A new device for online nanoscale sampling and capillary electrophoresis analysis of plant sap composition.

In this work, an online sampling of plant xylem sap combined with an efficient (CE)-based method was developed and applied to study the kinetics of changes in the sap composition and to assess plant fitness under stress conditions comprehensively. A laboratory-built CE device was developed to provide online sampling and CE analysis of various ionogenic species in the sap during plant stress response. The rapid online sampling and short CE analysis time allow for real-time monitoring of changes in sap constituents in the living plant during the stress response. The developed device was successfully used to analyze chloride, nitrate, and sulfate ions in the plant xylem during the salt stress or stress caused by nitrate deficiency within short time scales.

Do angiosperm tree species adjust intervessel lateral contact in response to soil drought?

During soil drought (i.e. limited soil water availability to plants), woody species may adjust the structure of their vessel network to improve their resistance against future soil drought stress. Impacts of soil drought on intervessel lateral contact remain poorly understood despite of its significance to xylem transport efficiency and safety. Here, we analysed drought-induced modifications in xylem structures of temperate angiosperm trees with a focus on intervessel lateral contact. Anatomical analyses were performed both in stems of seedlings cultivated under different substrate water availability and annual rings of mature individuals developed during years of low and high soil drought intensities. In response to limited water availability, a decrease in vessel diameter (up to -20%) and simultaneous increase in vessel density (up to +60%) were observed both in seedlings and mature trees. Conversely, there were only small and inconsistent drought-induced changes in intervessel contact frequency and intervessel contact fraction (typically up to ±15%) observed across species, indicating that intervessel lateral contact is a conservative trait. The small adjustments in intervessel lateral contacts were primarily driven by changes in the contact frequencies between neighbouring vessels (i.e. vessel grouping) rather than by changes in proportions of shared cell walls. Our results demonstrate that angiosperm tree species, despite remarkable adjustments in vessel dimensions and densities upon soil drought, exhibit surprisingly invariant intervessel lateral contact architecture.

Early Changes in Nitrate Uptake and Assimilation Under Drought in Relation to Transpiration.

Soil drying combined with nitrogen (N) deficiency poses a grave threat to agricultural crop production. The rate at which nitrate (NO3 -) is taken up depends partly on the uptake and transpiration of water. Rapid changes in nitrate assimilation, in contrast to other N forms, may serve as a component of the plant stress response to drought because nitrate assimilation may lead to changes in xylem pH. The modulation of xylem sap pH may be relevant for stomata regulation via the delivery of abscisic acid (ABA) to guard cells. In several factorial experiments, we investigated the interactions between nitrate and water availability on nitrate fate in the plant, as well as their possible implications for the early drought-stress response. We monitored the short-term response (2-6 days) of nitrate in biomass, transport to shoot and reduction in Pisum sativum, Hordeum vulgare, Vicia faba, and Nicotiana tabacum and correlated this with sap pH and transpiration rates (TRs). Cultivation on inorganic substrate ensured control over nutrient and water supply and prevented nodulation in legume species. NO3 - content in biomass decreased in most of the species under drought indicating significant decline in NO3 - uptake. Hordeum vulgare had the highest NO3 - concentrations in all organs even under drought and low NO3 - treatment. This species can likely respond much better to the combined adverse effects of low NO3 - and water scarcity. Nitrate reductase activity (NRA) was reduced in both roots and leaves of water deficient (WD) plants in all species except H. vulgare, presumably due to its high NO3 - contents. Further, transient reduction in NO3 - availability had no effect on sap pH. Therefore, it seems unlikely that NRA shifts from shoot root leading to the supposed alkalization of sap. We also did not observe any interactive effects of NO3 - and water deficiency on transpiration. Hence, as long as leaf NO3 - content remains stable, NO3 - availability in soil is not linked to short-term modulation of transpiration.

Steady-State Levels of Cytokinins and Their Derivatives May Serve as a Unique Classifier of Arabidopsis Ecotypes.

We determined steady-state (basal) endogenous levels of three plant hormones (abscisic acid, cytokinins and indole-3-acetic acid) in a collection of thirty different ecotypes of Arabidopsis that represent a broad genetic variability within this species. Hormone contents were analysed separately in plant shoots and roots after 21 days of cultivation on agar plates in a climate-controlled chamber. Using advanced statistical and machine learning methods, we tested if basal hormonal levels can be considered a unique ecotype-specific classifier. We also explored possible relationships between hormone levels and the prevalent environmental conditions in the site of origin for each ecotype. We found significant variations in basal hormonal levels and their ratios in both root and shoot among the ecotypes. We showed the prominent position of cytokinins (CK) among the other hormones. We found the content of CK and CK metabolites to be a reliable ecotype-specific identifier. Correlation with the mean temperature at the site of origin and the large variation in basal hormonal levels suggest that the high variability may potentially be in response to environmental factors. This study provides a starting point for ecotype-specific genetic maps of the CK metabolic and signalling network to explore its contribution to the adaptation of plants to local environmental conditions.

Ion-mediated increases in xylem hydraulic conductivity: seasonal differences between coexisting ring- and diffuse-porous temperate tree species.

Ion-mediated changes in hydraulic conductivity (ΔKh) represent a mechanism allowing plants to regulate the rate of xylem transport. However, the significance of ΔKh for ring-porous (RPS) and diffuse-porous tree species (DPS) remains unknown. Here, we examined ΔKh in young branches of three coexisting, temperate RPS (Fraxinus excelsior, Quercus robur, Robinia pseudoacacia) and three DPS (Acer pseudoplatanus, Carpinus betulus, Fagus sylvatica) across the whole year, and assessed the relationships of ΔKh to branch anatomy. Ring-porous species exhibited twice as high ΔKh (10.3% vs 5.3%) within the growing season (i.e., during wood production) compared with DPS, and the production of the annual ring was identified as a crucial process affecting maximum ΔKh within the season. In addition, xylem in branches of RPS generally contained more axial parenchyma (AP; 18% vs 7%) and was characterized by a greater relative contact fraction between vessels and parenchyma (FVP; 59% vs 18%) than xylem in DPS. Simultaneously, ΔKh measured within the growing season was positively correlated with AP, FVP and bark proportions, suggesting that parenchyma in branches may be important for high ΔKh. Significant increase in ΔKh observed during the growing season may help RPS to restore conductive capacity after winter, better compensate transport loss by drought-induced embolism and thereby improve water delivery to leaves.

Contrasting below- and aboveground responses of two deciduous trees to patchy nitrate availability.

We investigated how patchy nitrate availability influences growth and functioning of plant roots and generates, through vascular constraints on long-distance transport, aboveground heterogeneity in plant growth and chemistry. We examined two broadleaf tree species, Acer rubrum L. and Betula papyrifera Marsh. Plants were grown either in a split-root setup where a single root received full nutrient supply and the rest of the root system received all nutrients except nitrogen (patchy treatment), or in a single pot with full nutrient supply (homogeneous treatment). In both species, fine roots proliferated in the nitrogen patch, but B. papyrifera produced twice as much fine root biomass in response to patchy nitrate availability as did A. rubrum. There was no difference between treatments in nitrogen uptake rate in either species. In general, specific water uptake was higher in A. rubrum than in B. papyrifera, especially in the nitrogen-rich side pot. When nitrate availability was patchy, nitrate reductase activity in roots and leaves was unaffected in either species. In A. rubrum, but not in B. papyrifera, patchy nitrate supply resulted in aboveground heterogeneity, with leaves above the N-fertilized roots being larger and having a higher relative chlorophyll concentration than those inserted in the opposite quater of the stem.

Linking xylem water storage with anatomical parameters in five temperate tree species.

The release of water from storage compartments to the transpiration stream is an important functional mechanism that provides the buffering of sudden fluctuations in water potential. The ability of tissues to release water per change in water potential, referred to as hydraulic capacitance, is assumed to be associated with the anatomy of storage tissues. However, information about how specific anatomical parameters determine capacitance is limited. In this study, we measured sapwood capacitance (C) in terminal branches and roots of five temperate tree species (Fagus sylvatica L., Picea abies L., Quercus robur L., Robinia pseudoacacia L., Tilia cordata Mill.). Capacitance was calculated separately for water released mainly from capillary (CI; open vessels, tracheids, fibres, intercellular spaces and cracks) and elastic storage compartments (CII; living parenchyma cells), corresponding to two distinct phases of the moisture release curve. We found that C was generally higher in roots than branches, with CI being 3-11 times higher than CII Sapwood density and the ratio of dead to living xylem cells were most closely correlated with C In addition, the magnitude of CI was strongly correlated with fibre/tracheid lumen area, whereas CII was highly dependent on the thickness of axial parenchyma cell walls. Our results indicate that water released from capillary compartments predominates over water released from elastic storage in both branches and roots, suggesting the limited importance of parenchyma cells for water storage in juvenile xylem of temperate tree species. Contrary to intact organs, water released from open conduits in our small wood samples significantly increased CI at relatively high water potentials. Linking anatomical parameters with the hydraulic capacitance of a tissue contributes to a better understanding of water release mechanisms and their implications for plant hydraulics.

Partitioning of vessel resistivity in three liana species.

Vessels with simple perforation plates, found in the majority of angiosperms, are considered the evolutionarily most advanced conduits, least impeding the xylem sap flow. Nevertheless, when measured, their hydraulic resistivity (R, i.e., inverse value of hydraulic conductivity) is significantly higher than resistivity predicted using Hagen-Poiseuille equation (RHP). In our study we aimed (i) to quantify two basic components of the total vessel resistivity - vessel lumen resistivity and end wall resistivity, and (ii) to analyze how the variable inner diameter of the vessel along its longitudinal axis affects resistivity. We measured flow rates through progressively shortened stems of hop (Humulus lupulus L.), grapevine (Vitis vinifera L.), and clematis (Clematis vitalba L.) and used elastomer injection for identification of open vessels and for measurement of changing vessel inner diameters along its axis. The relative contribution of end wall resistivity to total vessel resistivity was 0.46 for hop, 0.55 for grapevine, and 0.30 for clematis. Vessel lumen resistivity calculated from our measurements was substantially higher than theoretical resistivity - about 43% for hop, 58% for grapevine, and 52% for clematis. We identified variation in the vessel inner diameter as an important source of vessel resistivity. The coefficient of variation of vessel inner diameter was a good predictor for the increase of the ratio of integral RHP to RHP calculated from the mean value of inner vessel diameter. We discuss the fact that we dealt with the longest vessels in a given stem sample, which may lead to the overestimation of vessel lumen resistivity, which consequently precludes decision whether the variable vessel inner diameter explains fully the difference between vessel lumen resistivity and RHP we observed.

An improved method for the visualization of conductive vessels in Arabidopsis thaliana inflorescence stems.

Dye perfusion is commonly used for the identification of conductive elements important for the study of xylem development as well as precise hydraulic estimations. The tiny size of inflorescence stems, the small amount of vessels in close arrangement, and high hydraulic resistivity delimit the use of the method for quantification of the water conductivity of Arabidopsis thaliana, one of the recently most extensively used plant models. Here, we present an extensive adjustment to the method in order to reliably identify individual functional (conductive) vessels. Segments of inflorescence stems were sealed in silicone tubes to prevent damage and perfused with a dye solution. Our results showed that dyes often used for staining functional xylem elements (safranin, fuchsine, toluidine blue) failed with Arabidopsis. In contrast, Fluorescent Brightener 28 dye solution perfused through segments stained secondary cell walls of functional vessels, which were clearly distinguishable in native cross sections. When compared to identification based on the degree of development of secondary cell walls, identification with the help of dye perfusion revealed a significantly lower number of functional vessels and values of theoretical hydraulic conductivity. We found that lignified but not yet functional vessels form a substantial portion of the xylem in apical and basal segments of Arabidopsis and, thus, significantly affect the analyzed functional parameters of xylem. The presented methodology enables reliable identification of individual functional vessels, allowing thus estimations of hydraulic conductivities to be improved, size distributions and vessel diameters to be refined, and data variability generally to be reduced.

Dynamic changes in root hydraulic properties in response to nitrate availability.

Changes in root hydraulic resistance in response to alterations in nitrate supply were explored in detail as a potential mechanism that allows plants to respond rapidly to changes in their environment. Sunflower (Helianthus annuus cv. Holiday) plants grown hydroponically with limited nitrate availability (200 micromol l(-1)) served as our model system. Experimental plants were 6-9-weeks-old with total dry mass of 2-4 g. Root pressurization of intact plants and detached root systems was used to elucidate the temporal dynamics of root hydraulic properties in sunflower plants following changes in external nitrate availability. The response was rapid, with a 20% decrease in hydraulic resistance occurring within the first hour after the addition of 5 mM nitrate and the magnitude of the effect was dependent on nitrate concentration. The change in root hydraulic resistance was largely reversible, although the temporal dynamics of the response to nitrate addition versus nitrate withdrawal was not symmetric (a gradual decrease in resistance versus its fast increase), raising the possibility that the underlying mechanisms may also differ. Evidence is presented that the observed changes in root hydraulic properties require the assimilation of nitrate by root cells. The hydraulic resistance of roots, previously stimulated by the addition of nitrate, increased more than in control plants in low nitrate under anoxia and that suggests a key role of aquaporin activity in this response. It is proposed that a rapid decrease in root hydraulic resistance in the presence of increased nitrate availability is an important trait that could enhance a plant's ability to compete for nitrate in the soil.

The use of internal nitrogen stores in the rhizomatous grass Calamagrostis epigejos during regrowth after defoliation.

BACKGROUND AND AIMS: The regrowth dynamics after defoliation of the invasive grass Calamagrostis epigejos were studied. As nitrogen (N) reserves have been shown to play an important role during plant regrowth, the identity, location and relative importance for regrowth of N stores were determined in this rhizomatous grass. METHODS: Plant growth, nitrate uptake and root respiration were followed during recovery from defoliation. Water soluble carbohydrates, nitrate, free amino acids and soluble proteins were analysed in the remaining organs. KEY RESULTS: Nitrate uptake and root respiration were severely reduced during the first days of regrowth. Roots were the main net source of mobilized N. The quantitatively dominant N storage compounds were free amino acids. Free amino acids and soluble proteins in the roots decreased by 55 and 50%, respectively, and a substantial (approximately 38%) decrease in stubble protein was also observed. Although the relative abundance of several soluble proteins in roots decreased during the initial recovery from defoliation, no evidence was found for vegetative storage protein (VSP). Furthermore, rhizomes did not act as a N storage compartment. CONCLUSIONS: Production of new leaf area was entirely reliant, during the first week after defoliation, on N stores present in the plant. Mobilized N originated mainly from free amino acids and soluble proteins located in roots, and less so from proteins in stubble. Presence of VSP in the roots was not confirmed. The data suggest that rhizomes played an important role in N transport but not in N storage.