, LOC_Os10g35370) and a transcription factor associated with chloroplast biogenesis (GOLDEN2‐LIKE 1, GLK1, LOC_Os06g24070). Genes involved in leaf cuticular wax biosynthesis, including hydroxysteroid dehydrogenase (LGF1, LOC_Os11g30560) and deposition (ATP binding cassette transporter RCN1, LOC_Os03g17350), are also upregulated early by SUB1A‐1