Root O2 consumption, CO2 production and tissue concentration profiles in chickpea, as influenced by environmental hypoxia.

Roots in flooded soils experience hypoxia, with the least O2 in the vascular cylinder. Gradients in CO2 across roots had not previously been measured. The respiratory quotient (RQ; CO2 produced : O2 consumed) is expected to increase as O2 availability declines. A new CO2 microsensor and an O2 microsensor were used to measure profiles across roots of chickpea seedlings in aerated or hypoxic conditions. Simultaneous, nondestructive flux measurements of O2 consumption, CO2 production, and thus RQ, were taken for roots with declining O2 . Radial profiling revealed severe hypoxia and c. 0.8 kPa CO2 within the root vascular cylinder. The distance penetrated by O2 into the roots was shorter at lower O2 . The gradient in CO2 was in the opposite direction to that of O2 , across the roots and diffusive boundary layer. RQ increased as external O2 was lowered. For chickpea roots in solution at air equilibrium, O2 was very low and CO2 was elevated within the vascular cylinder; the extent of the severely hypoxic core increased as external O2 was reduced. The increased RQ in roots in response to declining external O2 highlighted the shift from respiration to ethanolic fermentation as the severely hypoxic/anoxic core became a progressively greater proportion of the root tissues.

Physiology, gene expression, and metabolome of two wheat cultivars with contrasting submergence tolerance.

Responses of wheat (Triticum aestivum) to complete submergence are not well understood as research has focused on waterlogging (soil flooding). The aim of this study was to characterize the responses of 2 wheat cultivars differing vastly in submergence tolerance to test if submergence tolerance was linked to shoot carbohydrate consumption as seen in rice. Eighteen-day-old wheat cultivars Frument (intolerant) and Jackson (tolerant) grown in soil were completely submerged for up to 19 days while assessing responses in physiology, gene expression, and shoot metabolome. Results revealed 50% mortality after 9.3 and 15.9 days of submergence in intolerant Frument and tolerant Jackson, respectively, and significantly higher growth in Jackson during recovery. Frument displayed faster leaf degradation as evident from leaf tissue porosity, chlorophylla , and metabolomic fingerprinting. Surprisingly, shoot soluble carbohydrates, starch, and individual sugars declined to similarly low levels in both cultivars by day 5, showing that cultivar Jackson tolerated longer periods of low shoot carbohydrate levels than Frument. Moreover, intolerant Frument showed higher levels of phytol and the lipid peroxidation marker malondialdehyde relative to tolerant Jackson. Consequently, we propose to further investigate the role of ethylene sensitivity and deprivation of reactive O2 species in submerged wheat.

Leaf gas films contribute to rice (Oryza sativa) submergence tolerance during saline floods.

Floods and salinization of agricultural land adversely impact global rice production. We investigated whether gas films on leaves of submerged rice delay salt entry during saline submergence. Two-week-old plants with leaf gas films (+GF) or with gas films experimentally removed (-GF) were submerged in artificial floodwater with 0 or 50 mm NaCl for up to 16 d. Gas films were present >9 d on GF plants after which gas films were diminished. Tissue ion analysis (Na+ , Cl- and K+ ) showed that gas films caused some delay of Na+ entry, as leaf Na+ concentration was 36-42% higher in -GF leaves than +GF leaves on days 1-5. However, significant net uptakes of Na+ and Cl- , and K+ net loss, occurred despite the presence of gas films, indicating the likely presence of some leaf-to-floodwater contact, so that the gas layer must not have completely separated the leaf surfaces from the water. Natural loss and removal of gas films resulted in severe declines in growth, underwater photosynthesis, chlorophylla and tissue porosity. Submergence was more detrimental to leaf PN and growth than the additional effect of 50 mm NaCl, as salt did not significantly affect underwater PN at 200 µm CO2 nor growth.

Contrasting oxygen dynamics in Limonium narbonense and Sarcocornia fruticosa during partial and complete submergence.

Terrestrial saltmarsh plants inhabiting flood-prone habitats undergo recurrent and prolonged flooding driven by tidal regimes. In this study, the role of internal plant aeration in contrasting hypoxic/anoxic conditions during submergence was investigated in the two halophytes Limonium narbonense Mill. and Sarcocornia fruticosa (L.) A.J. Scott. Monitoring of tissue O2 dynamics was performed in shoots and roots using microelectrodes under drained conditions, waterlogging, partial and complete submergence, in light or darkness. For both species, submergence in darkness resulted in significant declines in tissue O2 status and when in light, in rapid O2 increases first in shoot tissues and subsequently in roots. During partial submergence, S. fruticosa benefitted from snorkelling and efficiently transported O2 to roots, whereas the O2 concentration in roots of L. narbonense declined by more than 90%. Significantly thinner leaves and articles were recorded under high degree of flooding stress and both species showed considerably high tissue porosity. The presence of aerenchyma seemed to support internal aeration in S. fruticosa whereas O2 diffusion in L. narbonense seemed impeded, despite the higher porosity (up to 50%). Thus, the results obtained for L. narbonense, being well adapted to flooding, suggests that processes other than internal aeration could be involved in better flooding tolerance e.g. fermentative processes, and that traits resulting in flooding tolerance in plants are not yet fully understood.

Leaf gas film retention during submergence of 14 cultivars of wheat (Triticum aestivum).

Flooding of fields after sudden rainfall events can result in crops being completely submerged. Some terrestrial plants, including wheat (Triticum aestivum L.), possess superhydrophobic leaf surfaces that retain a thin gas film when submerged, and the gas films enhance gas exchange with the floodwater. However, the leaves lose their hydrophobicity during submergence, and the gas films subsequently disappear. We tested gas film retention time of 14 different wheat cultivars and found that wheat could retain the gas films for a minimum of 2 days, whereas the wild wetland grass Glyceria fluitans (L.) R.Br. had thicker gas films and could retain its gas films for a minimum of 4 days. Scanning electron microscopy showed that the wheat cultivars and G. fluitans possessed high densities of epicuticular wax platelets, which could explain their superhydrophobicity. However, G. fluitans also had papillae that contributed to higher hydrophobicity during the initial submergence and could explain why G. fluitans retained gas films for a longer period of time. The loss of gas films was associated with the leaves being covered by an unidentified substance. We suggest that leaf gas film is a relevant trait to use as a selection criterion to improve the flood tolerance of crops that become temporarily submerged.

Flood tolerance of wheat - the importance of leaf gas films during complete submergence.

Submergence invokes a range of stressors to plants with impeded gas exchange between tissues and floodwater being the greatest challenge. Many terrestrial plants including wheat (Triticum aestivum L.), possess superhydrophobic leaf cuticles that retain a thin gas film when submerged, and the gas films enhance gas exchange with the floodwater. However, leaf hydrophobicity is lost during submergence and the gas films disappear accordingly. Here, we completely submerged wheat (with or without gas films) for up to 14 days and found that plants with gas films survived significantly longer (13 days) than plants without (10 days). Plants with gas films also had less dead tissue following a period of recovery. However, this study also revealed that reflections by gas films resulted in a higher light compensation point for underwater net photosynthesis for leaves with gas films compared with leaves without (IC=52 vs 35µmol photons m-2 s-1 with or without gas films, respectively). Still, already at ~5% of full sunlight the beneficial effect of gas films overcame the negative under ecologically relevant CO2 concentrations. Our study showed that dryland crops also benefit from leaf gas films during submergence and that this trait should be incorporated to improve flood tolerance of wheat.

Leaf gas films, underwater photosynthesis and plant species distributions in a flood gradient.

Traits for survival during flooding of terrestrial plants include stimulation or inhibition of shoot elongation, aerenchyma formation and efficient gas exchange. Leaf gas films form on superhydrophobic cuticles during submergence and enhance underwater gas exchange. The main hypothesis tested was that the presence of leaf gas films influences the distribution of plant species along a natural flood gradient. We conducted laboratory experiments and field observations on species distributed along a natural flood gradient. We measured presence or absence of leaf gas films and specific leaf area of 95 species. We also measured, gas film retention time during submergence and underwater net photosynthesis and dark respiration of 25 target species. The presence of a leaf gas film was inversely correlated to flood frequency and duration and reached a maximum value of 80% of the species in the rarely flooded locations. This relationship was primarily driven by grasses that all, independently of their field location along the flood gradient, possess gas films when submerged. Although the present study and earlier experiments have shown that leaf gas films enhance gas exchange of submerged plants, the ability of species to form leaf gas films did not show the hypothesized relationship with species composition along the flood gradient.

Visualisation by high resolution synchrotron X-ray phase contrast micro-tomography of gas films on submerged superhydrophobic leaves.

Floods can completely submerge terrestrial plants but some wetland species can sustain O2 and CO2 exchange with the environment via gas films forming on superhydrophobic leaf surfaces. We used high resolution synchrotron X-ray phase contrast micro-tomography in a novel approach to visualise gas films on submerged leaves of common cordgrass (Spartina anglica). 3D tomograms enabled a hitherto unmatched level of detail regarding the micro-topography of leaf gas films. Gas films formed only on the superhydrophobic adaxial leaf side (water droplet contact angle, Φ=162°) but not on the abaxial side (Φ=135°). The adaxial side of the leaves of common cordgrass is plicate with a longitudinal system of parallel grooves and ridges and the vast majority of the gas film volume was found in large ∼180µm deep elongated triangular volumes in the grooves and these volumes were connected to each neighbouring groove via a fine network of gas tubules (∼1.7µm diameter) across the ridges. In addition to the gas film retained on the leaf exterior, the X-ray phase contrast micro-tomography also successfully distinguished gas spaces internally in the leaf tissues, and the tissue porosity (gas volume per unit tissue volume) ranged from 6.3% to 20.3% in tip and base leaf segments, respectively. We conclude that X-ray phase contrast micro-tomography is a powerful tool to obtain quantitative data of exterior gas features on biological samples because of the significant difference in electron density between air, biological tissues and water.

Gas film retention and underwater photosynthesis during field submergence of four contrasting rice genotypes.

Floods can completely submerge some rice (Oryza sativa L.) fields. Leaves of rice have gas films that aid O2 and CO2 exchange under water. The present study explored the relationship between gas film persistence and underwater net photosynthesis (PN) as influenced by genotype and submergence duration. Four contrasting genotypes (FR13A, IR42, Swarna, and Swarna-Sub1) were submerged for 13 days in the field and leaf gas films, chlorophyll, and the capacity for underwater PN at near ambient and high CO2 were assessed with time of submergence. At high CO2 during the PN assay, all genotypes initially showed high rates of underwater PN, and this rate was not affected by time of submergence in FR13A. This superior photosynthetic performance of FR13A was not evident in Swarna-Sub1 (carrying the SUB1 QTL) and the declines in underwater PN in both Swarna-Sub1 and Swarna were equal to that in IR42. At near ambient CO2 concentration, underwater PN declined in all four genotypes and this corresponded with loss of leaf gas films with time of submergence. FR13A retained leaf gas films moderately longer than the other genotypes, but gas film retention was not linked to SUB1. Diverse rice germplasm should be screened for gas film persistence during submergence, as this trait could potentially increase carbohydrate status and internal aeration owing to increased underwater PN, which contributes to submergence tolerance in rice.

Internal aeration of paddy field rice (Oryza sativa) during complete submergence---importance of light and floodwater O2.

Flash floods can submerge paddy field rice (Oryza sativa), with adverse effects on internal aeration, sugar status and survival. Here, we investigated the in situ aeration of roots of rice during complete submergence, and elucidated how underwater photosynthesis and floodwater pO(2) influence root aeration in anoxic soil. In the field, root pO(2) was measured using microelectrodes during 2 d of complete submergence. Leaf gas films that formed on the superhydrophobic leaves were left intact, or experimentally removed, to elucidate their effect on internal aeration. In darkness, root pO(2) declined to very low concentrations (0.24 kPa) and was strongly correlated with floodwater pO(2). In light, root pO(2) was high (14 kPa) and primarily a function of the incident light determining the rates of underwater net photosynthesis. Plants with intact leaf gas films maintained higher underwater net photosynthesis relative to plants without gas films when the submerged shoots were in light. During complete submergence, internal aeration of rice in the field relies on underwater photosynthesis during the day and entry of O(2) from the floodwater during the night. Leaf gas films enhance photosynthesis during submergence leading to improved O(2) production and sugar status, and therefore contribute to the submergence tolerance of rice.

Leaf gas films of Spartina anglica enhance rhizome and root oxygen during tidal submergence.

Gas films on hydrophobic surfaces of leaves of some wetland plants can improve O(2) and CO(2) exchange when completely submerged during floods. Here we investigated the in situ aeration of rhizomes of cordgrass (Spartina anglica) during natural tidal submergence, with focus on the role of leaf gas films on underwater gas exchange. Underwater net photosynthesis was also studied in controlled laboratory experiments. In field experiments, O(2) microelectrodes were inserted into rhizomes and pO(2) measured throughout two tidal submergence events; one during daylight and one during night-time. Plants had leaf gas films intact or removed. Rhizome pO(2) dropped significantly during complete submergence and most severely during night. Leaf gas films: (1) enhanced underwater photosynthesis and pO(2) in rhizomes remained above 10 kPa during submergence in light; and (2) facilitated O(2) entry from the water into leaves so that rhizome pO(2) was about 5 kPa during darkness. This study is the first in situ demonstration of the beneficial effects of leaf gas films on internal aeration in a submerged wetland plant. Leaf gas films likely contribute to submergence tolerance of S. anglica and this feature is expected to also benefit other wetland plant species when submerged.

A perspective on underwater photosynthesis in submerged terrestrial wetland plants.

BACKGROUND AND AIMS: Wetland plants inhabit flood-prone areas and therefore can experience episodes of complete submergence. Submergence impedes exchange of O(2) and CO(2) between leaves and the environment, and light availability is also reduced. The present review examines limitations to underwater net photosynthesis (P(N)) by terrestrial (i.e. usually emergent) wetland plants, as compared with submerged aquatic plants, with focus on leaf traits for enhanced CO(2) acquisition. SCOPE: Floodwaters are variable in dissolved O(2), CO(2), light and temperature, and these parameters influence underwater P(N) and the growth and survival of submerged plants. Aquatic species possess morphological and anatomical leaf traits that reduce diffusion limitations to CO(2) uptake and thus aid P(N) under water. Many aquatic plants also have carbon-concentrating mechanisms to increase CO(2) at Rubisco. Terrestrial wetland plants generally lack the numerous beneficial leaf traits possessed by aquatic plants, so submergence markedly reduces P(N). Some terrestrial species, however, produce new leaves with a thinner cuticle and higher specific leaf area, whereas others have leaves with hydrophobic surfaces so that gas films are retained when submerged; both improve CO(2) entry. CONCLUSIONS: Submergence inhibits P(N) by terrestrial wetland plants, but less so in species that produce new leaves under water or in those with leaf gas films. Leaves with a thinner cuticle, or those with gas films, have improved gas diffusion with floodwaters, so that underwater P(N) is enhanced. Underwater P(N) provides sugars and O(2) to submerged plants. Floodwaters often contain dissolved CO(2) above levels in equilibrium with air, enabling at least some P(N) by terrestrial species when submerged, although rates remain well below those in air.

Use of sediment CO2 by submersed rooted plants.

BACKGROUND AND AIMS: Submersed plants have different strategies to overcome inorganic carbon limitation. It is generally assumed that only small rosette species (isoetids) are able to utilize the high sediment CO(2) availability. The present study examined to what extent five species of submersed freshwater plants with different morphology and growth characteristics (Lobelia dortmanna, Lilaeopsis macloviana, Ludwigia repens, Vallisneria americana and Hydrocotyle verticillata) are able to support photosynthesis supplied by uptake of CO(2) from the sediment. METHODS: Gross photosynthesis was measured in two-compartment split chambers with low inorganic carbon availability in leaf compartments and variable CO(2) availability (0 to >8 mmol L(-1)) in root compartments. Photosynthetic rates based on root-supplied CO(2) were compared with maximum rates obtained at saturating leaf CO(2) availability, and (14)C experiments were conducted for two species to localize bottlenecks for utilization of sediment CO(2). KEY RESULTS: All species except Hydrocotyle were able to use sediment CO(2), however, with variable efficiency, and with the isoetid, Lobelia, as clearly the most effective and the elodeid, Ludwigia, as the least efficient. At a water column CO(2) concentration in equilibrium with air, Lobelia, Lilaeopsis and Vallisneria covered >75% of their CO(2) requirements by sediment uptake, and sediment CO(2) contributed substantially to photosynthesis at water CO(2) concentrations up to 1000 micromol L(-1). For all species except Ludwigia, the shoot to root ratio on an areal basis was the single factor best explaining variability in the importance of sediment CO(2). For Ludwigia, diffusion barriers limited uptake or transport from roots to stems and transport from stems to leaves. CONCLUSIONS: Submersed plants other than isoetids can utilize sediment CO(2), and small and medium sized elodeids with high root to shoot area in particular may benefit substantially from uptake of sediment CO(2) in low alkaline lakes.