Under limited oxygen concentration, mitochondrial respiration is inversely proportional to glycosylate metabolism, which produces NADH. Following entry into mitochondria, NADH undergoes re-oxidation at the start of fermentative pathways. The lactate and toxic substrate ethanol produced with a minimum gain of ATP is a bottleneck for germination under water
[22][36]. Lactate, a substrate of lactate dehydrogenase (LDH), is typically toxic to the aleurone membrane in inducing amylase activities by GA, whereas PCD produces ethanol along with the activity of ADH. Seeds may be tolerant to anoxia by reverting the central glycolytic pathways. Ethanol may either be oxidized to acetyl dehydrogenase or diffused out of the seed coat, accumulating within spaces of grains and glumes
[23][37]. This also induces dormancy in terms of ecological consideration for submergence tolerance. The minimum amount of ATP (2 moles) over the normal tricarboxylic acid cycle (32 moles) is a limitation for anaerobic respiration
[24][38]. Therefore, a specific set of protein expression and their corresponding regulation are selection criteria for the better germination of seeds in the anaerobic mode. Distinct proteins like sucrose-phosphate synthase, PDC, and ADH are the most important in accessing breeding programs where submergence induces anoxia
[25][39]. Carbohydrate metabolism in rice seeds under prolonged anoxic conditions creates the most effective screening index. There are specific modalities of the regulation of genes on the anaerobic response elements upstream of the promoters, characterizing the coding of anaerobic proteins
[26][40]. These, in turn, become the factors to control the paths of fermentation for specific genes. From the sequence alignment,
cis-elements also bear the homology for anoxia-inducible genes in plants, similar to bacteria. Rice plants tolerant to anoxia can accommodate ATPs; however, for a long time, this has occurred via fermentative catabolism, as long as the hypoxia is maintained without affecting the embryo tissues.
6. Molecular Regulation of Major Glycolytic Flux: Amylase Activity with GA Induction
Major glycolytic flux is based on the availability of soluble sugars, which determines the rate of germination and seed growth in rice under submergence. The regulation of α-amylase expression by GA is a major control for soluble sugars as a chief fuel for glycolysis
[27][47]. Hypoxia-induced coleoptile growth is thus matched with GA biosynthesis, catabolism, and the regulation of amylase activity
[28][48]. The expression of the specific α amylase gene is also under activation when embryos deplete sugars from endosperm. This was well established from the expression of α amylase in rice embryos, as derived from suspension cells in aleurone, which becomes a major source of α amylases. Under two sugar-depleted culture sets of genes,
α-Amy3 and
α-Amy8 [29][49] are major amylases. This is responsible for starch breakdown below the threshold level for sugar content, which supports the germination of embryos. The sugar repression of
AM3 and
AM8 is based on the regulation of the transcription rate, as well as the stability of transcripts. Typical TA box (5′-TATCCA-3′) is the main sugar response element in rice embryos
[30][50].
7. Constraints and Remediation of Pre-Harvest Sprouting
Under different environmental stresses, the production of cereal seeds is physiologically affected by grain germination on plants, called pre-harvest sprouting. It results in a significant loss of yield, reduced grain quality, and other physiological traits in a wide coverage of cereals including maize, barley, wheat, rye, oats, jowar, etc.
[31][53]. Pre-harvest sprouting is favored with suitable temperature and moisture for seed germination. This is also associated with the energy exhausted by grains and the breakdown of high-energy-producing starch and lipids. The energy released by metabolites may allow shoots to expand under an environmental condition that supports sprouting
[32][54]. Moreover, genetic and environmental factors independently or by interactive means may influence pre-harvest sprouting. Specific varieties have been investigated for sprouting resistance that minimize the loss of yield and grain quality. A selection procedure for the characteristics that aid sprouting resistance from a rice data base has already been identified.
8. Metabolomic Approaches for Hypoxia Tolerance in Rice
In the recent past, a detailed comparative multi-omics analysis suggested the involvement of different pathways to tolerate hypoxia for embryos and coleoptiles. Rice seeds, with their in-built tolerance to grow under oxygen deficiency, lead to a conversion to pyruvate following alcohol. From metabolomic studies, a complete set of enzymes for starch mobilization under hypoxic stress remains active
[33][58]. Agronomically, this tolerance demonstrates rice as being a crop for direct seedlings in cultivation. The cell-wall-specific high expression of wall-modifying enzymes like trans-glycosylase as well as proteins for coleoptile growth are important
[34][59]. The turnover of glycolytic pathways induces a higher survival of hypersensitive rice seeds to hypoxia than other intolerant ones.
9. Regulation of Transcripts in Seed Germination under Submergence
As already reported, rice seeds favor some sort of anoxia which is moderated by the adoption of a quiescence strategy. A number of bio-metabolites and hormonal influences are pre-requisites for quiescence strategy under the regulation of some genes from the whole transcriptome
[35][65]. These genes include the enzyme system for the fermentative mechanism, where key enzymes for glycolysis are pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), etc.
[36][66]. At the metabolome level, alcoholic fermentation can provide adequate energy for seed germination, coleoptile growth, and ATP synthesis to facilitate the quiescence mechanism. In alcoholic fermentation, pyruvate is catalyzed into acetaldehyde by PDC. This is followed by ADH and other constitutive genes responsible for alcoholic fermentation
[37][67]. ADH becomes a key gene for submergence tolerance, which is regulated in a feedback manner with
slender rice 1 (SLR1) and
slender rice like 1 (SLRL1). However, for quiescence strategies, ADH is inhibited or less active for the
adh1 mutant associated with a decrease in the NADP
+/NADPH ratio
[38][68]. In this mutant, coleoptile growth and its elongation are hindered in the molecular regulation of anaerobic genes.
10. Biochemical Implications of Anoxic Seed Germination
Rice seeds, due to their anaerobic germination, show two distinct modalities for biotechnology implications, particularly in anaerobic conditions. Primarily, they have minimum or basal oxygen requirements for seed germination for dormancy-related pathways. Secondarily, the use of specific devices to maintain minimum oxygen tension and its diffusion for developing embryos is important
[39][71]. A number of chemical elicitors to influence the metabolic expression from developing embryos have been well addressed. Seed-coat-residing phenolics and their oxidation into other residues are related with the removal of dormancy under anoxic conditions. For aerobic rice, where oxygen concentration is not a constraint, seed coat phenolic residues are also affected. In some cases, established physiological and biochemical pathways are clear, but the signaling mechanism for the soil moisture deficit, ROS, and growth regulators is not explained. Therefore, the biotechnological implication of anaerobic seed germination requires a consolidated background with plant physiology, biochemistry, ecology, and cellular and molecular biology of seeds in the plasticity of environmental constraints.
11. Conclusions
Rapid seed germination and coleoptile elongation are primary traits of anoxia and hypoxia tolerance. Seeds can overcome anerobic stress when oxidative phosphorylation can provide adequate ATPs. These are required for energy conservation when seeds are to sustain their viability by arresting its growth. This is physiologically manifested into quiescence strategies, where growth is regulated under submergence. Sugar and amino acid metabolism were features of hypoxia-tolerant cultivars that support tissue development in coleoptiles. Multi-omics research suggests that genes functioning in a differential manner for potential transcript(s) may provide tolerance. These genes are genetic resources for the mobilization of nutrients under oxygen deficits in seeds. From existing land races of rice tolerant to submergence, significant functional gene(s) are recovered using the reference genome of FR13A, etc. Therefore, hypoxic germination shares the same background of gene pools concerning oxidative redox, and these must be employed in breeding programs for direct seeding in rice.