Genetic diversity and candidate genes for transient waterlogging tolerance in mungbean at the germination and seedling stages.

Mungbean [Vigna radiata var. radiata (L.) Wilczek] production in Asia is detrimentally affected by transient soil waterlogging caused by unseasonal and increasingly frequent extreme precipitation events. While mungbean exhibits sensitivity to waterlogging, there has been insufficient exploration of germplasm for waterlogging tolerance, as well as limited investigation into the genetic basis for tolerance to identify valuable loci. This research investigated the diversity of transient waterlogging tolerance in a mini-core germplasm collection of mungbean and identified candidate genes for adaptive traits of interest using genome-wide association studies (GWAS) at two critical stages of growth: germination and seedling stage (i.e., once the first trifoliate leaf had fully-expanded). In a temperature-controlled glasshouse, 292 genotypes were screened for tolerance after (i) 4 days of waterlogging followed by 7 days of recovery at the germination stage and (ii) 8 days of waterlogging followed by 7 days of recovery at the seedling stage. Tolerance was measured against drained controls. GWAS was conducted using 3,522 high-quality DArTseq-derived SNPs, revealing five significant associations with five phenotypic traits indicating improved tolerance. Waterlogging tolerance was positively correlated with the formation of adventitious roots and higher dry masses. FGGY carbohydrate kinase domain-containing protein was identified as a candidate gene for adventitious rooting and mRNA-uncharacterized LOC111241851, Caffeoyl-CoA O-methyltransferase At4g26220 and MORC family CW-type zinc finger protein 3 and zinc finger protein 2B genes for shoot, root, and total dry matter production. Moderate to high broad-sense heritability was exhibited for all phenotypic traits, including seed emergence (81%), adventitious rooting (56%), shoot dry mass (81%), root dry mass (79%) and SPAD chlorophyll content (70%). The heritability estimates, marker-trait associations, and identification of sources of waterlogging tolerant germplasm from this study demonstrate high potential for marker-assisted selection of tolerance traits to accelerate breeding of climate-resilient mungbean varieties.

Comparative transcriptome analysis reveals molecular regulation of salt tolerance in two contrasting chickpea genotypes.

Salinity is a major abiotic stress that causes substantial agricultural losses worldwide. Chickpea (Cicer arietinum L.) is an important legume crop but is salt-sensitive. Previous physiological and genetic studies revealed the contrasting response of two desi chickpea varieties, salt-sensitive Rupali and salt-tolerant Genesis836, to salt stress. To understand the complex molecular regulation of salt tolerance mechanisms in these two chickpea genotypes, we examined the leaf transcriptome repertoire of Rupali and Genesis836 in control and salt-stressed conditions. Using linear models, we identified categories of differentially expressed genes (DEGs) describing the genotypic differences: salt-responsive DEGs in Rupali (1,604) and Genesis836 (1,751) with 907 and 1,054 DEGs unique to Rupali and Genesis836, respectively, salt responsive DEGs (3,376), genotype-dependent DEGs (4,170), and genotype-dependent salt-responsive DEGs (122). Functional DEG annotation revealed that the salt treatment affected genes involved in ion transport, osmotic adjustment, photosynthesis, energy generation, stress and hormone signalling, and regulatory pathways. Our results showed that while Genesis836 and Rupali have similar primary salt response mechanisms (common salt-responsive DEGs), their contrasting salt response is attributed to the differential expression of genes primarily involved in ion transport and photosynthesis. Interestingly, variant calling between the two genotypes identified SNPs/InDels in 768 Genesis836 and 701 Rupali salt-responsive DEGs with 1,741 variants identified in Genesis836 and 1,449 variants identified in Rupali. In addition, the presence of premature stop codons was detected in 35 genes in Rupali. This study provides valuable insights into the molecular regulation underpinning the physiological basis of salt tolerance in two chickpea genotypes and offers potential candidate genes for the improvement of salt tolerance in chickpeas.

Tolerance of four grain legume species to waterlogging, hypoxia and anoxia at germination and recovery.

Legume seeds, when relay sown following rice, may suffer from soil waterlogging and the associated hypoxia or even anoxia. This study evaluated the tolerance of grain legume species, grass pea (three genotypes), lentil (two genotypes), faba bean (two genotypes) and field pea (one genotype), to soil waterlogging in a glasshouse, to anoxia and hypoxia in temperature-controlled room at germination and seedling stages. Changes in oxygen in the surface layers of soil, with time after waterlogging, were measured by microelectrode profiling. The soil profiling showed that soil oxygen declined and then stabilized by the fourth day after waterlogging and oxygen was not detected at 8 mm below the soil surface. Germination of seeds under waterlogging for up to 12 days and seedling survival after the soil was drained for up to 36 days were measured in pot experiments. Seed germination and/or survival in anoxia (N2-flushed solutions) and hypoxia (1.0 and 2.5 kPa oxygen) were evaluated, and so were post-anoxia or post-hypoxia recoveries, all in comparison with aerated controls. Lentil had higher seedling emergence (55 %) than the other species during soil waterlogging. However, lentil had lower seedling survival (9 %) than grass pea (28 %) during recovery following soil drainage. Grass pea seeds were more tolerant of anoxia and of hypoxia than the seeds of the three other species. In conclusion, grass pea, with higher percent germination and seedling survival during recovery, is more tolerant to waterlogging and subsequent soil drainage than the three other grain legume species. Grass pea was also more tolerant of hypoxia and of anoxia at the seed germination stage. These findings demonstrate the superior waterlogging tolerance of grass pea in relay sowing, as compared with the other grain legumes.

Response of Mungbean (cvs. Celera II-AU and Jade-AU) and Blackgram (cv. Onyx-AU) to Transient Waterlogging.

Mungbean [Vigna radiata (L.) Wilczek] and blackgram [Vigna mungo (L.) Hepper] are important crops for smallholder farmers in tropical and subtropical regions. Production of both crops is affected by unexpected and increasingly frequent extreme precipitation events, which result in transient soil waterlogging. This study aimed to compare the waterlogging tolerance of mungbean and blackgram genotypes under the varying duration of waterlogging stress at germination and seedling stages. We evaluated the responses to different durations of transient waterlogging in a sandy clay loam under temperature-controlled glasshouse conditions. Waterlogging durations were 0, 1, 2, 3, 4, 5, 6, 7, and 8 days during germination and 0, 2, 4, 8, and 16 days during the seedling stage. We used two mungbean genotypes (green testa), Celera II-AU (small-seeded), and Jade-AU (large-seeded), contrasting in seed size and hypocotyl pigmentation, and a blackgram genotype (black testa), Onyx-AU. Waterlogging reduced soil redox potential, delayed or even prevented germination, decreased seedling establishment, and affected shoot and root development. In the seedlings waterlogged (WL) at 15 days after sowing (DAS), adventitious root formation and crown nodulation varied between the genotypes, and 16 days of waterlogging substantially reduced growth but did not result in plant death. Plants in soil with waterlogging for 8-16 days followed by drainage and sampling at 39 DAS had reduced shoot and root dry mass by 60-65% in mungbean and 40% in blackgram compared with continuously drained controls, due at least in part to fewer lateral roots. Soil plant analysis development (SPAD) chlorophyll content was also reduced. Onyx-AU, a blackgram genotype, was more tolerant to transient waterlogging than Jade-AU and Celera II-AU in both growth stages. Of the two mungbean genotypes, Celera II-AU had a greater seedling establishment than Jade-AU post waterlogging imposed at sowing. In contrast, Jade-AU had more plant biomass and greater recovery growth than Celera II-AU after waterlogging and recovery during the seedling stage. Both species were delayed in emergence in response to the shorter periods of transient waterlogging at germination, and with the longer waterlogging germination and emergence failed, whereas at the seedling stage both showed adaptation by the formation of adventitious roots.

Regulation of root adaptive anatomical and morphological traits during low soil oxygen.

Flooding causes oxygen deprivation in soils. Plants adapt to low soil oxygen availability by changes in root morphology, anatomy, and architecture to maintain root system functioning. Essential traits include aerenchyma formation, a barrier to radial oxygen loss, and outgrowth of adventitious roots into the soil or the floodwater. We highlight recent findings of mechanisms of constitutive aerenchyma formation and of changes in root architecture. Moreover, we use modelling of internal aeration to demonstrate the beneficial effect of increasing cortex-to-stele ratio on sustaining root growth in waterlogged soils. We know the genes for some of the beneficial traits, and the next step is to manipulate these genes in breeding in order to enhance the flood tolerance of our crops.

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.

Rice acclimation to soil flooding: Low concentrations of organic acids can trigger a barrier to radial oxygen loss in roots.

Waterlogged soils contain monocarboxylic acids produced by anaerobic microorganisms. These "organic acids" can accumulate to phytotoxic levels and promote development of a barrier to radial O2 loss (ROL) in roots of some wetland species. Environmental cues triggering root ROL barrier induction, a feature that together with tissue gas-filled porosity facilitates internal aeration, are important to elucidate for knowledge of plant stress physiology. We tested the hypothesis that comparatively low, non-toxic, concentrations of acetic, propionic, butyric, and/or hexanoic acids might induce root ROL barrier formation in rice. Each organic acid, individually, triggered the ROL barrier in roots but with no effect (acetic or butyric acids) or with only slight effects (propionic or hexanoic acids) on root extension. Transcripts of four genes related to suberin biosynthesis were increased by some of the organic acid treatments. Respiration in root tissues was not, or moderately, inhibited. Beyond a narrow concentration range, however, respiration declined exponentially and the order (least to greatest) for EC50 (effective concentration for 50% inhibition) was butyric, propionic, acetic, then hexanoic acid. An understanding of the environmental cue for root ROL barrier induction should enhance future work to elucidate the molecular regulation of this root trait contributing to plant flooding tolerance.

Diel O2 Dynamics in Partially and Completely Submerged Deepwater Rice: Leaf Gas Films Enhance Internodal O2 Status, Influence Gene Expression and Accelerate Stem Elongation for 'Snorkelling' during Submergence.

Deepwater rice has a remarkable shoot elongation response to partial submergence. Shoot elongation to maintain air-contact enables 'snorkelling' of O2 to submerged organs. Previous research has focused on partial submergence of deepwater rice. We tested the hypothesis that leaf gas films enhance internode O2 status and stem elongation of deepwater rice when completely submerged. Diel patterns of O2 partial pressure (pO2) were measured in internodes of deepwater rice when partially or completely submerged, and with or without gas films on leaves, for the completely submerged plants. We also took measurements for paddy rice. Deepwater rice elongated during complete submergence and the shoot tops emerged. Leaf gas films improved O2 entry during the night, preventing anoxia in stems, which is of importance for elongation of the submerged shoots. Expressions of O2 deprivation inducible genes were upregulated in completely submerged plants during the night, and more so when gas films were removed from the leaves. Diel O2 dynamics showed similar patterns in paddy and deepwater rice. We demonstrated that shoot tops in air enabled 'snorkelling' and increased O2 in internodes of both rice ecotypes; however, 'snorkelling' was achieved only by rapid shoot elongation by deepwater rice, but not by paddy rice.

Corrigendum: Waterlogging of Winter Crops at Early and Late Stages: Impacts on Leaf Physiology, Growth and Yield.

[This corrects the article DOI: 10.3389/fpls.2018.01863.].

Rice leaf hydrophobicity and gas films are conferred by a wax synthesis gene (LGF1) and contribute to flood tolerance.

Floods impede gas (O2 and CO2 ) exchange between plants and the environment. A mechanism to enhance plant gas exchange under water comprises gas films on hydrophobic leaves, but the genetic regulation of this mechanism is unknown. We used a rice mutant (dripping wet leaf 7, drp7) which does not retain gas films on leaves, and its wild-type (Kinmaze), in gene discovery for this trait. Gene complementation was tested in transgenic lines. Functional properties of leaves as related to gas film retention and underwater photosynthesis were evaluated. Leaf Gas Film 1 (LGF1) was identified as the gene determining leaf gas films. LGF1 regulates C30 primary alcohol synthesis, which is necessary for abundant epicuticular wax platelets, leaf hydrophobicity and gas films on submerged leaves. This trait enhanced underwater photosynthesis 8.2-fold and contributes to submergence tolerance. Gene function was verified by a complementation test of LGF1 expressed in the drp7 mutant background, which restored C30 primary alcohol synthesis, wax platelet abundance, leaf hydrophobicity, gas film retention, and underwater photosynthesis. The discovery of LGF1 provides an opportunity to better understand variation amongst rice genotypes for gas film retention ability and to target various alleles in breeding for improved submergence tolerance for yield stability in flood-prone areas.

Waterlogging tolerance, tissue nitrogen and oxygen transport in the forage legume Melilotus siculus: a comparison of nodulated and nitrate-fed plants.

Background and Aims: Soil waterlogging adversely impacts most plants. Melilotus siculus is a waterlogging-tolerant annual forage legume, but data were lacking for the effects of root-zone hypoxia on nodulated plants reliant on N2 fixation. The aim was to compare the waterlogging tolerance and physiology of M. siculus reliant on N2 fixation or with access to NO3-. Methods: A factorial experiment imposed treatments of water level (drained or waterlogged), rhizobia (nil or inoculated) and mineral N supply (nil or 11 mm NO3-) for 21 d on plants in pots of vermiculite in a glasshouse. Nodulation, shoot and root growth and tissue N were determined. Porosity (gas volume per unit tissue volume) and respiration rates of root tissues and nodules, and O2 microelectrode profiling across nodules, were measured in a second experiment. Key Results: Plants inoculated with the appropriate rhizobia, Ensifer (syn. Sinorhizobium) medicae, formed nodules. Nodulated plants grew as well as plants fed NO3-, both in drained and waterlogged conditions. The growth and total N content of nodulated plants (without any NO3- supplied) indicated N2 fixation. Respiration rates (mass basis) were highest in nodules and root tips and lowest in basal root tissues. Secondary aerenchyma (phellem) formed along basal root parts and a thin layer of this porous tissue also covered nodules, which together enhanced gas-phase diffusion of O2 to the nodules; O2 was below detection within the infected zone of the nodule interior. Conclusions: Melilotus siculus reliant on N2 fixation grew well both in drained and waterlogged conditions, and had similar tissue N concentrations. In waterlogged conditions the relatively high respiration rates of nodules must rely on O2 movement via the aerenchymatous phellem in hypocotyl, roots and the outer tissue layers of nodules.

Waterlogging of Winter Crops at Early and Late Stages: Impacts on Leaf Physiology, Growth and Yield.

Waterlogging is expected to increase as a consequence of global climate change, constraining crop production in various parts of the world. This study assessed tolerance to 14-days of early- or late-stage waterlogging of the major winter crops wheat, barley, rapeseed and field pea. Aerenchyma formation in adventitious roots, leaf physiological parameters (net photosynthesis, stomatal and mesophyll conductances, chlorophyll fluorescence), shoot and root growth during and after waterlogging, and seed production were evaluated. Wheat produced adventitious roots with 20-22% of aerenchyma, photosynthesis was maintained during waterlogging, and seed production was 86 and 71% of controls for early- and late-waterlogging events. In barley and rapeseed, plants were less affected by early- than by late-waterlogging. Barley adventitious roots contained 19% aerenchyma, whereas rapeseed did not form aerenchyma. In barley, photosynthesis was reduced during early-waterlogging mainly by stomatal limitations, and by non-stomatal constraints (lower mesophyll conductance and damage to photosynthetic apparatus as revealed by chlorophyll fluorescence) during late-waterlogging. In rapeseed, photosynthesis was mostly reduced by non-stomatal limitations during early- and late-waterlogging, which also impacted shoot and root growth. Early-waterlogged plants of both barley and rapeseed were able to recover in growth upon drainage, and seed production reached ca. 79-85% of the controls, while late-waterlogged plants only attained 26-32% in seed production. Field pea showed no ability to develop root aerenchyma when waterlogged, and its photosynthesis (and stomatal and mesophyll conductances) was rapidly decreased by the stress. Consequently, waterlogging drastically reduced field pea seed production to 6% of controls both at early- and late-stages with plants being unable to resume growth upon drainage. In conclusion, wheat generates a set of adaptive responses to withstand 14 days of waterlogging, barley and rapeseed can still produce significant yield if transiently waterlogged during early plant stages but are more adversely impacted at the late stage, and field pea is not suitable for areas prone to waterlogging events of 14 days at either growth stage.

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.

Energetics of acclimation to NaCl by submerged, anoxic rice seedlings.

BACKGROUND AND AIMS: Our aim was to elucidate how plant tissues under a severe energy crisis cope with imposition of high NaCl, which greatly increases ion fluxes and hence energy demands. The energy requirements for ion regulation during combined salinity and anoxia were assessed to gain insights into ion transport processes in the anoxia-tolerant coleoptile of rice. METHODS: We studied the combined effects of anoxia plus 50 or 100 mm NaCl on tissue ions and growth of submerged rice (Oryza sativa) seedlings. Excised coleoptiles allowed measurements in aerated or anoxic conditions of ion net fluxes and O2 consumption or ethanol formation and by inference energy production. KEY RESULTS: Over 80 h of anoxia, coleoptiles of submerged intact seedlings grew at 100 mm NaCl, but excised coleoptiles, with 50 mm exogenous glucose, survived only at 50 mm NaCl, possibly due to lower energy production with glucose than for intact coleoptiles with sucrose as substrate. Rates of net uptake of Na+ and Cl- by coleoptiles in anoxia were about half those in aerated solution. Ethanol formation in anoxia and O2 uptake in aerobic solution were each increased by 13-15 % at 50 mm NaCl, i.e. ATP formation was stimulated. For acclimation to 50 mm NaCl, the anoxic tissues used only 25 % of the energy that was expended by aerobic tissues. Following return of coleoptiles to aerated non-saline solution, rates of net K+ uptake recovered to those in continuously aerated solution, demonstrating there was little injury during anoxia with 50 mm NaCl. CONCLUSION: Rice seedlings survive anoxia, without the coleoptile incurring significant injury, even with the additional energy demands imposed by NaCl (100 mm when intact, 50 mm when excised). Energy savings were achieved in saline anoxia by less coleoptile growth, reduced ion fluxes as compared to aerobic coleoptiles and apparent energy-economic ion transport systems.

A major locus involved in the formation of the radial oxygen loss barrier in adventitious roots of teosinte Zea nicaraguensis is located on the short-arm of chromosome 3.

A radial oxygen loss (ROL) barrier in roots of waterlogging-tolerant plants promotes oxygen movement via aerenchyma to the root tip, and impedes soil phytotoxin entry. The molecular mechanism and genetic regulation of ROL barrier formation are largely unknown. Zea nicaraguensis, a waterlogging-tolerant wild relative of maize (Zea mays ssp. mays), forms a tight ROL barrier in its roots when waterlogged. We used Z. nicaraguensis chromosome segment introgression lines (ILs) in maize (inbred line Mi29) to elucidate the chromosomal region involved in regulating root ROL barrier formation. A segment of the short-arm of chromosome 3 of Z. nicaraguensis conferred ROL barrier formation in the genetic background of maize. This chromosome segment also decreased apoplastic solute permeability across the hypodermis/exodermis. However, the IL and maize were similar for suberin staining in the hypodermis/exodermis at 40 mm and further behind the root tip. Z. nicaraguensis contained suberin in the hypodermis/exodermis at 20 mm and lignin at the epidermis. The IL with ROL barrier, however, did not contain lignin in the epidermis. Discovery of the Z. nicaraguensis chromosomal region responsible for root ROL barrier formation has improved knowledge of this trait and is an important step towards improvement of waterlogging tolerance in maize.

Evaluation of root porosity and radial oxygen loss of disomic addition lines of Hordeum marinum in wheat.

Hordeum marinum Huds. is a waterlogging-tolerant wild relative of wheat (Triticum aestivum L.). Greater root porosity (gas volume per root volume) and formation of a barrier to reduce root radial O2 loss (ROL) contribute to the waterlogging tolerance of H. marinum and these traits are evident in some H. marinum-wheat amphiploids. We evaluated root porosity, ROL patterns and tolerance to hypoxic stagnant conditions for 10 various H. marinum (two accessions) disomic chromosome addition (DA) lines in wheat (two varieties), produced from two H. marinum-wheat amphiploids and their recurrent wheat parents. None of the DA lines had a barrier to ROL or higher root porosity than the wheat parents. Lack of a root ROL barrier in the six DA lines for H. marinum accession H21 in Chinese Spring (CS) wheat indicates that the gene(s) for this trait do not reside on one of these six chromosomes; unfortunately, chromosome 3 of H. marinum has not been isolated in CS. Unlike the H21-CS amphiploid, which formed a partial ROL barrier in roots, the H90-Westonia amphiploid and the four derived DA lines available did not. The unaltered root aeration traits in the available DA lines challenge the strategy of using H. marinum as a donor of these traits to wheat.

Tissue-specific root ion profiling reveals essential roles of the CAX and ACA calcium transport systems in response to hypoxia in Arabidopsis.

Waterlogging is a major abiotic stress that limits the growth of plants. The crucial role of Ca(2+) as a second messenger in response to abiotic and biotic stimuli has been widely recognized in plants. However, the physiological and molecular mechanisms of Ca(2+) distribution within specific cell types in different root zones under hypoxia is poorly understood. In this work, whole-plant physiological and tissue-specific Ca(2+) changes were studied using several ACA (Ca(2+)-ATPase) and CAX (Ca(2+)/proton exchanger) knock-out Arabidopsis mutants subjected to waterlogging treatment. In the wild-type (WT) plants, several days of hypoxia decreased the expression of ACA8, CAX4, and CAX11 by 33% and 50% compared with the control. The hypoxic treatment also resulted in an up to 11-fold tissue-dependent increase in Ca(2+) accumulation in root tissues as revealed by confocal microscopy. The increase was much higher in stelar cells in the mature zone of Arabidopsis mutants with loss of function for ACA8, ACA11, CAX4, and CAX11 In addition, a significantly increased Ca(2+) concentration was found in the cytosol of stelar cells in the mature zone after hypoxic treatment. Three weeks of waterlogging resulted in dramatic loss of shoot biomass in cax11 plants (67% loss in shoot dry weight), while in the WT and other transport mutants this decline was only 14-22%. These results were also consistent with a decline in leaf chlorophyll fluorescence (F v/F m). It is suggested that CAX11 plays a key role in maintaining cytosolic Ca(2+) homeostasis and/or signalling in root cells under hypoxic conditions.

Salt sensitivity in chickpea: Growth, photosynthesis, seed yield components and tissue ion regulation in contrasting genotypes.

Chickpea is a relatively salt sensitive species but shows genotypic variation for salt tolerance, measured as grain yield per plant in mild-to-moderately saline soil. This experiment was designed to evaluate some physiological responses to salinity in three contrasting genotypes. One tolerant (Genesis836), one moderately tolerant (JG11) and one sensitive (Rupali) genotype were grown for 108d in non-saline nutrient solution (controls) and two levels of salinity treatment (30 and 60mM NaCl). No plants survived to maturity in the 60mM NaCl treatment; however, Genesis836 survived longer (87d) than JG11 (67d) while Rupali died after 27d; only Genesis836 flowered, but no pods were filled. At 30mM NaCl, Genesis836 produced a few filled pods, whereas JG11 and Rupali did not. Genotypic differences in plant dry mass at the vegetative stage were evident only at 60mM NaCl, while at maturity differences were evident at 30mM NaCl. Photosynthesis was maintained to different degrees by the three genotypes (e.g. at 30mM NaCl, 35-81% of controls; highest in Genesis836); photosynthesis was restricted predominately due to non-stomatal limitations as the intercellular CO2 concentration was only modestly affected (94-99% of controls). Photosystem II damage was evident in the less tolerant genotypes (e.g. at 30mM NaCl, actual quantum efficiency of photosystem II values were 63-96% of controls). Across treatments, shoot dry mass was negatively correlated with both Na(+) and Cl(-) shoot concentrations. However, the sensitive genotype (Rupali) had equal or lower concentrations of these ions in green leaves, stems or roots compared to tolerant genotypes (JG11 and Genesis836); ion 'exclusion' does not explain variation for salt tolerance among these three chickpea genotypes. The large difference between Rupali (sensitive) and Genesis836 (tolerant) in the salt-induced reduction in net photosynthesis via non-stomatal limitations and the assessed damage to photosystem II, but with similar leaf ion concentrations, provides evidence that variation in 'tissue tolerance' of Na(+) and/or Cl(-) in leaves contributes to the differential salt tolerance of these chickpea genotypes.

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.

Linking oxygen availability with membrane potential maintenance and K+ retention of barley roots: implications for waterlogging stress tolerance.

Oxygen deprivation is a key determinant of root growth and functioning under waterlogging. In this work, changes in net K(+) flux and membrane potential (MP) of root cells were measured from elongation and mature zones of two barley varieties under hypoxia and anoxia conditions in the medium, and as influenced by ability to transport O2 from the shoot. We show that O2 deprivation results in an immediate K(+) loss from roots, in a tissue- and time-specific manner, affecting root K(+) homeostasis. Both anoxia and hypoxia induced transient membrane depolarization; the extent of this depolarization varied depending on severity of O2 stress and was less pronounced in a waterlogging-tolerant variety. Intact roots of barley were capable of maintaining H(+) -pumping activity under hypoxic conditions while disrupting O2 transport from shoot to root resulted in more pronounced membrane depolarization under O2 -limited conditions and in anoxia a rapid loss of the cell viability. It is concluded that the ability of root cells to maintain MP and cytosolic K(+) homeostasis is central to plant performance under waterlogging, and efficient O2 transport from the shoot may enable operation of the plasma membrane H(+) -ATPase in roots even under conditions of severe O2 limitation in the soil solution.