SUB1A‐dependent and ‐independent mechanisms are involved in the flooding tolerance of wild rice species

Crop tolerance to flooding is an important agronomic trait. Although rice (Oryza sativa) is considered a flood‐tolerant crop, only limited cultivars display tolerance to prolonged submergence, which is largely attributed to the presence of the SUB1A gene. Wild Oryza species have the potential to unveil adaptive mechanisms and shed light on the basis of submergence tolerance traits. In this study, we screened 109 Oryza genotypes belonging to different rice genome groups for flooding tolerance. Oryza nivara and Oryza rufipogon accessions, belonging to the A‐genome group, together with Oryza sativa, showed a wide range of submergence responses, and the tolerance‐related SUB1A‐1 and the intolerance‐related SUB1A‐2 alleles were found in tolerant and sensitive accessions, respectively. Flooding‐tolerant accessions of Oryza rhizomatis and Oryza eichingeri, belonging to the C‐genome group, were also identified. Interestingly, SUB1A was absent in these species, which possess a SUB1 orthologue with high similarity to O. sativa SUB1C. The expression patterns of submergence‐induced genes in these rice genotypes indicated limited induction of anaerobic genes, with classical anaerobic proteins poorly induced in O. rhizomatis under submergence. The results indicated that SUB1A‐1 is not essential to confer submergence tolerance in the wild rice genotypes belonging to the C‐genome group, which show instead a SUB1A‐independent response to submergence.     

Differential metabolic regulation governed by the rice SUB1A gene during submergence stress and identification of alanylglycine by 1H NMR spectroscopy

Although the genetic mechanism of submergence survival for rice varieties containing the SUB1A gene has been elucidated, the downstream metabolic effects have not yet been evaluated. In this study, the metabolomes of Oryza sativa ssp. japonica cv. M202 and cv. M202(Sub1) were profiled using (1)H NMR spectroscopy to compare the metabolic effect of submergence stress and recovery on rice in the presence or absence of SUB1A. Significant changes were observed in the NMR resonances of compounds in pathways important for carbohydrate metabolism. The presence of SUB1A in M202(Sub1) was correlated with suppression of carbohydrate metabolism in shoot tissue, consistent with the role of SUB1A in limiting starch catabolism to fuel elongation growth. The absence of SUB1A in M202 was correlated with greater consumption of sucrose stores and accumulation of amino acids that are synthesized from glycolysis intermediates and pyruvate. Under submergence conditions, alanine, a product of pyruvate metabolism, showed the largest difference between the two varieties, but elevated levels of glutamine, glutamate, leucine, isoleucine, threonine, and valine were also higher in M202 compared with the M202(Sub1) variety. The identification and characterization of alanylglycine (AlaGly) in rice is also reported. After 3 days of submergence stress, AlaGly levels decreased significantly in both genotypes but did not recover within 1 day of desubmergence with the other metabolites evaluated. The influence of SUB1A on dynamic changes in the metabolome during complete submergence provides new insights into the functional roles of a single gene in invoking a quiescence strategy that helps stabilize crop production in submergence-prone fields.     

Rice with SUB1 QTL possesses greater initial leaf gas film thickness leading to delayed perception of submergence stress

Background and AimsSubmergence tolerance in rice is primarily attributed to the action of the SUB1 gene, but other associated traits such as leaf gas film (LGF) thickness, leaf hydrophobicity, porosity and leaf density have been known to aid submergence tolerance in rice. However, association of these traits with SUB1 quantitative trait locus (QTL) has not been demonstrated. In this study, we aim to investigate (1) whether the presence of the SUB1 QTL in the genetic background has any influence on the thickness of the LGF and (ii) whether its removal has any impact on stress perception and submergence tolerance in Sub1 and non-Sub1 rice.MethodsWe examined 12 genotypes (including both Sub1 and non-Sub1 types) for different leaf traits such as initial LGF thickness, leaf hydrophobicity, tissue porosity and leaf density in order to work out the relatioship of these traits to the SUB1 QTL in rice. Furthermore, we investigated the changes in the gene expression profile and different metabolic processes in selected genotypes in the presence and absence of their LGF to study its impact on stress perception and adaptation.Key ResultsThe initial thickness of the LGF and hydrophobicity seemed to have a highly positive correlation with the presence of the SUB1 QTL in the genetic background of rice; however, other leaf traits such as porosity and density seemed to be independent of it. Artificial removal of the LGF resulted in partial loss of tolerance, showing increased ethylene production and early induction of anoxia-related genes (SUB1A-1, ACS5, Ramy3D and ADH1) which manifested symptoms such as increased stem elongation, faster chlorophyll and starch breakdown, and partial loss of quiescence in SUB1-containing rice genotypes. Stripping of the LGF resulted in early and enhanced induction of SUB1A-1, indicating a quicker perception of stress.ConclusionsThe presence of SUB1 in the genetic background positively influences surface hydrophobicity and the concomitant LGF thickness of rice. Furthermore, LGF helps in terms of providing better ethylene dissipation and reduced in planta accumulation, owing to the slowing down of ethylene-induced leaf senescence under submergence stress.     

Ethylene influences in vitro regeneration frequency in the FR13A rice harbouring the SUB1A gene

Many studies have examined the effects of ethylene on in vitro plant growth and development, often with controversial results. Ethylene accumulates in culture vessels due to both the release from the tissues and the physical entrapment due to the need for closed containers. This hormone has several effects on plant regeneration, depending on the plant species and even the cultivar. A prerequisite for ethylene use for in vitro culture is thus to formulate a specific protocol for the genotype of interest. In rice, ethylene is a key regulator of adaptation strategies to low oxygen environments. In particular, the SUBMERGENCE1A (SUB1A) gene, when present, drives the acclimation response which when activated by ethylene produced by submerged plants leads to adaptation through reduced plant growth and ethanolic fermentation enhancement. This gene is restricted to a limited number of rice for which a very specific response to ethylene is expected, whatever the source. This paper reports the regeneration differences between a SUB1A rice landrace (indica-aus, FR13A) and a non-SUB1A variety (japonica, Nipponbare). Our results suggest that regeneration protocols with exogenous ethylene precursors supply are required for the FR13A rice harbouring the SUB1A gene to overcome the problem of low regeneration efficiency.     

The key regulator of submergence tolerance,SUB1A,promotes photosynthetic and metabolic recovery from submergence damage in rice leaves

The submergence‐tolerance regulator, SUBMERGENCE1A (SUB1A), of rice (Oryza sativa L.) modulates gene regulation, metabolism and elongation growth during submergence. Its benefits continue during desubmergence through protection from reactive oxygen species and dehydration, but there is limited understanding of SUB1A's role in physiological recovery from the stress. Here, we investigated the contribution of SUB1A to desubmergence recovery using the two near‐isogenic lines, submergence‐sensitive M202 and tolerant M202(Sub1). No visible damage was detected in the two genotypes after 3 d of submergence, but the sublethal stress differentially altered photosynthetic parameters and accumulation of energy reserves. Submergence inhibited photosystem II photochemistry and stimulated breakdown of protein and accumulation of several amino acids in both genotypes at similar levels. Upon desubmergence, however, more rapid return to homeostasis of these factors was observed in M202(Sub1). Submergence considerably restrained non‐photochemical quenching (NPQ) in M202, whereas the value was unaltered in M202(Sub1) during the stress. Upon reaeration, submerged plants encounter sudden exposure to higher light. A greater capability for NPQ‐mediated photoprotection can benefit the rapid recovery of photosynthetic performance and energy reserve metabolism in M202(Sub1). Our findings illuminate the significant role of SUB1A in active physiological recovery upon desubmergence, a component of enhanced tolerance to submergence.     

Molecular Events Underlying Coordinated Hormone Action in Submergence Escape Response of Deepwater Rice

Recent studies revealed that some rice varieties adopt opposite strategies to overcome flooding stress. While certain varieties hold metabolism and stay stunted until floodwater recedes, deepwater rice varieties undergo rapid stem elongation and do not suffer drowning problems. Both varieties use the same signaling agents, the ethylene response factors, as key factors even though they display opposite submergence responses. In deepwater rice, ethylene response factor genes SNORKEL1 and SNORKEL2 are believed to play a major role in submergence escape by mediating ethylene signaling, which leads to rapid stem elongation. These genes connect hormone signaling cascades from ethylene to ABA and gibberellins (GAs). Submergence increases ethylene levels in the internodal space, ethylene upregulates an ABA inactivating enzyme gene, OsCYP707A5 or OsABA8ox1, and some GA metabolism genes such as OsGA20ox genes and OsGA3ox genes. As a result of gene regulation by ethylene, internodal ABA levels decrease while GA levels increase, finally upregulating growth-related genes like expansin genes (OsEXPs). Along with the ethylene signaling in submergence, it is necessary to consider an alternative signaling pathway induced by hypoxia. Taken together, study on the submergence responses of rice plants will lead to improvement of crop production and contribution to basic research on plant growth.     

Mapping QTLs for submergence tolerance in rice using a population fixed for <Emphasis Type="Italic">SUB1A</Emphasis> tolerant allele

Submergence is a widespread problem of rice production, especially in low-lying areas in South and Southeast Asia. Despite the success of Sub1 mega varieties, repeated instances of prolonged and severe flooding in stress-prone areas suggests that the SUB1 gene is no longer sufficient in those regions and requires improved varieties with increased tolerance. A study was conducted to identify quantitative trait loci (QTLs) associated with submergence tolerance using 115 F₇ recombinant inbred lines (RILs) derived from the cross of Ciherang-Sub1, a popular Indonesian cultivar carrying the SUB1 gene that has relatively higher tolerance to submergence compared to the performance of most other Sub1 lines and the submergence and stagnant flooding tolerant IR10F365. As the tolerant allele at SUB1A on chromosome 9 was fixed in this mapping population, additional QTLs responsible for submergence tolerance were expected to be revealed. Genotyping with an Infinium 6K SNP chip resulted in 469 polymorphic markers that were then used for QTL mapping. Phenotyping was performed under complete submergence with two replicates. A major QTL for submergence derived from Ciherang-Sub1, named qSUB8.1, was detected on chromosome 8 with a LOD score of 10.3 and phenotypic variance of 27.5%. Additionally, a smaller QTL, also derived from Ciherang-Sub1, was detected on chromosome 2 with a LOD score of 3.5 and phenotypic variance of 12.7%. There was no digenic interaction detected between these QTLs suggesting their independent action. The QTLs detected in this study can be used in marker-assisted selection to further improve the tolerance of other Sub1 varieties.     

Epidermal cell death in rice is regulated by ethylene, gibberellin, and abscisic acid

Programmed cell death (PCD) of epidermal cells that cover adventitious root primordia in deepwater rice (Oryza sativa) is induced by submergence. Early suicide of epidermal cells may prevent injury to the growing root that emerges under flooding conditions. Induction of PCD is dependent on ethylene signaling and is further promoted by gibberellin (GA). Ethylene and GA act in a synergistic manner, indicating converging signaling pathways. Treatment of plants with GA alone did not promote PCD. Treatment with the GA biosynthesis inhibitor paclobutrazol resulted in increased PCD in response to ethylene and GA presumably due to an increased sensitivity of epidermal cells to GA. Abscisic acid (ABA) was shown to efficiently delay ethylene-induced as well as GA-promoted cell death. The results point to ethylene signaling as a target of ABA inhibition of PCD. Accumulation of ethylene and GA and a decreased ABA level in the rice internode thus favor induction of epidermal cell death and ensure that PCD is initiated as an early response that precedes adventitious root growth.     

GID1 modulates stomatal response and submergence tolerance involving abscisic acid and gibberellic acid signaling in rice

Plant responses to abiotic stresses are coordinated by arrays of growth and developmental programs.Gibberellic acid(CA) and abscisic acid(ABA) play critical roles in the developmental programs and environmental responses,respectively,through complex signaling and metabolism networks.However,crosstalk between the two phytohormones in stress responses remains largely unknown.In this study,we report that CIBBERELLIN-INSENSITIVE DWARF 1(GID1),a soluble receptor for GA,regulates stomatal development and patterning in rice(Oryza sativa L.).The gid1 mutant showed impaired biosynthesis of endogenous ABA under drought stress conditions,but it exhibited enhanced sensitivity to exogenous ABA.Scanning electron microscope and infrared thermal image analysis indicated an increase in the stomatal conductance in the gid1 mutant under drought conditions.Interestingly,the gid1 mutant had increased levels of chlorophyll and carbohydrates under submergence conditions,and showed enhanced reactive oxygen species(ROS)-scavenging ability and submergence tolerance compared with the wild-type.Further analyses suggested that the function of GID1 in submergence responses is partially dependent on ABA,and GA signaling by GID1 is involved in submergence tolerance by modulating carbohydrate consumption.Taken together,these findings suggest GID1 plays distinct roles in stomatal response and submergence tolerance through both the ABA and GA signaling pathways in rice.     

Overexpression of a rice diacylglycerol kinase gene OsBIDK1 enhances disease resistance in transgenic tobacco

A rice diacylglycerol kinase (DGK) gene, OsBIDK1, which encodes a 499-amino acid protein, was cloned and characterized. OsBIDK1 contains a conserved DGK domain, consisting of a diacylglycerol kinase catalytic subdomain and a diacylglycerol kinase accessory subdomain. Expression of OsBIDK1 in rice seedlings was induced by treatment with benzothiadiazole (BTH), a chemical activator of the plant defense response, and by infection with Magnaporthe grisea, causal agent of blast disease. In BTH-treated rice seedlings, expression of OsBIDK1 was induced earlier and at a higher level than in water-treated control seedlings after inoculation with M. grisea. Transgenic tobacco plants that constitutively express the OsBIDK1 gene were generated and disease resistance assays showed that overexpression of OsBIDK1 in transgenic tobacco plants resulted in enhanced resistance against infection by tobacco mosaic virus and Phytophthora parasitica var. nicotianae. These results suggest that OsBIDK1 may play a role in disease resistance responses.     

Examination of two lowland rice cultivars reveals that gibberellin-dependent early response to submergence is not necessarily mediated by ethylene

Using two lowland rice (Oryza sativa L.) cultivars we found that in both cases submerged-induced elongation early after germination depends on gibberellins (GAs). Submergence increases the content of the active GA₁ by enhancing the expression of GA biosynthesis genes, thus facilitating the seedlings to escape from the water and preventing asphyxiation. However, the two cultivars differ in their response to ethylene. The cultivar Senia (short), by contrast to cultivar Bomba (tall), does not elongate after ethylene application, and submerged-induced elongation is not negated by an inhibitor of ethylene perception. Also, while ethylene emanation in Senia is not altered by submergence, Bomba seedlings emanate more ethylene upon desubmergence, associated with enhanced expression of the ethylene biosynthesis gene OsACS5. The cultivar Senia thus allows the possibility of clarifying the role of ethylene and other factors as triggers of GA biosynthesis enhancement in rice seedlings under submergence.Key words: gibberellins, ethylene, submergence, lowland rice, Oryza sativa