Carbon and nitrogen metabolic regulation in freshwater plant Ottelia alismoides in response to carbon limitation: A metabolite perspective.

Carbon and nitrogen metabolism are basic, but pivotal metabolic pathways in plants and are tightly coupled. Maintaining the balance of carbon and nitrogen metabolism is critical for plant survival. Comprehensively revealing the metabolic balance of carbon-nitrogen interactions is important and helpful for understanding the adaptation of freshwater plants to CO2 limited aqueous environment. A comprehensive metabolomics analysis combined with physiological measurement was performed in the freshwater plant Ottelia alismoides acclimated to high and low CO2, respectively, for a better understanding of how the carbon and nitrogen metabolic adjustment in freshwater plants respond to carbon limitation. The present results showed that low CO2 acclimated O. alismoides exhibited significant diurnal titratable acidity and malate fluctuations, as well as an opposite diel pattern of starch change and high enzymatic activities required for crassulacean acid metabolism (CAM) photosynthesis, which indicates that CAM was induced under low CO2. Moreover, the metabolomic analysis showed that most intermediates of glycolysis, pentose phosphate pathway (PPP) and tricarboxylic acid (TCA) cycle, were increased under low CO2, indicative of active respiration in low-CO2-treated O. alismoides. Meanwhile, the majority of amino acids involved in pathways of glutamate and arginine metabolism, aspartate metabolism, and the branched-chain amino acids (BCAAs) metabolism were significantly increased under low CO2. Notably, γ-aminobutyric acid (GABA) level was significantly higher in low CO2 conditions, indicating a typical response with GABA shunt compensated for energy deprivation at low CO2. Taken together, we conclude that in low-CO2-stressed O. alismoides, CAM photosynthesis was induced, leading to higher carbon and nitrogen as well as energy requirements. Correspondingly, the respiration was greatly fueled via numerous starch degradation to ensure CO2 fixation in dark, while accompanied by linked promoted N metabolism, presumably to produce energy and alternative carbon sources and nitrogenous substances for supporting the operation of CAM and enhancing tolerance for carbon limitation. This study not only helps to elucidate the regulating interaction between C and N metabolism to adapt to different CO2 but also provides novel insights into the effects of CO2 variation on the metabolic profiling of O. alismoides.

Responses of Leaf Anatomy and CO2 Concentrating Mechanisms of the Aquatic Plant Ottelia cordata to Variable CO2.

Acclimation to variable CO2 was studied in floating leaves of the freshwater monocot Ottelia cordata grown in either low or high CO2. The most striking anatomical variations responding to high CO2 included the enlarged upper epidermal cells and the decreased area of epidermal chloroplasts. Stomata that distributed on the upper surface, and the stomatic chamber area, showed no significant response to high CO2. pH-drift experiments indicated that floating leaves of O. cordata were able to use bicarbonate regardless of CO2 concentrations. Photosynthetic enzyme activities and patterns of organic acids fluctuation confirmed that floating leaves of O. cordata can operate CAM only at low CO2, and perform C4-like metabolism at both high and low CO2. Overall, the present results imply that the floating leaves of O. cordata does not just rely on the atmospheric CO2 for its inorganic carbon, but is also dependent on CO2 and bicarbonate in the water. By showing these effects of CO2 variation, we highlight the need for further experimental studies on the regulatory mechanisms in O. cordata floating leaves, that prevent futile cycling among the three CO2 concentrating mechanisms (bicarbonate use, C4, and CAM metabolism) and the strategy for exploiting atmospheric CO2, as well as studies on the detailed biochemical pathway for C4 and CAM metabolism in this species.

External α-carbonic anhydrase and solute carrier 4 are required for bicarbonate uptake in a freshwater angiosperm.

The freshwater monocot Ottelia alismoides is the only known species to operate three CO2-concentrating mechanisms (CCMs): constitutive bicarbonate (HCO3-) use, C4 photosynthesis, and facultative Crassulacean acid metabolism, but the mechanism of HCO3- use is unknown. We found that the inhibitor of an anion exchange protein, 4,4'-diisothio-cyanatostilbene-2,2'-disulfonate (DIDS), prevented HCO3- use but also had a small effect on CO2 uptake. An inhibitor of external carbonic anhydrase (CA), acetazolamide (AZ), reduced the affinity for CO2 uptake but also prevented HCO3- use via an effect on the anion exchange protein. Analysis of mRNA transcripts identified a homologue of solute carrier 4 (SLC4) responsible for HCO3- transport, likely to be the target of DIDS, and a periplasmic α-carbonic anhydrase 1 (α-CA1). A model to quantify the contribution of the three different pathways involved in inorganic carbon uptake showed that passive CO2 diffusion dominates inorganic carbon uptake at high CO2 concentrations. However, as CO2 concentrations fall, two other pathways become predominant: conversion of HCO3- to CO2 at the plasmalemma by α-CA1 and transport of HCO3- across the plasmalemma by SLC4. These mechanisms allow access to a much larger proportion of the inorganic carbon pool and continued photosynthesis during periods of strong carbon depletion in productive ecosystems.

Structural basis for C4 photosynthesis without Kranz anatomy in leaves of the submerged freshwater plant Ottelia alismoides.

BACKGROUND AND AIMS: Ottelia alismoides (Hydrocharitaceae) is a freshwater macrophyte that, unusually, possesses three different CO2-concentrating mechanisms. Here we describe its leaf anatomy and chloroplast ultrastructure, how these are altered by CO2 concentration and how they may underlie C4 photosynthesis. METHODS: Light and transmission electron microscopy were used to study the anatomy of mature leaves of O. alismoides grown at high and low CO2 concentrations. Diel acid change and the activity of phosphoenolpyruvate carboxylase were measured to confirm that CAM activity and C4 photosynthesis were present. KEY RESULTS: When O. alismoides was grown at low CO2, the leaves performed both C4 and CAM photosynthesis whereas at high CO2 leaves used C4 photosynthesis. The leaf comprised an upper and lower layer of epidermal cells separated by a large air space occupying about 22 % of the leaf transverse-section area, and by mesophyll cells connecting the two epidermal layers. Kranz anatomy was absent. At low CO2, chloroplasts in the mesophyll cells were filled with starch even at the start of the photoperiod, while epidermal chloroplasts contained small starch grains. The number of chloroplasts in the epidermis was greater than in the mesophyll cells. At high CO2, the structure was unchanged but the thicknesses of the two epidermal layers, the air space, mesophyll and the transverse-section area of cells and air space were greater. CONCLUSIONS: Leaves of O. alismoides have epidermal and mesophyll cells that contain chloroplasts and large air spaces but lack Kranz anatomy. The high starch content of mesophyll cells suggests they may benefit from an internal source of CO2, for example via C4 metabolism, and are also sites of starch storage. The air spaces may help in the recycling of decarboxylated or respired CO2. The structural similarity of leaves at low and high CO2 is consistent with the constitutive nature of bicarbonate and C4 photosynthesis. There is sufficient structural diversity within the leaf of O. alismoides to support dual-cell C4 photosynthesis even though Kranz anatomy is absent.

Assessing interactions between environmental factors and aquatic toxicity: Influences of dissolved CO2 and light on Cd toxicity in the aquatic macrophyte Potamogeton crispus.

The objective of this study was to investigate the combined effects of varying dissolved CO2 concentration (ambient CO2, 3˜17 µmol L-1, elevated CO2, 48˜81 µmol L-1) and light intensity (high light, c. 150 µmol photon m-2 s-1, low light, c. 25 µmol photon m-2 s-1) on the bioaccumulation and phytotoxicity of cadmium (Cd) in a macrophyte Potamogeton crispus, under constant Cd exposure. The data confirmed that 100 µM Cd led to adverse changes in morphology, ultrastructure and biochemistry in P. crispus. The toxic effects depended strongly on CO2 concentration and light intensity: elevated CO2 and high light both increased Cd concentrations in P. crispus, and there was a significant interaction between the two factors. Compared to high light grown plants, the photochemical efficiency and chlorophyll content of low light grown P. crispus were much less affected and the MDA content was lower, when exposed to 100 µM Cd. In addition, an antioxidative response was observed with a significant increase in SOD, POD and GST activities, indicating that low light grown P. crispus are more protected against Cd toxicity. When compared with ambient CO2 concentrations, chlorophyll content, chlorophyll fluorescence, photosynthetic rate and starch content, as well as the activity of SOD and GST, were significantly enhanced in Cd treated P. crispus under elevated CO2. This suggests that elevated CO2 reduced Cd toxicity in P. crispus by increasing photosynthesis and enhancing the antioxidant system. Moreover, the statistical results showed that dissolved CO2 and light had additive effects on Cd toxicity in P. crispus, reflected by the physiological parameters of total chlorophyll content, SOD activity and MDA content, indicating that the combination of high CO2 and low light produced more protection against Cd toxicity than did the factors alone. Based on the results of this study, it appears clear that referring to a specific site in aquatic ecosystem, dissolved CO2 concentration and light availability should be considered when assessing and managing Cd impacts on aquatic plants.

Characterization of the Cd-resistant fungus Aspergillus aculeatus and its potential for increasing the antioxidant activity and photosynthetic efficiency of rice.

Considerable evidence exists that microorganisms play a significant role in the remediation of soil contaminated with heavy metals. Aspergillus aculeatus (A. aculeatus) isolated from Cd-polluted soil has been shown to increase the tolerance of turfgrasses to Cd stress. In this study, we assessed the tolerance, biosorption capacity for Cd and surface characteristics of this fungus and investigated the effect of plant inoculation with A. aculeatus on the lipid peroxidation, antioxidant activities and photosynthetic rates in rice cultivated in Cd-contaminated soil. The results indicated that the removal efficiency of A. aculeatus was 46.8% at a Cd concentration of 10 mg L-1. The A. aculeatus strains had the capacity to produce indole acetic acid, siderophore, and 1-aminocyclopropane-1-carboxylate deaminase and to solubilize phosphate. The O2- accumulation and the amount of MDA in rice roots inoculated with A. aculeatus were significantly lower than those in uninoculated plants. Nevertheless, no decrease in leaf ROS accumulation and photosynthetic activity was observed between the inoculated and uninoculated plants. Inoculation with A. aculeatus contained more of the ROS-scavenging metabolite GSH, a higher GSH/GSSG ratio, and higher antioxidative enzyme (SOD, POD, and CAT) activities, possibly explaining the lower ROS concentrations observed in inoculated roots in the presence of Cd. These results suggest that application of A. aculeatus has the potential to protect crops against Cd stress.

Cadmium Tolerance of Perennial Ryegrass Induced by Aspergillus aculeatus.

Cadmium (Cd) pollution is becoming increasingly prevalent, posing a global environmental hazard due to its negative effects on plants growth and human health. Phytoremediation is a green technology that involves uptake of Cd from the soil by a combination of plants and associated microbes. The objective of this study was to investigate the role of Aspergillus aculeatus in perennial ryegrass Cd tolerance. This fungus produced indole-3-acetic acid, siderophores, and 1-aminocyclopropane-1-carboxylate deaminase. Physiological traits including growth rate, turf quality and chlorophyll content were measured to evaluate the physiological responses of perennial ryegrass to Cd stress. These physiological traits were improved after inoculated with A. aculeatus. Inoculation of A. aculeatus actively reduced DTPA-Cd concentration in the soil and Cd translocation to plant shoots. Chlorophyll a fluorescence transient and the C/N ratio in shoots were elevated by A. aculeatus, which implied that the fungus could protect the photosystem II against Cd stress and increase the photosynthetic efficiency. These results suggested that A. aculeatus is beneficial in improving Cd tolerance of perennial ryegrass and reducing Cd-induced injuries, thus, it has promising potential for application of phytostabilization in Cd contaminated soil.

The Fungus Aspergillus aculeatus Enhances Salt-Stress Tolerance, Metabolite Accumulation, and Improves Forage Quality in Perennial Ryegrass.

Perennial ryegrass (Lolium perenne) is an important forage grass with high yield and superior quality in temperate regions which is widely used in parks, sport field, and other places. However, perennial ryegrass is moderately tolerant to salinity stress compared to other commercial cultivars and salt stress reduces their growth and productivity. Aspergillus aculeatus has been documented to participate in alleviating damage induced by salinity. Therefore, the objective of this study was to investigate the mechanisms underlying A. aculeatus-mediated salt tolerance, and forage quality of perennial ryegrass exposed to 0, 200, and 400 mM NaCl concentrations. Physiological markers and forage quality of perennial ryegrass to salt stress were evaluated based on the growth rate, photosynthesis, antioxidant enzymes activity, lipid peroxidation, ionic homeostasis, the nutritional value of forage, and metabolites. Plants inoculated with A. aculeatus exhibited higher relative growth rate (RGR), turf and forage quality under salt stress than un-inoculated plants. Moreover, in inoculated plants, the fungus remarkably improved plant photosynthetic efficiency, reduced the antioxidant enzymes activity (POD and CAT), and attenuated lipid peroxidation (decreased H2O2 and MDA accumulation) induced by salinity, compared to un-inoculated plants. Furthermore, the fungus also acts as an important role in maintaining the lower Na/K ratio and metabolites and lower the amino acids (Alanine, Proline, GABA, and Asparagine), and soluble sugars (Glucose and Fructose) for inoculated plants than un-inoculated ones. Our results suggest that A. aculeatus may be involved in modulating perennial ryegrass tolerance to salinity in various ways.

Effects of cadmium-resistant fungi Aspergillus aculeatus on metabolic profiles of bermudagrass [Cynodondactylon (L.)Pers.] under Cd stress.

Plants' tolerance to heavy metal stress may be induced by the exploitation of microbes. The objectives of this study were to investigate the effect of cadmium (Cd)-resistant fungus, Aspergillus aculeatus, on tolerance to Cd and alteration of metabolites in bermudagrass under Cd stress, and identify the predominant metabolites associated with Cd tolerance. Two genotypes of bermudagrass with contrasting Cd tolerance (Cd-sensitive 'WB92' and Cd-tolerant 'WB242') were exposed to 0, 50, 150 and 250 mg kg-1 Cd for 21 days. Physiological responses of bermudagrass to Cd stress were evaluated based on the relative growth rate (RGR) and normalized relative transpiration rate (NRT). Plants inoculated with A. aculeatus exhibited higher RGR and NRT under Cd stress than those of non-inoculated plants, regardless of genotypes. A total of 32 Cd-responsive metabolites in leaves and 21 in roots were identified in the two genotypes, including organic acids, amino acids, sugars, and fatty acids and others. Interestingly, under Cd stress, the leaves of inoculated 'WB92' accumulated less citric acid, aspartic acid, glutamic acid, sucrose, galactose, but more sorbose and glucose, while inoculated 'WB242' leaves had less citric acid, malic acid, sucrose, sorbose, but more fructose and glucose, compared to non-inoculated plants. In 'WB92' roots, the A. aculeatus reduced mannose content, but increased trehalose and citric acid content, while in 'WB242', it decreased sucrose, but enhanced citric acid content, compared to Cd regime. The results of this study suggest that A. aculeatus may induce accumulation of different metabolites associated with Cd tolerance in bermudagrass.

Amelioration of Salt Stress on Bermudagrass by the Fungus Aspergillus aculeatus.

There is considerable evidence that plant abiotic-stress tolerance can be evoked by the exploitation of a globally abundant microbe. A. aculeatus, which was initially isolated from the rhizosphere of bermudagrass, has been shown to increase heavy metal tolerance in turfgrasses. Here, we report on the potential of A. aculeatus to induce tolerance to salt stress in bermudagrass. Physiological markers for salt stress, such as plant growth rate, lipid peroxidation, photosynthesis, and ionic homeostasis were assessed. Results indicated that strain A. aculeatus produced indole-3-acetic acid (IAA) and siderophores and exhibited a greater capacity for Na+ absorption under salt stress. The plant inoculation by A. aculeatus increased plant growth and attenuated the NaCl-induced lipid peroxidation in roots and leaves of bermudagrass. The fungus significantly elevated the amount of IAA and glutathione and slightly enhanced photosynthetic efficiency of salt-treated bermudagrass. Tissues of inoculated plants had significantly increased concentrations of K+ but lower Na+ concentrations than those of uninoculated regimes. It appears that the role of A. aculeatus in alleviating bermudagrass salt stress is partly to produce IAA, to increase the activity of antioxidases, to absorb Na+ by fungal hyphae, and to prevent the plant from ionic homeostasis disruption.