Recent reports on responses to flooding, submergence, and low-oxygen stress have connected components in an essential regulatory network that underlies plasticity in growth and metabolism essential for the survival of distinct flooding regimes. Here, we discuss growth under severe oxygen-limited conditions (anaerobic growth) and less oxygen-deficient underwater conditions (ethylene-driven underwater growth). Low-oxygen stress causes an energy and carbohydrate crisis that must be controlled through regulated consumption of carbohydrates and energy reserves. In rice (Oryza sativa L.), lowoxygen stress, energy homeostasis and growth are connected by a calcineurin B-like interacting binding kinase (CIPK) in seeds germinated under water. In shoots, two opposing adaptive strategies to submergence, elongation (escape) and inhibition of elongation (quiescence), are controlled by related ethylene response factor (ERF) DNA binding proteins that act downstream of ethylene and modulate gibberellin-mediated shoot growth. Increased resolution of the flooding signaling network will require more precise investigation of the interactions between oxygen tension and cellular energy status in regulation of anaerobic metabolism and ethylene-driven growth, both essential to survival in variable flooding environments. Introduction Global warming is associated with increased flooding events adversarial to most plant species and thus affects both crop yield and plant distribution in natural ecosystems [1]. There is an urgent need to increase crop production, particularly rice, in flood-prone regions. Major steps toward breeding tolerant varieties have been made through the characterization of two multigenic loci that control the capacity to endure complete submergence (SUBMERGENCE 1, SUB1) or rapid outgrowth of adverse partial submergence (SNORKEL, SK) [2 ,3 , 4 ,5 ]. New SUB1 rice varieties, produced by markerassisted breeding, are high yielding even following two weeks of complete submergence [6 ,7]. Similarly, better yields of deepwater rice are anticipated, with other improvements to follow [8]. For example, rice and some other species have the remarkable capacity to germinate and elongate coleoptiles and stems under severe oxygen constraints [9,10] (i.e. under anoxia, See glossary). The recognition of rice CIPK15 as a regulator of underwater germination and early shoot elongation [11 ] provides a critical link between sugar sensing, starch utilization and coleoptile elongation under anoxia. The identification CIPK15 and other genes that regulate underwater seed establishment may enable breeding to reduce the practice of seedling transplantation and overall herbicide use. These findings in rice complement data from other genera (i.e. Arabidopsis, Lotus, Poplar, Potamogeton, and Rumex) suggesting there exists a conserved flooding response network in plants that includes ethylene-triggered alterations in gene expression leading to growth and stress-induced catabolism of stored or soluble carbohydrates for energy-efficient production and utilization of ATP. A key challenge is to decipher the interplay between hormones (i.e. ethylene, abscisic acid (ABA), and gibberellic acids (GA)), oxygen availability, and specific metabolites (i.e. ATP, sugars, and pyruvate) that drives a dynamic network balancing growth and quiescence to facilitate survival Ethylene-controlled growth Recently, a quantitative trait locus was discovered encoding two genes that trigger rapid internode elongation in rice varieties when cultivated under partially submerged 'deepwater' conditions. These genes, SNORKEL1 (SK1) and SNORKEL2 (SK2) encode nuclear-localized DNA binding proteins with a single APETALA2/ethylene response factor (ERF) domain [2 ,12]. The SKs are absent in non-deepwater varieties, including all japonicas; however when backcrossed into a japonica, 30% of the internode elongation capacity of the deepwater variety was transferred. Moreover, constitutive overexpression of SK1 or SK2 in a japonica increases the number of elongated internodes, even under non-submerged The presence or absence of submergence-induced shoot elongation relates to the selection pressures of different flooding regimes. Long lasting, relatively shallow floods strongly favor enhanced underwater elongation, whereas short, deep floods restrict underwater elongation and thus 490 Cell signalling and gene regulation Glossary anaerobic growth: growth in the absence of oxygen underwater growth: growth of tissues under water, often under conditions of reduced oxygen availability normoxia: oxygen levels at 1 atm, typically 20.6% anoxia: no oxygen available hypoxia: oxygen levels below the critical oxygen pressure for mitochondrial oxidative phosphorylation for the cell (or organ) Figure 1 Overview of the submergence regulatory network in rice. The network involves three key factors: increased cellular ethylene content, depletion of ATP and consumption of readily available sucrose reserves. Submergence of aerial organs results in an accumulation of ethylene that triggers GApromoted cell elongation. In deepwater rice, ethylene promotes induction of the ERF genes SK1 and SK2 and elevation of GA, driving rapid internode elongation and escape of leaves near the water surface [2 ]. In lines tolerant of deep submergence, ethylene activates the ERF SUB1A-1, which promotes a rise in SLR1 and SLRL1, two transcription factors that directly inhibit GA-mediated activation of gene expression. This enhances survival by a quiescence strategy that limits carbohydrate consumption and elongation growth [3 ,5 ]. In japonica rice, which has neither SK1/2 nor SUB1A-1, submergence promotes underwater elongation of shoots until energy reserves are exhausted. Ethylene-triggered submergence-induced GAresponsiveness promotes expression of SUB1C, which acts upstream of a subset of a-amylases that convert starch into glucose for ATP production. Submergence includes the deprivation of oxygen, which leads to a deficiency in ATP. Under low-oxygen stress, ATP is produced at the substrate-level via glycolysis coupled with NAD + regeneration through ethanolic fermentation [1]. The production of alanine, GABA and succinate also contribute to ATP production [1, Natural variation in ethylene-driven submergenceinduced shoot elongation is also observed in other semi-wetland plants. In the wild species Rumex palustris, this natural variation includes both distinctions in final petiole length following submergence and the timing of elongation [16]. Recently, Manzur et al. [19] presented evidence that shoots of Lotus tenuis elongate upon partial shoot submergence, but not when the entire shoot is submerged; thus, both antithetical escape and quiescence survival strategies can exist within a single species. This could reflect the requirement for a threshold in ethylene or another metabolite to be reached to trigger quiescence. Under water shoot elongation acts synergistically with other leaf traits such as aerenchyma development and efficient underwater photosynthesis [14]. Pierik et al. [20] showed that partial de-submergence leads to an increase in biomass in R. palustris, which displays submergence escape, but not in R. acetosa which invokes quiescence. The drawback of extending leaves above water in R. acetosa was associated with low petiole porosity, highlighting interdependency between adaptive traits. Another collaboration between traits is observed in shoots that elongate underwater toward better illumination and ultimately, the water surface. Improvements in carbon gain needed to sustain elongation growth are achieved near the water surface where higher light levels act synergistically with traits that improve gas exchange (e.g. thin leaves, thin cuticles, thin cell walls, mesophyll chloroplasts that re-orientate toward the epidermis [21], and leaf surface gas films [22 ]). Anaerobic germination and early shoot elongation Rice produces one of the few plant seeds that can germinate under strict anoxia. This so-called anaerobic germination and early growth capacity is accomplished by harnessing reserves to fuel shoot elongation at the expense of root development [10]. This growth strategy ultimately mediates oxygen diffusion via a porous coleoptile/stem to submerged tissues and promotes the transition to autotrophic growth. Work on several species has shown that coleoptile elongation under anoxia or severe hypoxia involves accelerated glycolysis (Pasteur Effect), intensive ethanolic fermentation, limited cytoplasmic acidification, and regulation of cell wall loosening proteins [10,23]. Under anoxia, this elongation growth is not driven by ethylene, which requires oxygen for its biosynthesis [23]. Recently, rice's capacity to germinate and extend its coleoptile under water was shown to involve sensing of cellular energy resources, most likely ATP or soluble sugars [11 ]. It requires SnRK1s, the plant's Snf1/ AMP kinases, shown to sense and adjust cellular homeostasis in response to limitations in cellular energy imposed by hypoxia in mesophyll protoplasts of Arabidopsis [24 ] and CIPK15, which phosphorylates SnRK1A, thereby activating the transcriptional activator MYBS1, which initiates production of a-amylases responsible for starch catabolism to glucose. Seeds with a cipk15 loss-of-function mutation germinate in air but not underwater unless provided sucrose. This implies that a change in energy homeostasis, most likely depletion of sugars, drives consumption of endosperm reserves for shoot growth. Indeed, rice cultivars with early and vigorous coleoptile underwater display higher amylase activity The data indicate that the catabolism of starch during anaerobic germination and in submerged shoots of established plants is triggered by distinct mechanisms. In the case of anaerobic germination, an energy deficiency promotes activation of a-amylase transcription. By contrast in submerged plants, ethylene-promoted GA-responsiveness regulates a-amylase expression. If the signaling that drives anaerobic germination and early shoot elongation and SUB1A-mediated submergence tolerance are indeed independent, then it should be feasible to combine these traits to benefit farmers. Adaptive energy management The reduced efficiency in ATP synthesized per mol of glucose during oxygen deprivation leads to a cellular energy crisis. Plants generally respond by elevating sucrose catabolism, glycolysis and ethanolic fermentation to increase substrate-level production of ATP [1]. Transcriptomic and metabolomic studies of diverse angiosperms report the elevation of mRNAs and enzymes that enable sucrose catabolism and the fermentation of pyruvate to ethanol as well as lactate, alanine and GABA during oxygen deprivation [18 ,24 ,27-34,35 ,36 ,37-40]. Anaerobic substrate-level ATP production may be further augmented though alanine metabolism to succinate via a bifurcation of the TCA pathway Oxygen availability varies in organs and cells both during normal development and periods of external oxygen deprivation because of diffusion barriers and distinctions in metabolic activities. Consistent with this, electrode measurements have resolved oxygen gradients across roots, tubers, and stems Conclusion and perspectives The depth, duration, frequency and seasonal timing of floods impose distinct selection pressures on adaptive traits that enable survival of flooding [14]. Recent advances have provided new insights regarding regulation in three interacting networks of response: firstly, ethylene-driven shoot elongation; secondly, anaerobic seed germination and coleoptile growth; and thirdly, maximized conservation of carbohydrates and energy when oxidative phosphorylation is limited. Natural selection favors traits in particular environments when benefits outweigh costs. Shoot elongation is therefore mainly relevant in relatively shallow but prolonged floods, whereas mobilization of seed reserves for rapid shoot elongation aids establishment of seeds buried in anaerobic mud. When either of these two energydemanding escape strategies is too costly, such as in environments with ephemeral and/or very deep floods, the energy conserving quiescence strategy proves more effective. A general network describing the key submergence-induced pathways in rice is presented in Our current challenges include elucidating downstream targets of relevant transcription factors and unraveling essential cell to whole-plant survival strategies. Additionally, it is of utmost importance to precisely link dynamics in oxygen and ATP to metabolic adjustments and survival strategies. The molecular characterization of genetic variation in flooding response strategies is guaranteed to further enable the breeding of crops that can endure or outgrow flooding, as achieved for rice [2 ,4 ,6 ,7]. This is not only essential for crops cultivated in flood-prone farmlands, but is generally relevant because hypoxia exists also in bulky tubers, meristems, and maturing seeds
