Accumulation of sugars in the xylem apoplast observed under water stress conditions is controlled by xylem pH

Severe water stress constrains, or even stops, water transport in the xylem due to embolism formation. Previously, the xylem of poplar trees was shown to respond to embolism formation by accumulating carbohydrates in the xylem apoplast and dropping xylem sap pH. We hypothesize that these two processes may be functionally linked as lower pH activates acidic invertases degrading sucrose and inducing accumulation of monosaccharides in xylem apoplast. Using a novel in vivo method to measure xylem apoplast pH, we show that pH drops from ~6.2 to ~5.6 in stems of severely stressed plants and rises following recovery of stem water status. We also show that in a lower pH environment, sugars are continuously accumulating in the xylem apoplast. Apoplastic carbohydrate accumulation was reduced significantly in the presence of a proton pump blocker (orthovanadate). These observations suggest that a balance in sugar concentrations exists between the xylem apoplast and symplast that can be controlled by xylem pH and sugar concentration. We conclude that lower pH is related to loss of xylem transport function, eventually resulting in accumulation of sugars that primes stems for recovery from embolism when water stress is relieved.     

Bicarbonate-induced alkalinization of the xylem sap in intact maize seedlings as measured in situ with a novel xylem pH probe

In higher plants the pH of the xylem sap plays an important role in drought signaling, growth regulation, and plant nutrition. However, the interpretation of the data is very controversial. The main reason for this is that the xylem pH in intact plants was not directly accessible hitherto. We present here a novel, minimally-invasive probe based on the xylem pressure-potential probe (used for measuring directly xylem pressure and the electrical potential between root xylem sap and medium). Single-tipped, double-barreled capillaries were used, one barrel served as H(+)-selective electrode, whereas pressure and electrical potential were recorded by the other one. Upon insertion of the probe into the root xylem of maize (Zea mays) seedlings, pH values ranging between about 4.2 and 4.9 were monitored when the roots were immersed in standard nutrient solution. The pH did not respond to changes in light irradiation (up to 300 micromol m(-2) s(-1)), but increased upon exposure of the root to 5 or 20 mm bicarbonate in the bath solution. Changes in pH could also be recorded in transpiring plants when the root was cut below the insertion point of the probe and placed in media with different pH. The data support the hypothesis of Mengel ([1994] Plant Soil 165: 275-283) that upon external supply with bicarbonate Fe is immobilized in the leaf apoplast via changes in xylem pH.     

Sugar demand of ripening grape berries leads to recycling of surplus phloem water via the xylem

We tested the common assumption that fleshy fruits become dependent on phloem water supply because xylem inflow declines at the onset of ripening. Using two distinct grape genotypes exposed to drought stress, we found that a sink‐driven rise in phloem inflow at the beginning of ripening was sufficient to reverse drought‐induced berry shrinkage. Rewatering accelerated berry growth and sugar accumulation concurrently with leaf photosynthetic recovery. Interrupting phloem flow through the peduncle prevented the increase in berry growth after rewatering, but interrupting xylem flow did not. Nevertheless, xylem flow in ripening berries, but not berry size, remained responsive to root or shoot pressurization. A mass balance analysis on ripening berries sampled in the field suggested that phloem water inflow may exceed growth and transpiration water demands. Collecting apoplastic sap from ripening berries showed that osmotic pressure increased at distinct rates in berry vacuoles and apoplast. Our results indicate that the decrease in xylem inflow at the onset of ripening may be a consequence of the sink‐driven increase in phloem inflow. We propose a conceptual model in which surplus phloem water bypasses the fruit cells and partly evaporates from the berry surface and partly moves apoplastically to the xylem for outflow.     

Effects of iron deficiency on the composition of the leaf apoplastic fluid and xylem sap in sugar beet. Implications for iron and carbon transport

The effects of iron deficiency on the composition of the xylem sap and leaf apoplastic fluid have been characterized in sugar beet (Beta vulgaris Monohil hybrid). pH was estimated from direct measurements in apoplastic fluid and xylem sap obtained by centrifugation and by fluorescence of leaves incubated with 5-carboxyfluorescein and fluorescein isothiocyanate-dextran. Iron deficiency caused a slight decrease in the pH of the leaf apoplast (from 6.3 down to 5.9) and xylem sap (from 6.0 down to 5.7) of sugar beet. Major organic acids found in leaf apoplastic fluid and xylem sap were malate and citrate. Total organic acid concentration in control plants was 4.3 mM in apoplastic fluid and 9.4 mM in xylem sap and increased to 12.2 and 50.4 mM, respectively, in iron-deficient plants. Inorganic cation and anion concentrations also changed with iron deficiency both in apoplastic fluid and xylem sap. Iron decreased with iron deficiency from 5.5 to 2.5 microM in apoplastic fluid and xylem sap. Major predicted iron species in both compartments were [FeCitOH](-1) in the controls and [FeCit(2)](-3) in the iron-deficient plants. Data suggest the existence of an influx of organic acids from the roots to the leaves via xylem, probably associated to an anaplerotic carbon dioxide fixation by roots.     

Metabolomic and proteomic changes in the xylem sap of maize under drought

Plants produce compounds in roots that are transported to shoots via the xylem sap. Some of these compounds are vital for signalling and adaptation to environmental stress such as drought. In this study, we screened the xylem sap using mass spectrometry to quantify the changes in new and previously identified sap constituents under extended drought. We detected and quantified the changes in the concentration of 31 compounds present in the xylem sap under progressively increasing drought stress. We found changes in the hormones abscisic acid (ABA) and cytokinin, and the presence of high concentrations of the aromatic cytokinin 6-benzylaminopurine (BAP). Several phenylpropanoid compounds (coumaric, caffeic and ferulic acids) were found in xylem sap. The concentrations of some of these phenylpropanoid compounds changed under drought. In parallel, an analysis of the xylem sap proteome was conducted. We found a higher abundance of cationic peroxidases, which with the increase in phenylpropanoids may lead to a reduction in lignin biosynthesis in the xylem vessels and could induce cell wall stiffening. The application of new methodologies provides insights into the range of compounds in sap and how alterations in composition may lead to changes in development and signalling during adaptation to drought.     

Seasonal bulk xylem pressure in temperate broadleaf eudicot trees: a case study for sugar long-distance transport and signaling

Sugars regulate growth, development, and defense in trees. Sugars are also important signaling molecules and are transported over long distances via xylem and phloem. Sucrose loading to tracheids and vessels is associated with bulk xylem pressure and occurs seasonally in temperate broadleaf eudicot trees. Following restoration of xylem hydraulic conductivity in spring, sugars are unloaded from xylem sap at apical branches and deposited as starch before growth of shoot apical meristems. Growth of cambia and shoot apical meristems leads to starch catabolism that yields hexose-phosphates to fuel cell growth and regulate other signal networks. The contrast between cell molecular biology of Arabidopsis and physiology of temperate broadleaf eudicot trees indicates the importance of phosphorylation in long-distance sugar signaling. Hexokinase, acting as a hub for signal and hormone networks, is likely an important regulator of sugar signaling in response to stimuli such as energy status, sugar status, and environmental conditions. The comparative analysis suggested here could help bridge physiology and detailed molecular mechanisms regarding physiology of trees.     

Capacitive effect of cavitation in xylem conduits: results from a dynamic model

Embolisms decrease plant hydraulic conductance and therefore reduce the ability of the xylem to transport water to leaves provided that embolized conduits are not refilled. However, as a xylem conduit is filled with gas during cavitation, water is freed to the transpiration stream and this transiently increases xylem water potential. This capacitive effect of embolism formation on plant function has not been explicitly quantified in the past. A dynamic model is presented that models xylem water potential, xylem sap flow and cavitation, taking into account both the decreasing hydraulic conductance and the water release effect of xylem embolism. The significance of the capacitive effect increases in relation to the decreasing hydraulic conductance effect when transpiration rate is low in relation to the total amount of water in xylem conduits. This ratio is typically large in large trees and during drought.     

Modification of leaf apoplastic pH in relation to stomatal sensitivity to root-sourced abscisic acid signals

The confocal microscope was used to determine the pH of the leaf apoplast and the pH of microvolumes of xylem sap. We quantified variation in leaf apoplast and sap pH in relation to changes in edaphic and atmospheric conditions that impacted on stomatal sensitivity to a root-sourced abscisic acid signal. Several plant species showed significant changes in the pH of both xylem sap and the apoplast of the shoot in response to environmental perturbation. Xylem sap leaving the root was generally more acidic than sap in the midrib and the apoplast of the leaf. Increasing the transpiration rate of both intact plants and detached plant parts resulted in more acidic leaf apoplast pHs. Experiments with inhibitors suggested that protons are removed from xylem sap as it moves up the plant, thereby alkalinizing the sap. The more rapid the transpiration rate and the shorter the time that the sap resided in the xylem/apoplastic pathway, the smaller the impact of proton removal on sap pH. Sap pH of sunflower (Helianthus annuus) and Commelina communis did not change significantly as soil dried, while pH of tomato (Lycopersicon esculentum) sap increased as water availability in the soil declined. Increasing the availability of nitrate to roots also significantly alkalinized the xylem sap of tomato plants. This nitrogen treatment had the effect of enhancing the sensitivity of the stomatal response to soil drying. These responses were interpreted as an effect of nitrate addition on sap pH and closure of stomata via an abscisic acid-based mechanism.     

Sugar accumulation in roots of two grape varieties with contrasting response to water stress

Root sugar accumulation was studied in two grapevine varieties contrasting in tolerance to water stress. During a 10‐day water withholding treatment, the drought‐tolerant variety, Grenache, sustained less negative predawn and midday leaf water potentials as well as root water potential compared with the sensitive variety, Semillon. Grenache vines also maintained lower stomatal conductance and transpiration than Semillon vines throughout the drying period. In both varieties there was accumulation of sucrose in the roots and concentrations were inversely correlated to leaf and root water status. In both Grenache and Semillon, elevated root osmolality was associated with decreased soil moisture indicating that sugar accumulation may play a role in osmotic protection. Petiole xylem sap abscisic acid (ABA) concentrations increased with water deficit in both varieties and were highest for vines with the most negative root and predawn leaf water potentials. Furthermore, root sucrose concentrations were positively correlated with leaf xylem sap ABA concentrations, indicative of integration between carbohydrate metabolism and the ABA signalling system. Similar root sugar accumulation patterns between the two varieties, however, demonstrate that other factors are likely influencing the ability of the drought‐tolerant variety to remain hydrated.     

Seasonal and spatial variation of carbohydrates in mistletoes (Viscum album) and the xylem sap of its hosts (Populus x euamericana and Abies alba)

In the present field study we analysed the seasonal pattern of carbohydrate composition and contents in the xylem sap of Viscum album and the xylem sap of a deciduous ( Populus × euramericana ) and a coniferous ( Abies alba ) host tree species. The results were compared with the soluble carbohydrate composition and contents of mistletoe tissues. On both hosts significant amounts of glucose, fructose, and sucrose were found in the xylem sap of Viscum throughout the seasons. The general seasonal pattern of sugar contents, i.e. high concentrations in spring and lower concentrations in other seasons on Populus , and intermediate concentrations throughout the year on Abies , largely reflected the xylem sap carbohydrate composition and contents of the respective host. These observations provide indirect evidence for carbohydrate flux from the xylem sap of the host into the mistletoe. However, in both hosts xylem sap seems to be deviated into the mistletoe without specific control of carbohydrate flux. Differences observed between the seasonal pattern of xylem sap carbohydrate concentrations in Viscum on Populus and Abies may originate from the different time of leaf development of these species. A clear-cut seasonal pattern of soluble carbohydrates was not observed in the leaves of Viscum on both hosts. Still soluble carbohydrates seem to be reallocated from the senescing to the newly developed leaves of Viscum indicating that the seasonal requirement of carbohydrate for growth and development can only completely be met by carbohydrate acquisition from the host and their own photosynthesis.     

Long-distance ABA Signaling and Its Relation to Other Signaling Pathways in the Detection of Soil Drying and the Mediation of the Plant’s Response to Drought

In this article we review evidence for a variety of long-distance signaling pathways involving hormones and nutrient ions moving in the xylem sap. We argue that ABA has a central role to play, at least in root-to-shoot drought stress signaling and the regulation of functioning, growth, and development of plants in drying soil. We also stress the importance of changes in the pH of the leaf cell apoplast as influenced both by edaphic and climatic variation, as a regulator of shoot growth and functioning, and we show how changes in xylem and apoplastic pH can affect the way in which ABA regulates stomatal behavior and growth. The sensitivity to drought of the pH/ABA sensing and signaling mechanism is emphasized. This allows regulation of plant growth, development and functioning, and particularly shoot water status, as distinct from stress lesions in growth and other processes as a reaction to perturbations such as soil drying.     

Arabidopsis leaf hydraulic conductance is regulated by xylem sap pH,controlled, in turn,by a P-type H+-ATPase of vascular bundle sheath cells

The leaf vascular bundle sheath cells (BSCs) that tightly envelop the leaf veins, are a selective and dynamic barrier to xylem sap water and solutes radially entering the mesophyll cells. Under normal conditions, xylem sap pH below 6 is presumably important for driving and regulating the transmembranal solute transport. Having discovered recently a differentially high expression of a BSC proton pump, AHA2, we now test the hypothesis that it regulates the xylem sap pH and leaf radial water fluxes. We monitored the xylem sap pH in the veins of detached leaves of wild-type Arabidopsis, AHA mutants and aha2 mutants complemented with AHA2 gene solely in BSCs. We tested an AHA inhibitor (vanadate) and stimulator (fusicoccin), and different pH buffers. We monitored their impact on the xylem sap pH and the leaf hydraulic conductance (K_leaf), and the effect of pH on the water osmotic permeability (P_f) of isolated BSCs protoplasts. We found that AHA2 is necessary for xylem sap acidification, and in turn, for elevating K_leaf. Conversely, AHA2 knockdown, which alkalinized the xylem sap, or, buffering its pH to 7.5, reduced K_leaf, and elevating external pH to 7.5 decreased the BSCs P_f. All these showed a causative link between AHA2 activity in BSCs and leaf radial hydraulic water conductance.     

Influence of sampling time and sap extraction methodology on xylem pH values in two grapevine varieties grown under drought conditions

The effect of differences in applied pressure and time of sampling on pH values of xylem sap collected using the leaf pressurization technique was examined in two grapevine varieties originating from contrasting habitats (Vitis vinifera L., cvs. Sabatiano and Mavrodafni) after subjecting them to drought. Three fractions of xylem sap exudates were collected from each leaf according to differences in applied pressure; fractions (I), (II) and (III) corresponding to 1 MPa, 2 MPa and 2.5 MPa pressure, respectively. The pH values in fraction (I) were significantly lower than those in fractions (II) and (III). The sap pH values in fraction (III) seemed to better correspond to changes in leaf apoplastic pH. The time of sampling was found to strongly influence xylem pH values. In particular, a positive relationship between predawn xylem pH values and soil drying was observed. Conversely, xylem pH values measured later during the day (i.e. at 8:00, 9:00 and 10:00 am) were not significantly affected by the reduction in soil water availability in both varieties. It is suggested that the most suitable period for sap sampling in order to better discriminate drought effects on xylem sap pH is at predawn. Furthermore, there were significant differences in pH values as well as in the sensitivity of stomatal conductance to pH between the two varieties. These differences might be related to strategy differences between grapevine varieties for adaptation to drought.     

木本植物木质部的冻融栓塞应对研究进展

冻融栓塞在中高纬度地区木本植物中普遍存在。抗冻融栓塞能力对在寒冷环境中木本植物的生长和安全越冬十分关键, 这直接决定植物分布范围。冻融栓塞是由于冰中气体溶解度低, 木质部水分在低温下冷冻, 使之前水中溶解的气体逸出到导管中, 随后木质部中的冰融化又使气泡扩张而引发的栓塞现象。木质部解剖结构的差异会影响植物的抗冻融栓塞能力, 植物还可以通过调节木质部正压、代谢耗能等方式主动修复冻融栓塞, 也可通过增加树液溶质含量等逃避冷冻, 以减少低温损伤。然而, 与干旱栓塞相比, 目前对木质部冻融栓塞的形成以及植物响应和调节机制的理解不足。为此, 该文首先综述了木质部冻融栓塞的形成机制和植物的逃避、忍耐、修复等3种冻融栓塞的应对策略, 然后总结了木质部抗低温胁迫能力的生理表现、影响因子和评价指标, 并在此基础上讨论了低温抗性、干旱抗性和水力效率之间的多元权衡关系, 最后提出今后该领域中的5个优先研究问题: (1)不同植物冰冻的最低温度阈值; (2)是否存在应对低温胁迫的水力脆弱性分割机制; (3)冻融栓塞修复与代谢消耗的关系; (4)低温抗性、干旱抗性和水力效率之间的权衡关系; (5)抗冻融栓塞性状是否能够纳入经济性状谱系。     

Seasonal changes in the efflux of sugars from parenchyma cells into the apoplast in poplar stems (Populus × canademis “robusta”)

Summary The efflux of sugars from parenchyma cells into the apoplast has been studied in the wood of Populus × canadensis robusta in relation to the sugar level of the tissue and the sugar content of the tracheal sap during all physiological stages of the year. In poplar, the spring maximum in sugar content of the tracheal sap is clearly not the result of continuous exosmosis during winter but is reached within a short period in spring. The seasonal patterns of starch and sugar content of the wood and of the sugar content of the tracheal sap are described. The efflux of sugars from tissue sections changes drastically over the year, i.e., from 0.3 to 8.3 g mg^–1DWT day^–1. In general, it is high in fall and winter, and low during late spring and summer. However, high tissue sugar levels proved not always to be correlated with high efflux rates or with high sugar levels in the tracheal sap, indicating that the intracellular compartmentalization of sugars, their passive and catalysed release into, and their re-uptake from the apoplast are all essentially involved in determining the actual sugar content of the sap. Sucrose, which is the dominant sugar in the tracheal sap during winter (pH 7.0–7.5) and in the efflux experiments at pH 7.5, in contrast to the hexoses which prevail in the spring sap (pH around 5.5) and also in the efflux experiments at pH 5.6, is considered to be preferentially released in poplar and to become extraplasmatically hydrolysed. The reasons for tree-specific differences are discussed.     

荷木树干CO_2释放通量与木质部液流和CO_2浓度的关系

采用红外气体分析仪,于2008年10月17-19日连续3个昼夜原位监测了荷木的树干CO_2释放通量、树干温度、木质部液流密度和CO_2浓度.结果表明:树干CO_2释放通量(EA)日变化呈S形曲线,不同径级间差异显著.EA与树干温度呈显著幂函数关系(0.24

Abstract：

By using a Li-820 infra-red CO_2 gas analyzer, an in situ measurement of Schima super-ba stem CO_2 efflux was conducted for three consecutive days from 17 to 19 October 2008. In the meantime, the stem temperature, xylem sap efflux density, and xylem CO_2 concentration were measured. The stem CO_2 efflux had a diurnal variation of "S" pattern, and differed significantly with stem diameter. There was a significant exponential relationship between stem CO_2 efflux and stem temperature (0. 24 < R~2 < 0. 78). The temperature coefficient (b) and regression coeffi-cient (R~2) were higher at nighttime than at daytime, and the Q_(10) value ranged from 2. 01 to 2. 79. The stem CO_2 efflux correlated significantly with the xylem CO_2 concentration, and the best regression curve was cubic (R~2= 0. 48). Excluding the effects of stem temperature, the stem CO_2 efflux showed a significant negative correlation with xylem sap flux density (r =-0.462). Therefore, only using simple temperature function to estimate stem CO_2 efflux would yield a significant error, and xylem sap flux should be taken into consideration in the stem CO_2 emux estimation.     

The apoplastic pH of the substomatal cavity of Vicia faba leaves and its regulation responding to different stress factors.

The apoplastic pH of the substomatal cavity is an essential determinant of stomatal movement. In detached leaves of Vicia faba substomatal apoplastic pH and its dependence on external (stress) factors was investigated using a non-invasive approach: pH-microsensors were inserted into open stomata, and upon contact with the apoplastic fluid, pH was measured continuously, as apoplastic pH was challenged by changed conditions of light, atmosphere (NH(3), CO(2)), and xylem sap (abscisic acid, cyanide, fusicoccin, pH, inorganic salts). Apoplastic pH proved extremely sensitive to infiltration and local flooding, which rapidly increased the apoplastic pH by more than 1.5 pH units. Recovery from infiltration took several hours, during which light effects on the apoplastic pH were strongly impeded. This indicates that pH tests carried out under such conditions may not be representative of the undisturbed leaf. NH(3), flushed across the stomata, yielded a rapid apoplastic alkalinization from which an apoplastic buffer capacity of 2-3 mM per pH unit was calculated. Fusicoccin, fed into the xylem sap acidified the apoplast, whereas cyanide alkalized it, thus underscoring the importance of the plasma membrane H(+) pump for apoplastic pH regulation. To address the question to what extent pH was a drought signal, the effect of iso-osmotic pH changes, fed into the xylem through the petiole were tested. It is demonstrated that the apoplastic response remained below 0.1 pH per pH unit imposed, regardless of the buffer capacity. An increase in the osmolarity of the bath solution (harbouring the cut petiole) using KCl, NaCl, CaCl(2) or sorbitol alkalized the substomatal apoplast. It is suggested that pH may only act as drought signal when accompanied by elevated osmolarity.     

Lipids in xylem sap of woody plants across the angiosperm phylogeny

Lipids have been observed attached to lumen-facing surfaces of mature xylem conduits of several plant species, but there has been little research on their functions or effects on water transport, and only one lipidomic study of the xylem apoplast. Therefore, we conducted lipidomic analyses of xylem sap from woody stems of seven plants representing six major angiosperm clades, including basal magnoliids, monocots and eudicots, to characterize and quantify phospholipids, galactolipids and sulfolipids in sap using mass spectrometry. Locations of lipids in vessels of Laurus nobilis were imaged using transmission electron microscopy and confocal microscopy. Xylem sap contained the galactolipids di- and monogalactosyldiacylglycerol, as well as all common plant phospholipids, but only traces of sulfolipids, with total lipid concentrations in extracted sap ranging from 0.18 to 0.63 nmol ml⁻¹ across all seven species. Contamination of extracted sap from lipids in cut living cells was found to be negligible. Lipid composition of sap was compared with wood in two species and was largely similar, suggesting that sap lipids, including galactolipids, originate from cell content of living vessels. Seasonal changes in lipid composition of sap were observed for one species. Lipid layers coated all lumen-facing vessel surfaces of L. nobilis, and lipids were highly concentrated in inter-vessel pits. The findings suggest that apoplastic, amphiphilic xylem lipids are a universal feature of angiosperms. The findings require a reinterpretation of the cohesion-tension theory of water transport to account for the effects of apoplastic lipids on dynamic surface tension and hydraulic conductance in xylem.