Reversible, Water Stress-Induced Non-Uniform Chlorophyll Fluorescence Quenching in Wilting Leaves of Potentilla reptans may not be due to Patchy Stomatal Responses

Abstract: After exposure to full sunlight under natural conditions, attached leaves of the common meadow weed Potentilla reptans show non-uniform ("patchy") chlorophyll fluorescence quenching in the early stages of fluorescence transients. These areas of bright fluorescence can be readily reproduced in detached leaves that are allowed to wilt on the laboratory bench in weak light. The extent and duration of the patchiness increases with increasing water stress (higher relative saturation deficits). Images captured during saturating flashes show that the patches also display slow development of non-photochemical quenching, consistent with the possibility that photosynthetic metabolism is impaired in these areas. Wilted Potentilla leaves readily regain full turgor when petioles are placed in water, and uniform chlorophyll fluorescence is recovered with in 30mm. Epidermal impressions reveal closed stomata over areas of both low and high fluorescence in wilted leaves. Because highly fluorescent patches also persist when wilted tissues are exposed to high CO₂ (i.e., patchiness is unlikely to be due to local differences in CO₂ supply) the data suggest direct effects of water stress on metabolism in wilted leaves. Leaf transverse sections show that although major veins may isolate areas of the lamina, minor veins do not. Relationships to leaf anatomy are discussed.     

白蜡虫7种寄主植物叶片解剖结构与寄主选择性的关系

用石蜡切片法在显微镜下观察白蜡虫[Ericerus pela(Chavannes)]7种寄主植物叶的解剖结构。结果表明,寄主植物的叶脉、表皮、栅栏组织、海绵组织、维管束解剖结构及数量性状特征在属、种间存在显著差异。7种寄主植物中,华南小蜡(Ligustrum calleryanum Decne.)和白蜡树(Fraxinus chinensis Roxb.)表皮被毛,华南小蜡表皮毛浓密,白蜡树表皮毛稀疏,其它寄主植物表皮无毛;女贞树(Ligustrum lucidum Ait.)栅栏组织、海绵组织和叶脉的厚度最厚、维管束直径最大,其它寄主植物相对较小。因此,叶片表面光滑、叶脉发达、叶片肥厚是白蜡虫优良寄主植物的重要解剖学特征。7种寄主植物中脉和侧脉发达,近轴面凹陷,远轴面突起,肉脉包埋在叶肉中,推测叶脉突起和凹槽可能是白蜡虫固定位点选择的关键线索。     

Hydraulic analysis of water flow through leaves of sugar maple and red oak

Leaves constitute a substantial fraction of the total resistance to water flow through plants. A key question is how hydraulic resistance within the leaf is distributed among petiole, major veins, minor veins, and the pathways downstream of the veins. We partitioned the leaf hydraulic resistance (R(leaf)) for sugar maple (Acer saccharum) and red oak (Quercus rubra) by measuring the resistance to water flow through leaves before and after cutting specific vein orders. Simulations using an electronic circuit analog with resistors arranged in a hierarchical reticulate network justified the partitioning of total R(leaf) into component additive resistances. On average 64% and 74% of the R(leaf) was situated within the leaf xylem for sugar maple and red oak, respectively. Substantial resistance-32% and 49%- was in the minor venation, 18% and 21% in the major venation, and 14% and 4% in the petiole. The large number of parallel paths (i.e. a large transfer surface) for water leaving the minor veins through the bundle sheath and out of the leaf resulted in the pathways outside the venation comprising only 36% and 26% of R(leaf). Changing leaf temperature during measurement of R(leaf) for intact leaves resulted in a temperature response beyond that expected from changes in viscosity. The extra response was not found for leaves with veins cut, indicating that water crosses cell membranes after it leaves the xylem. The large proportion of resistance in the venation can explain why stomata respond to leaf xylem damage and cavitation. The hydraulic importance of the leaf vein system suggests that the diversity of vein system architectures observed in angiosperms may reflect variation in whole-leaf hydraulic capacity.     

Leaf water 18O and 2H maps show directional enrichment discrepancy in Colocasia esculenta

Spatial patterns of leaf water isotopes are challenging to predict because of the intricate link between vein and lamina water. Many models have attempted to predict these patterns, but to date, most have focused on monocots with parallel veins. These provide a simple system to study, but do not represent the majority of plant species. Here, a new protocol is developed using a Picarro induction module coupled to a cavity ringdown spectrometer to obtain maps of the leaf water isotopes (¹⁸O and ²H). The technique is applied to Colocasia esculenta leaves. The results are compared with isotope ratio mass spectrometry. In C. esculenta, a large enrichment in the radial direction is observed, but not in the longitudinal direction. The string‐of‐lakes model fails to predict the observed patterns, while the Farquhar–Gan model is more successful, especially when enrichment is accounted for along the radial direction. Our results show that reticulate‐veined leaves experience a larger enrichment along the axis of the secondary veins than along the midrib. We hypothesize that this is due to the lower major/minor vein ratio that leads to longer pathways between major veins and sites of evaporation.     

Co-ordinated development of the leaf midrib xylem with the lamina in Nicotiana tabacum

Background and Aims

The water-transport capacity of leaf venation is positively related to the leaf-lamina area, because the number and diameter of vein-xylem conduits are controlled to match the lamina area. This study aimed to investigate how this co-ordinated relationship between the leaf-lamina area and vein-xylem characteristics is achieved by examining the midrib xylem of tobacco leaves.

Methods

The changes in the midrib-xylem characteristics over time were quantified using leaves with four different final lamina areas. The measured data were fitted to sigmoidal functions. From the constants of the fitted curves, the final values in mature leaves, maximal developmental rates (V_Dev) and developmental duration (T_Dev) were estimated for each of the xylem characteristics. Whether it is the lamina or the midrib xylem that drives the co-ordinated development was examined by lamina removal from unfolding leaves. The effects of the application of 0·1 % IAA (indole-3-acetic acid) to leaves with the laminas removed were also analysed.

Key Results

For both the leaf lamina and the midrib-xylem characteristics, the differences in final values among leaves with different lamina areas were more strongly associated with those in V_Dev. Notably, the V_Dev values of the midrib-xylem characteristics were related to those of the leaf-lamina area. By lamina removal, the conduit diameter was reduced but the number of conduits did not significantly change. By IAA application, the decrease in the conduit diameter was halted, and the number of conduits in the midrib xylem increased.

Conclusions

According to the results, the V_Dev values of the lamina area and the midrib-xylem characteristics changed in a co-ordinated manner, so that the water-transport capacity of the midrib xylem was positively related to the leaf-lamina area. The results also suggest that IAA derived from the leaf lamina plays a crucial role in the development of the leaf venation.     

Bundle sheath lignification mediates the linkage of leaf hydraulics and venation

The lignification of the leaf vein bundle sheath (BS) has been observed in many species and would reduce conductance from xylem to mesophyll. We hypothesized that lignification of the BS in lower‐order veins would provide benefits for water delivery through the vein hierarchy but that the lignification of higher‐order veins would limit transport capacity from xylem to mesophyll and leaf hydraulic conductance (K_leaf). We further hypothesized that BS lignification would mediate the relationship of K_leaf to vein length per area. We analysed the dependence of K_leaf, and its light response, on the lignification of the BS across vein orders for 11 angiosperm tree species. Eight of 11 species had lignin deposits in the BS of the midrib, and two species additionally only in their secondary veins, and for six species up to their minor veins. Species with lignification of minor veins had a lower hydraulic conductance of xylem and outside‐xylem pathways and lower K_leaf. K_leaf could be strongly predicted by vein length per area and highest lignified vein order (R² = .69). The light‐response of K_leaf was statistically independent of BS lignification. The lignification of the BS is an important determinant of species variation in leaf and thus whole plant water transport.     

Water Transport in Impaired Leaf Vein Systems

Abstract: The subject of our investigation was the water regime of broad bean leaves ( Vicia faba L.), especially after having mechanically severed parts of the leaf blade and the leaf venation. Under moderate conditions, 18 - 22 °C temperature and 50 - 70 % relative humidity, the leaves remained viable even after extensive damage. Only if more than 90 % of the xylem cross sectional area of a leaf was severed, the leaf wilted. Lesser damage to the xylem cross-sectional area only resulted in a reduced rate of transpiration and assimilation, compared to intact leaves. The cuts in larger veins were bypassed into small or even very small veins, as shown by xylem transport of dyes. In intact leaves, small veins have a negligible task in long-distance transport. Here, however, transport velocity in small veins was severalfold increased compared to the measurement of transport velocity in veins of the same size in intact leaves. Thereby, water transport to leaf areas distal from the cut was ensured.     

Are leaves ‘freewheelin'? Testing for a Wheeler‐type effect in leaf xylem hydraulic decline

A recent study found that cutting shoots under water while xylem was under tension (which has been the standard protocol for the past few decades) could produce artefactual embolisms inside the xylem, overestimating hydraulic vulnerability relative to shoots cut under water after relaxing xylem tension (Wheeler et al. 2013). That study also raised the possibility that such a ‘Wheeler effect’ might occur in studies of leaf hydraulic vulnerability. We tested for such an effect for four species by applying a modified vacuum pump method to leaves with minor veins severed, to construct leaf xylem hydraulic vulnerability curves. We tested for an impact on leaf xylem hydraulic conductance (K_x) of cutting the petiole and minor veins under water for dehydrated leaves with xylem under tension compared with dehydrated leaves after previously relaxing xylem tension. Our results showed no significant ‘cutting artefact’ for leaf xylem. The lack of an effect for leaves could not be explained by narrower or shorter xylem conduits, and may be due to lesser mechanical stress imposed when cutting leaf petioles, and/or to rapid refilling of emboli in petioles. These findings provide the first validation of previous measurements of leaf hydraulic vulnerability against this potential artefact.     

Ultrasound acoustic emissions from dehydrating leaves of deciduous and evergreen trees

While drying, detached leaves produced ultrasound acoustic emissions (UAE) comparable to emissions from stem and twig wood. Experiments on Ilex aquifolium L. showed that the main source of these signals was cavitation in the veins, to which conduits and fibres probably both contributed. Regions of the leaf blade with abundant mesophyll and only small veins emitted few signals. More signals were counted on the adaxial side of the midrib than on the abaxial one and on the proximal third than on the distal one, in accordance with the anatomical structure. Sound attenuation was pronounced. Eight species were compared with respect to cavitation behaviour, field water relations and pressure–volume curves, and the data showed differences in cumulative number of events and resistance of leaves to cavitation. Data were generally in good agreement with anatomical structure and habitat preferences. The number of signals per conduit counted on cross-sections was in some leaves much higher than unity, which suggests short xylem elements or an acoustic activity of cells other than conduits. There was no correlation between cavitation threshold or cumulative number of signals and the degree of sclerophylly; unexpectedly, there was a correlation between the cumulative number of signals at a water potential of -1.3 MPa and the bulk modulus of elasticity.     

STUDIES ON THE LEAF OF POPULUS DELTOIDES (SALICACEAE): MORPHOLOGY AND ANATOMY

Mature field- and growth-chamber-grown leaves of Populus deltoides Bartr. ex Marsh. were examined with light and scanning electron microscopes to determine their vasculature and the spatial relationships of the various orders of vascular bundles to the mesophyll. Three leaf traces, one median and two lateral, enter the petiole at the node. Progressing acropetally in the petiole these bundles are rearranged and gradually form as many as 13 tiers of vascular tissue in the petiole at the base of the lamina. (Most leaves contained seven vertically stacked tiers.) During their course through the midrib the tiers “unstack” and portions diverge outward and continue as secondary veins toward the margin on either side of the lamina. As the midvein approaches the leaf tip it is represented by a single vascular bundle which is a continuation of the original median bundle. Tertiary veins arise from the secondary veins or the midvein, and minor veins commonly arise from all orders of veins. All major veins–primaries, secondaries, intersecondaries, and tertiaries–are associated with rib tissue, while minor veins are completely surrounded by a parenchymatous bundle sheath. The bundle sheaths of tertiary, quaternary, and portions of quinternary veins are associated with bundle-sheath extensions. Minor veins are closely associated spatially with both ad- and abaxial palisade parenchyma of the isolateral leaf and also with one or two layers of paraveinal mesophyll that extend horizontally between the veins. The leaves of growth-chamber-grown plants had thinner blades, a higher proportion of air space, and greater interveinal distances than those of field-grown plants.     

Hydraulic architecture of leaf venation in Laurus nobilis L.

Veins are the main irrigation system of the leaf lamina and an understanding of the hydraulic architecture of the vein networks is essential for understanding leaf function. However, determination of leaf hydraulic parameters is challenging, because for most leaves the vein system is reticulate, contains a hierarchy of different vein sizes, and consists of leaky conduits. We present a new approach that allows for measurements of pressure differences between the petiole and any vein within the leaf. Measurements of Laurus nobilis leaves indicate that first‐ and second‐order veins have high axial conductance and relatively small radial permeability, thus allowing water to reach distal areas of the leaf with only a small loss of water potential. Higher order veins tend to be more hydraulically resistant and permit greater radial leakage. This design allows for a relatively equitable distribution of water potential and thus reflects the capacity of the venation to provide a relatively homogeneous water supply across the leaf lamina, with only the leaf margins being hydraulically disadvantaged relative to the rest of the leaf.     

Scaling of xylem vessels and veins within the leaves of oak species

General models of plant vascular architecture, based on scaling of pipe diameters to remove the length dependence of hydraulic resistance within the xylem, have attracted strong interest. However, these models have neglected to consider the leaf, an important hydraulic component; they assume all leaves to have similar hydraulic properties, including similar pipe diameters in the petiole. We examine the scaling of the leaf xylem in 10 temperate oak species, an important hydraulic component. The mean hydraulic diameter of petiole xylem vessels varied by 30% among the 10 oak species. Conduit diameters narrowed from the petiole to the midrib to the secondary veins, consistent with resistance minimization, but the power function scaling exponent differed from that predicted for stems. Leaf size was an organizing trait within and across species. These findings indicate that leaf vasculature needs to be included in whole-plant scaling models, for these to accurately reflect and predict whole-plant transport and its implications for performance and ecology.     

Distribution of chloride in tobacco laminae and midribs as influenced by the chloride content of the irrigation water

The high variability of the ratio of midrib to lamina chloride in tobacco leaves delivered to the curing plant prompted two experiments which aimed to identify some of the causes of such variability. In young tobacco plants chloride concentration was highest in the third or fourth leaf from the base of the plant, but in more mature plants (when the inflorescence began to appear) leaf chloride increased linearly from the apex to the base of the plant. The ratio of the concentration of midrib chloride to that of lamina chloride was always highest in the basal leaves, and decreased with increasing chloride concentration in the irrigation water,i.e. with increasing chloride supply more chloride went to the lamina than to the midrib per unit dry weight. This was verified with reported chloride contents for two irrigation experiments.     

The major veins of mesomorphic leaves revisited: tests for conductive overload in Acer saccharum (Aceraceae) and Quercus rubra (Fagaceae)

Many leaves survive the severing of their major veins in apparently excellent health. According to the classical explanation, the leaf minor veins provide "conductive overload," an excess of parallel conductive paths, rendering the major veins hydraulically dispensable. Whether such an excess of conductive paths exists has important implications for vascular design and for leaf response to vascular damage. We subjected leaves of Acer saccharum and Quercus rubra to cutting treatments that disrupted the major vein system and determined leaf survival, stomatal conductance (g), quantum yield of photosystem II (Φ(PSII)), and leaf hydraulic conductance (K(leaf)). For A. saccharum, the cuts led to the death of distal lamina. For Q. rubra, however, the treated leaves typically remained apparently healthy. Despite their appearance, the treated Q. rubra leaves had a strongly reduced K(leaf), relative to control leaves, and g and Φ(PSII) were reduced distal to the cuts, respectively, by 75-97% and 48-76%. Gas exchange proximal to the cuts was unaffected, indicating the independence of lamina regions and their local stomata. Analogous results were obtained with excised Q. rubra leaves. These studies demonstrate an indispensable, vital role of the major veins in conducting water throughout the lamina.     

Evidence for xylem embolism as a primary factor in dehydration-induced declines in leaf hydraulic conductance

Hydraulic conductance of leaves (K(leaf)) typically decreases with increasing water stress and recent studies have proposed different mechanisms responsible for decreasing K(leaf) . We measured K(leaf) concurrently with ultrasonic acoustic emissions (UAEs) in dehydrating leaves of several species to determine whether declining K(leaf) was associated with xylem embolism. In addition, we performed experiments in which the surface tension of water in the leaf xylem was reduced by using a surfactant solution. Finally, we compared the hydraulic vulnerability of entire leaves with the leaf lamina in three species. Leaf hydraulic vulnerability based on rehydration kinetics and UAE was very similar, except in Quercus garryana. However, water potentials corresponding to the initial decline in K(leaf) and the onset of UAE in Q. garryana were similar. In all species tested, reducing the surface tension of water caused K(leaf) to decline at less negative water potentials compared with leaves supplied with water. Microscopy revealed that as the fraction of embolized xylem increased, K(leaf) declined sharply in Q. garryana. Measurements on leaf discs revealed that reductions in lamina hydraulic conductance with dehydration were not as great as those observed in intact leaves, suggesting that embolism was the primary mechanism for reductions in K(leaf) during dehydration.     

Determination of the total chlorophyll distribution pattern in living leaves

Tradescantia albiflora-leaves were used in developing a determination method for chlorophyll in living leaves using a microscopic spectro photometer (MPV, Leitz).The wavelength of the maximal absorption for chlorophyll a was found to be at 670 nm and for chlorophyll b at 652 nm. To calculate the reference values the intensity of the transmitted light at 750 nm was measured. The absorption at 750 nm results only from the chlorophyll free structure in the leaves. To correct optical errors the two-wavelength method was used. The values gained in arbitrary units were calibrated against data measured in the conventional way. The resulting calibration line shows a very high correlation coefficient where r²=0.997. It was proved that the calibration line was also correct for determinations with leaves from other plant species.Using this determination method the chlorophyll content of small areas on the living leaf blade of Phaseolus vulgaris was determined.As consequence of the thicker mesophyll accompanying the veins the chlorophyll concentration is 30% higher in this tissue. A lower chlorophyll concentration was observed in the thinner leaf tip and in the oldest regions at the leaf ground.At the leaf tip, the leaf ground and in the tissue along the veins an iron deficit causes less bleaching than in the areoles. In the same leaf regions the bleaching herbicide atrazine induces rapid bleaching if supplied through transpiration stream. The application of Atrazine on the leaf surface gives rise to the contrary effect.All these phenomena seem to be a result of a differing water supply by the xylem vessels.     

Sink to Source Transition of Populus Leaves

Development of the Populus leaf is presented as a model system to illustrate the sequence of events that occur during the sink to source transition. A Populus leaf is served by three leaf traces, each of which consists of an original procambial trace bundle that differentiates acropetally and continuously from more mature procambium in the stem and a complement of subsidiary bundles that differentiates bidirectionally from a leaf basal meristem. During development these subsidiary bundles maintain continuity through the meristematic region of the node. The basipetally developing subsidiary bunles form phloem bridges that serve to integrate adjacent leaf traces of the stem vasculature. Distal to the node the acropetally developing bundles from all three leaf traces are reoriented in a precise and orderly sequence to form tiers of petiolar bundles. These tiers of bundles extend into the midrib where bundles diverge at intervals as the major lateral veins. The dorsal-most tier of bundles extends to the lamina tip and each successive tier of bundles contributes to lateral veins situated more proximally in the lamina. Although the midrib and the major vein system differentiate acropetally in the lamina, they mature basipetally. Maturation of the mesophyll and other lamina tissues also mature basipetally. As a consequence of the basi-petal maturation process, the lamina tip matures very early and begins exporting photosynthates while the lamina base is still importing from other leaves. The transition of a leaf from sink to source status must therefore be considered as a progression of structural and functional events that occur in synchrony.     

Hydraulic and Chemical Responses of Citrus Seedlings to Drought and Osmotic Stress

In this work we investigated the function of abscisic acid (ABA) as a long-distance chemical signal communicating water shortage from the root to the shoot in citrus plants. Experiments indicated that stomatal conductance, transpiration rates, and leaf water potential decline progressively with drought. ABA content in roots, leaves, and xylem sap was also increased by the drought stress treatment three- to sevenfold. The addition of norflurazon, an inhibitor of ABA biosynthesis, significantly decreased the intensity of the responses and reduced ABA content in roots and xylem fluid, but not in leaves. Polyethylene glycol (PEG)-induced osmotic stress caused similar effects and, in general, was counteracted only by norflurazon at the lowest concentration (10%). Partial defoliation was able to diminish only leaf ABA content (22.5%) at the highest PEG concentration (30%), probably through a reduction of the active sites of biosynthesis. At least under moderate drought (3–6 days without irrigation), mechanisms other than leaf ABA concentration were required to explain stomatal closure in response to limited soil water supply. Measurements of xylem sap pH revealed a progressive alkalinization through the drought condition (6.4 vs. 7.1), that was not counteracted with the addition of norflurazon. Moreover, in vitro treatment of detached leaves with buffers iso-osmotically adjusted at pH 7.1 significantly decreased stomatal conductance (more than 30%) as much as 70% when supplemented with ABA. Taken together, our results suggest that increased pH generated in drought-stressed roots is transmitted by the xylem sap to the leaves, triggering reductions in shoot water loss. The parallel rise in ABA concentration may act synergistically with pH alkalinization in xylem sap, with an initial response generated from the roots and further promotion by the stressed leaves.     

Effect of Excision on Leaf Water Potential

Improvements of thermocouple hygrometric techniques for in situleaf water potential measurement in the field now allows forcontinuous monitoring of water potential in response to an externalperturbation, such as leaf excision. Using Citrus jambhiri plants,measured leaf water potentials of completely excised leaf portionsimmediately increased when the petiole was excised or incisionswere made either transverse or parallel to the midrib. Incisionsparallel to the midrib were on the side nearest the hygrometeror opposite it if preceded by a petiole excision. Midrib incisionswere 100–150 mm long with the nearest cut edge being 20–50mm from the hygrometer cavity. All excisions were such thatleaf tissue was removed from the leaf with water potential onone of the leaf portions being measured continuously prior toand after excising. The peak increase in measured water potentialof the excised leaf portions ranged between 20 kPa and 80 kPabut averaged 50 kPa. In uncovered leaves, particularly underfield conditions with the associated high evaporative demand,measured leaf water potential declined rapidly after the initialincrease. The increase in measured water potential immediatelyfollowing various types of excision was confirmed for dark andlight conditions (laboratory and field respectively) using bothpsychrometric and dewpoint modes and occurred for secondaryexcisions, but to a lesser extent. Discovery of this phenomenonimplies that water potential measured on detached leaves maynot always represent accurately in situ leaf water potential. Key words: Leaf water potential, Thermocouple hygrometers, Leaf excision effects     

The flow and dispersion of water in the vascular network of dicotyledonous leaves

The relationships between the geometric characteristics of, the local flow rates of xylem sap in, and relative pressures in the reticulate anastomosing vascular network of dicotyledonous leaves of Populus Balsamifera L. are reported. The conducting channels of cellulosic microcapillaries are covered by sheaths of chloroplast free cells through the walls of which water withdrawn from vascular bundles percolates to reach evaporation sites. Along the mid-rib and branch generations, the population and cross-section areas of the microcapillaries decrease with distance but not in a monotonic manner. Lateral withdrawal rates from the veins were highest at the base of the leaf lamina. More than 50% of the inlet stream had dispersed out of the conduits within the first 25% of the leaf lamina area from the petiole junction. Absolute values of pressure gradients generally decreased in the apical direction along the vein.