Figure 18.12. Shoot elongation of rice cultivars during submergence relative to survival. Plants were submerged 14 d at a mean daily irradiance of 22.3 ML m-2. Open symbols, lowland rice; closed symbols, deepwater rice cultivars. Setter and Laureles (1996).
Leaves, petioles and stems of completely submerged herbaceous plants are generally not anoxic, although can become hypoxic. The internal O2 concentration in these submerged shoot organs is determined by the rate of photosynthesis under water, the rate of respiration of the tissue and the rate of gas exchange with the external water medium. This results, for example, in endogenous petiole O2 concentrations in fully submerged Arabidopsis thaliana of 17 kPa during the light period and 6 kPa during darkness (Lee et al. 2011; Vashisht et al. 2011). Due to the strong variation over time, O2 is seen as an unreliable indicator of submergence of shoot organs. Therefore, shoots make use of the entrapment of another gaseous component, the plant hormone ethylene, to sense the change of the outside environment from air to water. Similar to other gases, ethylene only very slowly diffuses in water. Since ethylene is produced by every plant cell, slow diffusion in water leads to a substantial increase of ethylene levels inside shoot tissues within less than one hour of submergence. This enhanced endogenous ethylene concentration is guaranteed in submerged shoots as long as some O2 is present to maintain the O2-dependent ethylene biosynthesis.
Flood tolerant plants exposed to complete submergence exploit two contrasting suites of traits, escape or quiescence, to survive this stress. In brief, plants with the escape strategy: (i) increase the growth rate of shoot organs, such as petioles and stems, so as to emerge above floodwaters, and (ii) initiate the development of aerenchyma to facilitate internal gas diffusion. Quiescent plants, on the other hand, “wait out the submergence event” and are characterized by: (i) conservation of energy and carbohydrates via, for example, a reduction of the underwater growth rate, and (ii) an increase of molecular components that prepare shoot and root organs for future conditions with low O2 and production of protective molecules that counteract harmful cellular changes associated with flooding, such as production of ROS.
A classical study with a range of rice cultivars revealed that shoot elongation under water without reaching the surface goes at the expense of survival and thereby demonstrating elongation is associated with costs (Figure 18.12). From an evolutionary point of view the escape strategy will only persist if these costs are outweighed by benefits such as improved aeration, energy generation and carbon production, and ultimately improved survival, growth and reproduction. Therefore, the escape strategy is restricted to plants in environments with shallow floods or with deeper floods that persist over longer durations as the case with deepwater and floating rice. Transient very deep or ephemeral floods, however, favour plants with the quiescence strategy.
Figure 18.13. Petiole elongation response in Rumex palustris upon submergence. Plant on the left-hand-side was in air, plant on the right-hand-side was submerged for the final 10 days. (Photograph courtesy of Liesje Mommer).
Fast extension of shoot organs in response to submergence is described for species from a wide range of families. As an example, Figure 18.13 illustrates submergence-induced petiole elongation in the semi-aquatic plant Rumex palustris.
Depending on the tissue type and the developmental stage of the shoot organ, fast underwater growth involves cell elongation only (e.g. petiole of Rumex palustris) or a combination of increased cell division and elongation (e.g. stem of deepwater rice). Fast cell elongation is regulated by specific transcription factors and by an interacting set of plant hormones. In deep water rice, characterized by an enormous stem elongation capacity upon submergence, ethylene regulates two important genes, SNORKEL1 and SNORKEL2, which encode nuclear-localized DNA binding proteins that belong to the Ethylene Response Factor (ERF) family of transcription factors. Non-deepwater rice varieties lack these ERF genes and their importance for elongation was demonstrated by introgression of these loci from deepwater rice into non-elongating varieties which then showed substantial elongation when submerged. Next to ethylene, three other downstream-operating plant hormones are involved in submergence-induced shoot elongation. Upon submergence, levels of abscisic acid are quickly reduced, whereas auxin and gibberellic acid increase. Ultimately these signal transduction components affect the rate limiting step for cell elongation: the cell wall. In order to allow turgor-driven cell expansion, cell walls must loosen by means of specific cell wall loosening proteins such as Expansins. The expression of Expansin genes is strongly upregulated and the abundance of Expansin proteins increases, soon after submergence of species with shoots that elongate.
An important trait for plants that survive flooding by means of the quiescence strategy is reduction of underwater growth to conserve carbohydrates and retention of chlorophyll to enable continued, albeit reduced, photosynthesis. Increased submergence tolerance in rice caused by reduced plant growth rates under water is regulated by the ethylene-induced expression of the SUB1A-1 gene. Interestingly, this gene belongs to the same ERF transcription factor family as the two SNORKEL genes. SUB1A-1 limits elongation growth by two mechanisms: (i) minimizing the decline in the gibberellin signaling repressor SLENDER RICE-1 and the related SLENDER RICE LIKE-1, and (ii) enhancing GA catabolism by differentially regulating genes associated with brassinosteroid synthesis in submerged shoots (Schmitz et al. 2013). On top of that, SUB1A-1 also inhibits synthesis of ethylene, expression of Expansins and reduces starch and sucrose reserve depletion. Recently, SUB1A-1 was crossed into high yielding rice varieties leading to more flood tolerant varieties that have recently been released to farmers in Asia. These varieties have yield advantages of 1 to over 3 tons/hectare over the varieties lacking SUB1A-1 following submergence for various durations (Mackill et al. 2012). Figure 18.14 demonstrates increased survival and yield after 15 d of complete submergence during the vegetative stage followed by recovery after de-submergence of a rice variety containing SUB1A-1 compared with the original variety that lacks it.
Figure 18.14. IR64 (left) and IR64-Sub1 (right) after 15 days of submergence during vegetative stage in the field. Two-week-old seedlings were transplanted into a field, grown for another two weeks then completely submerged for 15 days. The field was then drained and plants were allowed to recover under non-stress conditions. The photograph was taken about 90 days after de-submergence. (Photograph courtesy of AM Ismail).
In summary, most plants cannot withstand complete submergence lasting over a few days; however, semi-aquatic plants such as certain rice genotypes can survive complete submergence even for over two weeks. Tolerance of rice to transient submergence is mainly achieved by restricting growth and respiration, thus conserving carbohydrate reserves to enhance recovery when the floodwater recedes.
Figure 18.15. Leaf gas films on common reed (Phragmites australis) during complete submergence. Gas films form on hydrophobic leaf surfaces. Leaf gas films enhance gas-exchange with the surrounding floodwater: CO2 entry for photosynthesis (in light) and O2 entry for internal aeration for respiration (in dark), of submerged plants. (Photograph courtesy of Ole Pedersen).
Photosynthesis in completely submerged wetland plants is severely impeded by low light, and the slow diffusion of CO2 across the aqueous diffusive boundary layer (DBL) adjacent to leaves. Aquatic and amphibious plants have evolved a number of leaf traits to reduce the total resistance to CO2 uptake, including: (i) dissected leaves, (ii) undulating leaf edges to increase turbulence across the leaf, (iii) thin leaves, (iv) reduced cuticle, and (v) chloroplasts in epidermal cells. The first three traits (i, ii & iii) all serve to erode/reduce the DBL and thus decrease the distance of molecular diffusion (i.e. decreased total external resistance to CO2 uptake), and (iv & v) reduce the resistance within the tissue for the diffusion pathway to chloroplasts. Likewise, many submerged terrestrial wetland plants also display some acclimation to inundation such as thinner leaves, reduced cuticle and chloroplast orientation towards the source of CO2. Amphibious plants are positioned between the truly aquatic plants and the terrestrial wetland plants and display a suite of leaf acclimation traits that allows efficient gas exchange by leaves in air, as well as those formed under water.
Rates of net photosynthesis by submerged leaves are typically much lower than rates achieved in air, even for acclimated leaves. Underwater net photosynthesis by submerged terrestrial plants is generally lower than the rates achieved by aquatic plants. However, this is only true when photosynthetic rates are expressed on a per unit area basis, the units commonly used in terrestrial plant physiology. When net photosynthesis is expressed on a per unit dry mass basis, rates in aquatic plants > amphibious plants > terrestrial plants and this order reflects the higher carbon-return per unit of dry mass investment by the aquatic leaf types, as compared with terrestrial leaf types, when submerged.
Gas films on leaves of submerged terrestrial wetland plants have also been shown to facilitate underwater photosynthesis. Gas films form on hydrophobic leaf surfaces of many wetland plants when submerged (Figure 18.15); e.g., species of Phragmites, Typha, Spartina, Carex, Phalaris and Oryza (including cultivated rice), and the gas film forms a large gas-water interface that facilitates gas exchange with the surrounding water.
It is likely that the stomata remain open underneath the gas film. The gas films enable leaves of such terrestrial wetland plants to photosynthesize under water, albeit at rates much reduced when compared with in air, but without further acclimation and this strategy may therefore be particularly advantageous under short floods; as examples, frequent tidal submergence or short duration flash floods such as in some rice-growing areas and natural wetlands where water recedes after a week or two. The improved O2 and sugar status of submerged rice owing to the beneficial effects of leaf gas films would enhance survival during complete submergence.
Ole Pedersen, University of Copenhagen, Denmark
Development of microelectrodes robust enough for use in field conditions has enabled in situ measurements of O2 dynamics in submerged plants (Figure 1). Eco-physiological research on submerged plants has been a challenge; infrared gas analyzers do not work under water! Field studies (in situ) have revealed how fluctuating light, diurnal changes in temperature and water column O2 concentrations all influence internal aeration of submerged aquatic plants. In the case of seagrasses, application of sulphide microelectrodes has also added to the growing evidence of sulphide poisoning as a likely cause of extensive die-backs.
Figure 1. Diver operating O2 and H2S microelectrodes (left) in a Thallasia seagrass meadow in the Caribbean. Development of in situ equipment has enabled measurements of internal aeration under challenging field conditions in aquatic systems (right). (Photographs courtesy of Malene Hedegård Petersen (left) and Ole Pedersen (right).
Figure 2. Oxygen dynamics in the rhizome of the seagrass Zostera marina during a diurnal cycle. O2 in the rhizome closely follows incoming light during daytime, whereas during the night internal aeration relies on supply of O2 from the water column. The dependence on water column pO2 during the night for internal aeration becomes particularly clear when the tide carries water across the seagrass meadow with lower pO2 as the internal pO2 of the rhizomes immediately declines (see arrows). During the day, it is the other way around as fluctuations in incoming light is immediately reflected in rhizome pO2 as daytime rhizome pO2 follows underwater photosynthesis. Data from Greve et al. (2003).
Seagrasses are flowering plants with roots, rhizomes, sometimes stems, and almost always strap shaped leaves to reduce pressure drag and thus the uprooting forces created by wave action. Seagrasses are key marine ecosystem engineers and habitat for various marine animals, but seagrasses are under world-wide threat from human activities (eutrophication, dredging and other physical disturbances). Eutrophication impacts directly on seagrasses by decreasing the available light (stimulates growth of epiphytes and planktonic algae) but also indirectly by stimulating the decomposition of organic matter in the sediment (mineralization of algae and seagrass litter is often limited by N and P) and thus increases the O2 demand of the sediment. In marine sediments, sulphate reduction by microorganisms can be substantial under anoxic conditions, producing sulphide, a potent phytotoxin with toxicity and mode of action similar to that of cyanide. Sulphide exists in three different chemical forms in water (H2S, HS- and S2-) with the gaseous H2S dominating at low pH and S2- at high pH. Gaseous H2S can enter the root and rhizome aerenchyma and move via diffusion to other parts of the seagrass, such as to leaf meristems that are relatively sensitive to sulphide. The resulting sulphide poisoning is a major cause of the worldwide die back of seagrasses observed in temperate as well as in tropical seagrass meadows.
Mechanistic field studies employing in situ microelectrodes have improved our understanding of internal aeration and sulphide intrusion in natural seagrass meadows. During the day, internal aeration of roots and rhizomes relies on O2 production in underwater photosynthesis, as does radial O2 loss (ROL) from roots to the sediments. There is a strong relationship between incoming light and O2 partial pressure in roots (Figure 2). Clouds immediately lead to a decline in root O2, whereas during periods of high sunlight root O2 was highest. During the night time, however, internal aeration relies on a steady flux of O2 from the water and into the leaves, and via the aerenchyma, further down into the roots and rhizomes.
Figure 3. Oxygen dynamics in the rhizome of the seagrass Zostera marina during the day (A) and during the night (B). During the day, underwater photosynthesis produces O2 in the leaves and rhizome pO2 is thus a function of incoming light as O2 easily diffuses from the chloroplast and into the root via the porous tissues. In contrast, during the night, the water column is the only source of O2 for the plant and O2 diffuses from the water and into the leaves and further down into the belowground tissues. Consequently, rhizome pO2 is strongly correlated to water column pO2 during darkness. Re-analyzed data from Greve et al. (2003).
Figure 4. Lobelia dortmanna and other isoetids in a Norwegian oligotrophic lobelia lake. The water of such lakes contains only little dissolved CO2 but L. dortmanna can take up CO2 from the sediment where the concentration is typically 100‑fold higher. As a consequence, L. dortmanna has no barrier to radial O2 loss (ROL) in its roots and almost all O2 produced in underwater photosynthesis is lost to the sediment via the roots. (Photograph courtesy of Ole Pedersen).
Critically low water column O2 can occur during nights in areas with still, warm waters, resulting from net system respiration faster than inwards movement of O2 into the stagnant waters. Under such night time conditions, roots can experience anoxia. Cessation of ROL to the sediments means there is no longer chemical oxidation of H2S to SO42- in the rhizosphere, so that gaseous H2S enters the aerenchyma and spreads via gas phase diffusion to all parts of the seagrass. The very metabolically active leaf meristems are thought to be particularly sensitive to sulphide poisoning. It is thought provoking that during most sudden die backs of seagrasses, the shoots are found drifting in the water with apparently healthy leaves but detached from the vertical stem exactly where the basal leaf meristems is located.
In contrast to coastal marine habitats of seagrasses, lobelia lakes are highly transparent oligotrophic, low alkaline lakes of the northern hemisphere. The vegetation consist of several evergreen species that are morphologically strikingly similar: short stiff leaves arranged in a rosette and with unbranched roots that can make up more than 50% of the biomass. As a whole, the type of vegetation is referred to as isoetids from the genus Isoetes that occurs in most lobelia lakes. Although not present in all lobelia lakes, Lobelia dortmanna is key species (Figure 4). Isoetids take up CO2 from the sediment via the roots and some are even CAM plants, although conservation of water is probably the least of all concerns for these plants. CO2 concentrations are highest at night, so CAM enables storage of malate for subsequent decarboxylation providing CO2 for photosynthesis the next day.
Many sandy sediments in shallow lobelia lakes are permanently oxic. Oxic sediments are a consequence of inherently low mineralization rates as the oligotrophic conditions lead to very low production of organic matter that is subsequently decomposed in the sediment. Nevertheless, the CO2 concentration in these sediments can be 100-fold higher than in the water above and L. dortmanna, along with the other isoetids, tap into this rich source of CO2 with their large root systems. The large gradient in partial pressure of CO2 between sendiment and photosynthetic leaves drives a flux of CO2 from the sediment, into the root aerenchyma (radial CO2 uptake) and then upwards into the porous leaves. Interestingly, the leaves are covered with a relatively thick cuticle to prevent loss of CO2 to the surrounding water, and as a result up to 100% of the O2 produced in underwater photosynthesis is lost via ROL from the roots (Figure 5).
Figure 5. Oxygen dynamics in leaves and roots of Lobelia dortmanna and the surrounding water column and sediment in Lake Värsjö, Sweden. In light, underwater photosynthesis drives leaf pO2 up above 30 kPa and the steep gradient to roots results in a substantial O2 flux into roots. The roots have no barrier to ROL and thus the majority of O2 produced in photosynthesis is lost to the sediment resulting in large diurnal fluctuation in sediment pO2; in fact, the sediment remains permanently oxic. Data modified from Sand-Jensen et al. 2005.
In conclusion, the isoetids tested so far do not form a barrier to ROL in their roots and isoetids are thus restricted to sediments with very low O2 demand; any root barrier would also restrict CO2 uptake. In contrast, the few species of seagrasses studied all show a strong barrier to ROL and in the marine H2S rich environment the barrier would also reduce the inward flux of gaseous H2S.
Greve TM, Borum J, Pedersen O (2003) Meristematic oxygen variability in eelgrass (Zostera marina). Limnol Oceanogr 48: 210-216
Sand-Jensen K, Pedersen O, Binzer T, Borum J (2005) Contrasting oxygen dynamics in the freshwater isoetid Lobelia dortmanna and the marine seagrass Zostera marina. Ann Bot 96: 613-623
Pedersen O, Colmer TD, Sand-Jensen K (2013) Underwater photosynthesis of submerged plants – recent advances and methods. Front Plant Sci 4 DOI: 10.3389/fpls.2013.0014
During daytime, underwater photosynthesis not only provides sugars but it also produces O2 which results in significant aeration of belowground tissues. In light, the partial pressure gradient of O2 from shoot to root is huge (high O2 in the surrounding water, zero O2 in the anoxic soil) and can thus drive a substantial flux of O2 from shoot to root in well-developed aerenchyma as this pathway poses relatively little resistance to diffusion. In fact, O2 in the roots of rice displays a normal saturation curve relationship when plotted against light available to the shoots, a pattern also found in truly aquatic plants (see Case Study 4).
At night, the floodwater surrounding the shoot is the main source of O2 for internal aeration also of belowground tissues. The O2 “stored” in aerenchymous tissues cannot support night time respiration of belowground tissues as the O2 in the aerenchyma relatively quickly equilibrates with the environment (soil in the case of roots and water column in the case of shoot). However, with ample O2 in the floodwater a large partial pressure gradient exists for O2 movement from the floodwater into the leaves and further into the roots (both respiring and thus consuming O2). As a result, the relationship between floodwater O2 and root pO2 in darkness is often linear (see Case Study 4). The floodwater O2 threshold concentration required for O2 to enter and reach the root extremities is determined by the total resistance to molecular diffusion into (DBL, surface gas films, stomatal resistance, cuticular resistance) and through the plant body (tissue porosity, diffusion distance) plus loss of O2 along the route (respiration and any ROL). As with underwater photosynthesis, leaf gas films reduce the resistance to gas exchange between floodwater and leaves, so enhancing O2 entry at night-time. Plants with gas films have been shown to maintain better internal aeration as compared to plants with the gas films experimentally removed.