Comparative ionomics and metabolomics in extremophile and glycophytic Lotus species under salt stress challenge the metabolic pre-adaptation hypothesis

The legume genus Lotus includes glycophytic forage crops and other species adapted to extreme environments, such as saline soils. Understanding salt tolerance mechanisms will contribute to the discovery of new traits which may enhance the breeding efforts towards improved performance of legumes in marginal agricultural environments. Here, we used a combination of ionomic and gas chromatography‐mass spectrometry (GC‐MS)‐based metabolite profilings of complete shoots (pooling leaves, petioles and stems) to compare the extremophile Lotus creticus, adapted to highly saline coastal regions, and two cultivated glycophytic grassland forage species, Lotus corniculatus and Lotus tenuis. L. creticus exhibited better survival after exposure to long‐term lethal salinity and was more efficient at excluding Cl^‐ from the shoots than the glycophytes. In contrast, Na⁺ levels were higher in the extremophile under both control and salt stress, a trait often observed in halophytes. Ionomics demonstrated a differential rearrangement of shoot nutrient levels in the extremophile upon salt exposure. Metabolite profiling showed that responses to NaCl in L. creticus shoots were globally similar to those of the glycophytes, providing little evidence for metabolic pre‐adaptation to salinity. This study is the first comparing salt acclimation responses between extremophile and non‐extremophile legumes, and challenges the generalization of the metabolic salt pre‐adaptation hypothesis.     

Water Relations Only Partly Explain the Distributions of Three Perennial Plant Species in a Semi-arid Environment

The water relations and stomatal conductances of three perennial plant species, Stipa tenacissima L., Anthyllis cytisoides L., and Retama sphaerocarpa (L.) Boiss., dominant on the upper slopes, mid-slopes and floor of a valley, respectively, in semi-arid south-east Spain, were investigated to test the hypothesis that differences in plant-soil water relations could account for the different distributions of each species in the catena. Diurnal measurements of water potential (Ψ_w), relative water content (RWC) and stomatal conductance (g_s) of leaves were made over one year. Leaf temperature, air humidity, wind-speed and incident quantum flux density were measured concurrently. Soil water content was determined gravimetrically at 0 – 5 cm and 15 – 20 cm depths. Measurements of Ψ_w, RWC and g_s were analysed according to meteorological conditions, based on the maxima for daily air temperature and atmospheric saturation water vapour deficit and on soil moisture content. The hypothesis that plant-soil water relations can explain the distribution of the three species along the catena from valley side to valley floor was rejected for Anthyllis and Stipa but confirmed for Retama. This revised version was published online in July 2006 with corrections to the Cover Date.     

Photosynthetic parameters and leaf water potential of five common bean genotypes under mild water deficit

The leaf water potential, gas exchange and chlorophyll fluorescence were evaluated in five common bean (Phaseolus vulgaris) genotypes A222, A320, BAT477, Carioca and Ouro Negro subjected to moderate water deficit. At the maximum water deficit (10 d of water withholding), the leaf water potential of genotypes A320 and A222 was higher (−0.35 and −0.50 MPa) when compared to the other genotypes (−0.67 to −0.77 MPa). The stomatal conductance and net photosynthetic rate were significantly reduced in all genotypes due to the water deficit. The greater reduction in stomatal conductance of A320 under drought resulted in high intrinsic water use efficiency. Mild water deficit affected the photochemical apparatus in bean genotypes probably by down-regulation since plants did not show photoinhibition. The photochemical apparatus of A222 and A320 genotypes was more sensitive to drought stress, showing reduced apparent electron transport even after the recovery of plant water status. On the other hand, even after 10 d of water withholding, the maximum efficiency of photosystem 2 was not affected, what suggest efficiency of the photoprotection mechanisms.     

Growth and Water Relations of Lotus Creticus Creticus Plants as Affected by Salinity

Young plants of Lotus creticus creticus growing in a hydroponic culture were submitted to 0, 70 and 140 mM NaCl treatments for 28 d and the growth and ecophysiological characteristics of these plants have been studied. The growth of Lotus plants was not affected by salinity when applied for a short period (about 15 d); however, 140 mM NaCl induced a decrease in shoot RGR at the end of the treatment. The root growth was not decreased, even it was stimulated by 140 mM NaCl. The osmotic adjustment of Lotus plants at 70 and 140 mM NaCl maintained constant pressure potential, avoiding the visual wilting. For a similar leaf water potential, cuticular transpiration of salinized plants was lower than in control plants due to the salinity effect on the cuticle. Moreover, the presence of hairy leaves (60 and 160 trichomes per mm² in young and adult leaves, respectively) allows keeping almost 81 % of sprayed water and absorbing the 9 % of the water retained, decreased the epidermal conductance to water vapour diffusion.     

Diurnal Gas Exchange and Superior Resources Use Efficiency of Typical C4 Species in Hunshandak Sandland,China

Net photosynthetic rate (P _N), transpiration rate (E), stomatal conductance (g _s), leaf water potential (ψ_leaf), leaf nitrogen content, and photosynthetic nitrogen use efficiency (PNUE) were compared between a typical C₄ plant, Agriophyllum squarrosum and a C₃ plant, Leymus chinensis, in Hunshandak Sandland, China. The plant species showed different diurnal gas exchange patterns on June 12–14 when photosynthetic photon flux density (PPFD), air temperature (T _air), and water potential were moderate. P _N, E, and g _s of A. squarrosum showed distinct single peak while those of L. chinensis were depressed at noon and had two peaks in their diurnal courses. Gas exchange traits of both species showed midday depression under higher photosynthetic photon flux density (PPFD) and T _air when Ψ_leaf was significantly low down on August 6–8. However, those of A. squarrosum were depressed less seriously. Moreover, A. squarrosum had higher P _N, Ψ_leaf, water use efficiency (WUE), and PNUE than L. chinensis. Thus A. squarrosum was much more tolerant to heat and high irradiance and could utilise the resources on sand area more efficiently than L. chinensis. Hence species like A. squarrosum may be introduced and protected to reconstruct the degraded sand dunes because of their higher tolerance to stress and higher resource use efficiency. This revised version was published online in August 2006 with corrections to the Cover Date.     

Carbon isotope discrimination and stomatal responses of mature Pinus sylvestris L. trees exposed in situ for three years to elevated CO2 and temperature

The Climate Change Experiment (CLIMEX) is a unique large scale facility in which an entire undisturbed catchment of boreal vegetation has been exposed to elevated CO₂ (560 ppm) and temperature (+3°C summer, +5°C winter) for the past three years with all the soil-plant-atmosphere linkages intact. Here, carbon isotope composition and stomatal density have been analysed from sequential year classes of needles of mature Scots pine trees (Pinus sylvestris L.) to investigate the response of time-integrated water-use efficiency (UWE) and stomatal density to CO₂ enrichment and climate change. Carbon isotope discrimination decreased and WUE increased in cohorts of needles developing under increased CO₂ and temperature, compared to needles on the same trees developing in pretreatment years. Mid-season instantaneous gas exchange, measured on the same trees for the past four years, indicated that these responses resulted from higher needle photosynthetic rates and reduced stomatal conductance. Needles of P. sylvestris developing under increased CO₂ and temperature had consistently lower stomatal densities than their ambient grown counterparts on the same trees. The stomatal density of P. sylvestris needles was inversely correlated with δ¹³C-derived WUE, implying some effect of this morphological response on leaf gas exchange. Future atmospheric CO₂ and temperature increases are therefore likely to improve the water economy of P. sylvestris, at least at the scale of individual needles, by affecting stomatal density and gas exchange processes.     

Water Stress Induced Changes in Anatomy of Tomato Leaf Epidermes

Anatomical changes of leaf epidermes of tomato plants (Lycopersicon esculentum Mill. cv. INCA 9) submitted to water stress in the preflowering stage were studied. 20 d after germination, plants were subjected to three treatments: 1) 100 % of evapotranspired water was applied every day, 2) from 100 up to 10 % of evapotranspired water was applied every day, and 3) water supply was completely suppressed. Trichome density was similar in apical, middle and basal zones, and adaxial and abaxial leaf surfaces. Stomatal density and length, and epidermal cell length and width had similar values on the same leaf surface, but the values were higher on the abaxial than on the adaxial leaf surface. The water deficit had little effect on number of trichomes, length and width of epidermal cells and length of stomata, and decreased the stomatal density especially on adaxial surface.     

In vitro propagation ofMedicago andLotus species by node culture

Summary Nodes ofMedicago sativa, Lotus corniculatus, Lotus tenuis, andLotus pedunculatus were cultured on MS basal media with different growth regulators. InM. sativa each node produced one shoot and the apical dominance was unaffected by high levels of cytokinins, and subsequent cycles of culture. Shoot development was stimulated by the presence ofN ⁶-isopentenyl-adenine in the culture medium and was dependent on the genotype of the explant. Shoot development was not affected by the original position of the node on the plant nor by the plant age. Shoots rooted in MS medium gelled with starch and containing 2 mg·liter⁻¹ indol-3-acetic acid. In the threeLotus species, node culture was a more effective technique than inM. sativa. The number of shoots per node increased with the time of culture and with the presence of 0.05 mg·liter⁻¹ of 6-benzylaminopurine. The highest number of shoots derived from one node was achieved inL. pedunculatus and inL. tenuis by culturing single nodes, whereas inL. corniculatus stem segments had to be totally covered by the medium for success. Rooting was easily achieved in MS medium with or without auxins.     

Stomatal patchiness in Mediterranean evergreen sclerophylls

Midday depression of net photosynthesis and transpiration in the Mediterranean sclerophylls Arbutus unedo L. and Quercus suber L. occurs with a depression of mesophyll photosynthetic activity as indicated by calculated carboxylation efficiency (CE) and constant diurnal calculated leaf intercellular partial pressure of CO₂ (C_i). This work examines the hypothesis that this midday depression can be explained by the distribution of patches of either wide-open or closed stomata on the leaf surface, independent of a coupling mechanism between stomata and mesophyll that results in a midday depression of photosynthetic activity of the mesophyll. Pressure infiltration of four liquids differing in their surface tension was used as a method to show the occurrence of stomatal patchiness and to determine the status of stomatal aperture within the patches. Liquids were selected such that the threshold leaf conductance necessary for infiltration through the stomatal pores covered the expected diurnal range of calculated leaf conductance (g) for these species. Infiltration experiments were carried out with leaves of potted plants under simulated Mediterranean summer conditions in a growth chamber. For all four liquids, leaves of both species were found to be fully infiltratable in the morning and in the late afternoon while during the periods leading up to and away from midday the leaves showed a pronounced patchy distribution of infiltratable and non-infiltratable areas. Similar linear relationships between the amount of liquid infiltrated and g (measured by porometry prior to detachment and infiltration) for all liquids clearly revealed the existence of pneumatically isolated patches containing only wide-open or closed stomata. The good correspondence between the midday depression of CE, calculated under the assumption of no stomatal patchiness, and the diurnal changes in non-infiltratable leaf area strongly indicates that the apparent reduction in mesophyll activity results from assuming no stomatal patchiness. It is suggested that simultaneous responses of stomata and mesophyll activity reported for other species may also be attributed to the occurrence of stomatal patchiness. In Quercus coccifera L., where the lack of constant diurnal calculated C_i and major depression of measured CE at noontime indicates different stomatal behavior, non-linear and dissimilar relationships between g and the infiltratable quantities of the four liquids were found. This indicates a wide distribution of stomatal aperture on the leaf surface rather than only wide-open or closed stomata.Dedicated to Professor Otto L. Lange on the occasion of his 65th birthday     

Effects of Di-n-Butyl Phthalate on Mycorrhizal and Non-Mycorrhizal Cowpea Plants

This study examined the effect of mycorrhizal colonization with Glomus intraradices on physiological parameters and foliar nutrient concentrations in Olea europaea L. subsp. sylvestris and Rhamnus lycioides L. seedlings subjected to well-watered or drought-stressed conditions. Under drought stress, mycorrhizal O. europaea seedlings showed significantly higher photosynthetic and transpiration rates, stomatal conductance and foliar P concentration than its similarly-sized non-mycorrhizal counterpart. The intrinsic water use efficiency (photosynthetic rate to stomatal conductance ratio) was not change in O. europaea and decreased in R. lycioides seedlings due to mycorrhizal colonization under both well-watered and drought-stress conditions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Contrasting strategies to cope with drought by invasive and endemic species of Lantana in Galapagos

This study compares how Lantana camara, an invasive species, and L. peduncularis, an autochthonous one, cope with drought in Galapagos. Soil surface temperature was the abiotic environmental parameter that best explained variations in photosynthetic stress. Higher soil surface temperatures were recorded in the lowlands and in rain-shadow areas, which were also the driest areas. L. peduncularis, with a shallow root system, behaved as a drought-tolerant species, showing lower relative growth rates, which decreased with leaf water content and higher photosynthetic stress levels in the lowlands and in a northwest rain-shadow area in comparison with higher and wetter locations. Its basal and maximal fluorescences decreased at lower altitudes, reflecting the recorded drops in chlorophyll concentration. In contrast, L. camara with a deep root system behaved as a drought-avoiding species, showing leaf and relative water contents higher than 55% and avoiding permanent damage to its photosynthetic apparatus even in the driest area where it showed very low chlorophyll content. Its relative growth rate decreased more in dry areas in comparison to wetter zones than did that of L. peduncularis, even though it had greater water content. Furthermore, L. camara showed higher water contents, growth rate, and lower photosynthetic stress levels than L. peduncularis in the arid lowlands. Thus, L. peduncularis maintained lower maximum quantum efficiency of photosystem II photochemistry (F _v/F _m) than L. camara even at sunrise, due to higher basal fluorescence values with similar maximal fluorescence, which indicated permanent damage to PSII reaction centres. Our results help to explain the success and limitations of L. camara in the invasion of arid and sub-arid environments.     

Enhanced UV-B radiation increases the reproductive effort in the Mediterranean shrub Cistus creticus under field conditions

Seedlings of the Mediterranean shrub Cistus creticus L. were grown in the field under ambient or ambient plus supplemental UV-B radiation (simulating a 15% ozone depletion over Patras, 38.3°W, 29.1°E) for 20 months. During this period, measurements of photosynthetic capacity, photochemical efficiency of PS II, chlorophylls and carotenoids were performed once per season. Supplemental UV-B radiation had no significant effect on these parameters nor on the total, above ground biomass accumulation, plant height and leaf specific mass measured at plant harvest. It was observed, however, that UV-B supplementation increased the number of seeds per fruit as well as mean individual seed mass. As a result, seed number and total seed mass per plant were considerably increased. Germination rates of produced seeds were not affected. We may conclude that C. creticus is a UV-B resistant plant whose competitive ability may be improved by enhanced UV-B radiation through an increase in its reproductive effort and a higher contribution to the seed bank.     

Effect of soil drying on growth,biomass allocation and leaf gas exchange of two annual grass species

Influence of short-term water stress on plant growth and leaf gas exchange was studied simultaneously in a growth chamber experiment using two annual grass species differing in photosynthetic pathway type, plant architecture and phenology:Triticum aestivum L. cv. Katya-A-1 (C₃, a drought resistant wheat cultivar of erect growth) andTragus racemosus (L.) All. (C₄, a prostrate weed of warm semiarid areas). At the leaf level, gas exchange rates declined with decreasing soil water potential for both species in such a way that instantaneous photosynthetic water use efficiency (PWUE, mmol CO₂ assimilated per mol H₂O transpired) increased. At adequate water supply, the C₄ grass showed much lower stomatal conductance and higher PWUE than the C₃ species, but this difference disappeared at severe water stress when leaf gas exchange rates were similarly reduced for both species. However, by using soil water more sparingly, the C₄ species was able to assimilate under non-stressful conditions for a longer time than the C₃ wheat did. At the whole-plant level, decreasing water availability substantially reduced the relative growth rate (RGR) ofT. aestivum, while biomass partitioning changed in favour of root growth, so that the plant could exploit the limiting water resource more efficiently. The change in partitioning preceded the overall reduction of RGR and it was associated with increased biomass allocation to roots and less to leaves, as well as with a decrease in specific leaf area. Water saving byT. racemosus sufficiently postponed water stress effects on plant growth occurring only as a moderate reduction in leaf area enlargement. For unstressed vegetative plants, relative growth rate of the C₄ T. racemosus was only slightly higher than that of the C₃ T. aestivum, though it was achieved at a much lower water cost. The lack of difference in RGR was probably due to growth conditions being relatively suboptimal for the C₄ plant and also to a relatively large investment in stem tissues by the C₄ T. racemosus. Only 10% of the plant biomass was allocated to roots in the C₄ species while this was more than 30% for the C₃ wheat cultivar. These results emphasize the importance of water saving and high WUE of C₄ plants in maintaining growth under moderate water stress in comparison with C₃ species.     

Water relations of differentially salinized ash-tree in view of the effect of a protective nutrient solution

Summary Young potted ash-trees,Fraxinus excelsior L. were differentially salinized with NaCl and then treated with a nutrient solution. Due to the application of NaCl, higher leaf water potentials and increased stomatal diffusive resistances were recorded. Simultaneously with a drop of the stomatal diffusive resistance in midsummer, leaf necrosis began to spread rapidly. This stomatal behaviour could not be observed in those trees treated with the nutrient solution whose protective effect probably results herein.     

Gas Exchange and Chlorophyll Fluorescence in Symbiotic and Non-Symbiotic Ryegrass Under Water Stress

The symbiotic association of endophyte fungus, Neotyphodium lolii, and ryegrass improves the ryegrass resistance to drought. This is shown by a 30 % increase in the number of suckers in infected plants (E+), compared to plants lacking endophyte (E−), and by a higher water potential in the E+ than E− plants. The E+ plants have higher stomatal conductance (g _s), transpiration rate, net photosynthetic rate (P _N), and photorespiratory electron transport rate than the E− plants. The maximal photochemical efficiency (F_v/F_m) and the actual photochemical efficiency (Φ_PS2) are not affected by the endophyte fungus. The increase in P _N of the E+ plants subjected to water stress was independent from internal CO₂ concentration. An increased P _N was observed in E+ plants also in optimal water supply. Hence the drought resistance of E+ plants results in increased g _s, P _N, and photorespiratory electron transport rate. This revised version was published online in June 2006 with corrections to the Cover Date.     

Artichoke Leaf Morphology and Surface Features in Different Micropropagation Stages

Artichoke (Cynara scolymus L.) leaf size and shape, glandular and covering trichomes, stomatal density, stomata shape, pore area and epicuticular waxes during micropropagation stages were studied by scanning electron microscopy (SEM) and morphometric analysis with the aim to improve the survival rate after transfer to greenhouse conditions. Leaves from in vitro shoots at the proliferation stage showed a spatular shape, ring-shaped stomata, a large number of glandular trichomes and juvenile covering hairs, but failed to show any epicuticular waxes. Leaves from in vitro plants at the root elongation stage showed a lanceolated elliptic shape with a serrated border, elliptical stomata, decreased pore area percentage, stomatal density, and mature covering trichomes. One week after transfer to ex vitro conditions, epicuticular waxes appeared on the leaf surface and stomata and pore area were smaller as compared to in vitro plants. Artichoke acclimatization may be improved by hormonal stimulation of root development, since useful morphological changes on leaves occurred during root elongation.     

Stomatal numbers of soybean and response to water stress

The relationship among stomatal density, photosynthetic rate, leaf conductance, plant growth, bean yield and kaempferol triglucoside (K9) in the leaves of soybean (Glycine max (L.) Merr.) was examined in two field tests. K9 in the leaves was associated with reduced stomatal density, reduced photosynthetic rate, reduced stomatal conductance, reduced plant weight and lower bean yield. Plants with high stomatal frequency (lacking K9) were better able to take advantage of increased water supply by increasing stomatal conductance (upper surface), transpiration and bean yield. Plants with low stomatal frequency (with K9) were unresponsive to irrigation and in this sense were more tolerant of water stress, but their overall yield was low.     

Growth and ion relations in response to combined salinity and waterlogging in the perennial forage legumes Lotus corniculatus and Lotus tenuis

Lotus tenuis (Wadst. & Kit.) is a perennial legume widely grown for pasture in the flood-prone and salt affected Pampa region of Argentina. The physiology of salt and waterlogging tolerance in L. tenuis (four cultivars) was evaluated, and compared with Lotus corniculatus (three cultivars); the most widely cultivated Lotus species. Overall, L. tenuis cultivars accumulated less Na⁺ and Cl⁻, and more K⁺ in shoots than L. corniculatus cultivars, when exposed to 200 mM NaCl for 28 days in aerated or in stagnant solutions. Root porosity was higher in L. tenuis cultivars due to greater aerenchyma formation. In a NaCl dose–response experiment (0–400 mM NaCl in aerated solution), L. tenuis (cv. Chaja) accumulated half as much Cl⁻ in its shoots than L. corniculatus (cv. San Gabriel) at all external NaCl concentrations, and about 30% less shoot Na⁺ in treatments above 250 mM NaCl. Ion distributions in shoots were determined for plants at 200 mM NaCl. L. tenuis (cv. Chaja) again accumulated about half as much Cl⁻ in old leaves, young leaves and stems, compared with concentrations in L. corniculatus (cv. San Gabriel). There were not, however, significant differences between the two species for Na⁺ concentrations in the various shoot tissues. The higher root porosity, and maintenance of lower shoot Cl⁻ and Na⁺ concentrations in L. tenuis, compared with L. corniculatus, contributes to the greater tolerance to combined salt and waterlogging stress in L. tenuis. Moreover, significant variation for tolerance to combined salinity and waterlogging stress was identified within both L. tenuis and L. corniculatus.     

Comparison of proline accumulation in two mediterranean shrubs subjected to natural and experimental water deficit

The effect of water stress on proline accumulation was tested in two contrasted species of Mediterranean scrub: Halimium halimifolium (L.) Willk and Pistacia lentiscus L. Leaf water potential, stomatal resistance and proline content have been measured both in experimental and in natural water stress conditions. Both species accumulated proline in their leaves when leaf water potential dropped below a threshold value of –3.0 MPa, under natural as well as under experimental conditions. In the field, however, a time-lag between decrease of leaf water potential and proline accumulation could be observed. In Halimium halimifolium, proline accumulation appeared to be associated with severe stress conditions as most plants with high proline contents suffered irreversible wilting, especially in the greenhouse. P. lentiscus showed a different pattern, accumulating proline at two different times of the year, as a response to cold or to drought. The results of our study indicated that the role of proline in this species, rather than an osmotic agent, seems to be more related to a protective action in cases of severe stress conditions.     

Influence of vesicular-arbuscular mycorrhizal infection and P addition on growth and P nutrition of Anthyllis cytisoides L. and Brachypodium retusum (Pers.) Beauv.

The effect of P applications and mycorrhizal inoculation on the growth and P nutrition of Anthyllis cytisoides L. (Fabaceae) and Brachypodium retusum (Pers.) Beauv. (Poaceae) was studied. Both plants are widely distributed and well adapted to semi-arid habitats in southern Spain. In all treatments, even with high P doses, mycorrhizal plants showed a higher concentration of phosphorus in their tissues than non-mycorrhizal plants. Mycorrhizal inoculation enhanced the growth of the plants when no P was applied. At high P addition, non-mycorrhizal plants showed higher growth than mycorrhizal plants. The response of each plant type to P application was somewhat different.