Thorsten Knipfer

Water uptake along the length of grapevine fine roots: developmental anatomy, tissue specific aquaporin expression, and pathways of water transport

Peak aquaporin expression/activity and hydraulic conductivity occurred in root tips and interior tissues; contrary to theoretical predictions, low aquaporin expression and activity in suberized secondary growth portions of fine roots suggests a limited role in controlling water uptake in this region of the root. To better understand water uptake patterns in root systems of woody perennial crops, we detailed the developmental anatomy and hydraulic physiology along the length of grapevine (Vitis berlandieri × Vitis rupestris) fine roots from the tip to secondary growth zones. Our characterization included the localization of suberized structures and aquaporin gene expression and the determination of hydraulic conductivity (Lpr) and aquaporin protein activity (via chemical inhibition) in different root zones under both osmotic and hydrostatic pressure gradients. Tissue-specific messenger RNA levels of the plasma membrane aquaporin isogenes (VvPIPs) were quantified using laser-capture microdissection and quantitative polymerase chain reaction. Our results highlight dramatic changes in structure and function along the length of grapevine fine roots. Although the root tip lacked suberization altogether, a suberized exodermis and endodermis developed in the maturation zone, which gave way to the secondary growth zone containing a multilayer suberized periderm. Longitudinally, VvPIP isogenes exhibited strong peaks of expression in the root tip that decreased precipitously along the root length in a pattern similar to Arabidopsis (Arabidopsis thaliana) roots. In the radial orientation, expression was always greatest in interior tissues (i.e. stele, endodermis, and/or vascular tissues) for all root zones. High Lpr and aquaporin protein activity were associated with peak VvPIP expression levels in the root tip. This suggests that aquaporins play a limited role in controlling water uptake in secondary growth zones, which contradicts existing theoretical predictions. Despite having significantly lower Lpr, woody roots can constitute the vast majority of the root system surface area in mature vines and thus provide for significant water uptake potential.

Grapevine species from varied native habitats exhibit differences in embolism formation/repair associated with leaf gas exchange and root pressure.

Drought induces xylem embolism formation, but grapevines can refill non-functional vessels to restore transport capacity. It is unknown whether vulnerability to embolism formation and ability to repair differ among grapevine species. We analysed in vivo embolism formation and repair using x-ray computed microtomography in three wild grapevine species from varied native habitats (Vitis riparia, V. arizonica, V. champinii) and related responses to measurements of leaf gas exchange and root pressure. Vulnerability to embolism formation was greatest in V. riparia, intermediate in V. arizonica and lowest in V. champinii. After re-watering, embolism repair was rapid and pronounced in V. riparia and V. arizonica, but limited or negligible in V. champinii even after numerous days. Similarly, root pressure measured after re-watering was positively correlated with drought stress severity for V. riparia and V. arizonica (species exhibiting embolism repair) but not for V. champinii. Drought-induced reductions in transpiration were greatest for V. riparia and least in V. champinii. Recovery of transpiration after re-watering was delayed for all species, but was greatest for V. champinii and most rapid in V. arizonica. These species exhibit varied responses to drought stress that involve maintenance/recovery of xylem transport capacity coordinated with root pressure and gas exchange responses.

In Situ Visualization of the Dynamics in Xylem Embolism Formation and Removal in the Absence of Root Pressure: A Study on Excised Grapevine Stems

In excised grapevine stems, temporal changes in xylem embolism formation and removal are driven by vessel-associated tissue. Gas embolisms formed during drought can disrupt long-distance water transport through plant xylem vessels, but some species have the ability to remove these blockages. Despite evidence suggesting that embolism removal is linked to the presence of vessel-associated parenchyma, the underlying mechanism remains controversial and is thought to involve positive pressure generated by roots. Here, we used in situ x-ray microtomography on excised grapevine stems to determine if embolism removal is possible without root pressure, and if the embolism formation/removal affects vessel functional status after sample excision. Our data show that embolism removal in excised stems was driven by water droplet growth and was qualitatively identical to refilling in intact plants. When stem segments were rehydrated with H2O after excision, vessel refilling occurred rapidly (<1 h). The refilling process was substantially slower when polyethylene glycol was added to the H2O source, thereby providing new support for an osmotically driven refilling mechanism. In contrast, segments not supplied with H2O showed no refilling and increased embolism formation. Dynamic changes in liquid/wall contact angles indicated that the processes of embolism removal (i.e. vessel refilling) by water influx and embolism formation by water efflux were directly linked to the activity of vessel-associated living tissue. Overall, our results emphasize that root pressure is not required as a driving force for vessel refilling, and care should be taken when performing hydraulics measurements on excised plant organs containing living vessel-associated tissue, because the vessel behavior may not be static.

Mechanical Failure of Fine Root Cortical Cells Initiates Plant Hydraulic Decline during Drought

Drought-induced mechanical failure (lacunae formation) in fine root cortical cells initiates hydraulic decline under drought stress and precedes xylem embolism and root shrinkage. Root systems perform the crucial task of absorbing water from the soil to meet the demands of a transpiring canopy. Roots are thought to operate like electrical fuses, which break when carrying an excessive load under conditions of drought stress. Yet the exact site and sequence of this dysfunction in roots remain elusive. Using in vivo x-ray computed microtomography, we found that drought-induced mechanical failure (i.e. lacunae formation) in fine root cortical cells is the initial and primary driver of reduced fine root hydraulic conductivity (Lpr) under mild to moderate drought stress. Cortical lacunae started forming under mild drought stress (−0.6 MPa Ψstem), coincided with a dramatic reduction in Lpr, and preceded root shrinkage or significant xylem embolism. Only under increased drought stress was embolism formation observed in the root xylem, and it appeared first in the fine roots (50% loss of hydraulic conductivity [P50] reached at −1.8 MPa) and then in older, coarse roots (P50 = −3.5 MPa). These results suggest that cortical cells in fine roots function like hydraulic fuses that decouple plants from drying soil, thus preserving the hydraulic integrity of the plant’s vascular system under early stages of drought stress. Cortical lacunae formation led to permanent structural damage of the root cortex and nonrecoverable Lpr, pointing to a role in fine root mortality and turnover under drought stress.

Patterns of drought-induced embolism formation and spread in living walnut saplings visualized using X-ray microtomography

Embolism formation and spread are dependent on conduit structure and xylem network connectivity. Detailed spatial analysis has been limited due to a lack of non-destructive methods to visualize these processes in living plants. We used synchrotron X-ray computed tomography (microCT) to visualize these processes in vivo for Juglans microcarpa Berl. saplings subjected to drought, and also evaluated embolism repair capability after re-watering. Cavitation was not detected in vivo until stem water potentials (Ψ(stem)) reached -2.2 MPa, and loss of stem hydraulic conductivity as derived from microCT images predicted that 50% of conductivity was lost at Ψ(stem) of ∼ -3.5 MPa; xylem vulnerability as determined with the centrifuge method was comparable only in the range of Ψ(stem) from -2.5 to -3.5 MPa. MicroCT images showed that cavitation appeared initially in isolated vessels not connected to other air-filled conduits. Once embolized vessels were present, multiple vessels in close proximity cavitated, and 3-D analysis along the stem axis revealed some connections between cavitated vessels. A tomography-derived automated xylem network analysis found that only 36% of vessels had one or more connections to other vessels. Cavitation susceptibility was related to vessel diameter, with large diameter vessels (>40 μm, mean diameter 25-30 μm) cavitating mainly under moderate stress (Ψ(stem) > -3 MPa) and small diameter vessels (<30 μm) under severe stress. After re-watering there was no evidence for short or longer term vessel refilling over 2 weeks despite a rapid recovery of plant water status. The low embolism susceptibility in 1-year-old J. microcarpa may aid sapling survival during establishment.

Water Transport Properties of the Grape Pedicel during Fruit Development: Insights into Xylem Anatomy and Function Using Microtomography

Xylem flow into the grape fruit declines before the onset of ripening, and losses in pedicel hydraulic conductivity could be attributed to xylem vessel blockages. Xylem flow of water into fruits declines during fruit development, and the literature indicates a corresponding increase in hydraulic resistance in the pedicel. However, it is unknown how pedicel hydraulics change developmentally in relation to xylem anatomy and function. In this study on grape (Vitis vinifera), we determined pedicel hydraulic conductivity (kh) from pressure-flow relationships using hydrostatic and osmotic forces and investigated xylem anatomy and function using fluorescent light microscopy and x-ray computed microtomography. Hydrostatic kh (xylem pathway) was consistently 4 orders of magnitude greater than osmotic kh (intracellular pathway), but both declined before veraison by approximately 40% and substantially over fruit development. Hydrostatic kh declined most gradually for low (less than 0.08 MPa) pressures and for water inflow and outflow conditions. Specific kh (per xylem area) decreased in a similar fashion to kh despite substantial increases in xylem area. X-ray computed microtomography images provided direct evidence that losses in pedicel kh were associated with blockages in vessel elements, whereas air embolisms were negligible. However, vessel elements were interconnected and some remained continuous postveraison, suggesting that across the grape pedicel, a xylem pathway of reduced kh remains functional late into berry ripening.

Root hydraulics in salt-stressed wheat

The aim of the present study was to test whether salinity, which can impact through its osmotic stress component on the ability of plants to take up water, affects root water transport properties (hydraulic conductivity) in bread wheat (Triticum aestivum L). Hydroponically grown plants were exposed to 100mM NaCl when they were 10-11 days old. Plants were analysed during the vegetative stage of development when they were 15-17 days old and the root system consisted entirely of seminal roots, and when they were 22-24 days old, by which time adventitious roots had developed. Root hydraulic conductivity (Lp) was determined through exudation experiments (osmotic Lp) on individual roots and the entire plant root system, and through experiments involving intact, transpiring plants (hydrostatic Lp). Salt stress caused a general reduction (40-80%) in Lp, irrespective of whether individual seminal and adventitious roots, entire root systems or intact, transpiring plants were analysed. Osmotic and hydrostatic Lp were in the same range. The data suggest that most radial root water uptake in wheat grown in the presence and absence of NaCl occurs along a pathway that involves the crossing of membranes. As wheat plants develop, a nonmembraneous (apoplast) pathway contributes increasingly to radial water uptake in control but not in NaCl-stressed plants.

Aquaporins and Root Water Uptake

Water is one of the most critical resources limiting plant growth and crop productivity, and root water uptake is an important aspect of plant physiology governing plant water use and stress tolerance. Pathways of root water uptake are complex and are affected by root structure and physiological responses of the tissue. Water travels from the soil to the root xylem through the apoplast (i.e., cell wall space) and/or cell-to-cell, but hydraulic barriers in the apoplast (e.g., suberized structures in the endodermis) can force water to traverse cell membranes at some points along this path. Anytime water crosses a cell membrane, its transport can be affected by the activity of membrane-intrinsic water channel proteins (aquaporins). We review how aquaporins can play an important role in affecting root water transport properties (hydraulic conductivity, Lp), and thus alter water uptake, plant water status, nutrient acquisition, growth, and transpiration. Plants have the capacity to regulate aquaporin activity through a variety of mechanisms (e.g., pH, phosphorylation, internalization, oxidative gating), which may provide a rapid and reversible means of regulating root Lp. Changes in root Lp via the modulation of aquaporin activity is thought to contribute to root responses to a broad range of stresses including drought, salt, nutrient deficiency, and cold. Given their role in contributing to stress tolerance, aquaporins may serve as future targets for improving crop performance in stressful environments.

Storage compartments for capillary water rarely refill in an intact woody plant

Visualization of refilling dynamics in Laurus nobilis revealed that both capillary water storage and vessel refilling play minor roles under in vivo conditions. Water storage is thought to play an integral role in the maintenance of whole-plant water balance. The contribution of both living and dead cells to water storage can be derived from rehydration and water-release curves on excised plant material, but the underlying tissue-specific emptying/refilling dynamics remain unclear. Here, we used x-ray computed microtomography to characterize the refilling of xylem fibers, pith cells, and vessels under both excised and in vivo conditions in Laurus nobilis. In excised stems supplied with water, water uptake exhibited a biphasic response curve, and x-ray computed microtomography images showed that high water storage capacitance was associated with fiber and pith refilling as driven by capillary forces: fibers refilled more rapidly than pith cells, while vessel refilling was minimal. In excised stems that were sealed, fiber and pith refilling was associated with vessel emptying, indicating a link between tissue connectivity and water storage. In contrast, refilling of fibers, pith cells, and vessels was negligible in intact saplings over two time scales, 24 h and 3 weeks. However, those compartments did refill slowly when the shoot was covered to prevent transpiration. Collectively, our data (1) provide direct evidence that storage compartments for capillary water refill in excised stems but rarely under in vivo conditions, (2) highlight that estimates of capacitance from excised samples should be interpreted with caution, as certain storage compartments may not be utilized in the intact plant, and (3) question the paradigm that fibers play a substantial role in daily discharge/recharge of stem capacitance in an intact tree.

Polarity of Water Transport across Epidermal Cell Membranes in Tradescantia virginiana

An outward bias polarizing of water transport, which appears to be a property of the intact membrane/protein complex of epidermal cells in situ. Using the automated cell pressure probe, small and highly reproducible hydrostatic pressure clamp (PC) and pressure relaxation (PR) tests (typically, applied step change in pressure = 0.02 MPa and overall change in volume = 30 pL, respectively) were applied to individual Tradescantia virginiana epidermal cells to determine both exosmotic and endosmotic hydraulic conductivity (LpOUT and LpIN, respectively). Within-cell reproducibility of measured hydraulic parameters depended on the method used, with the PR method giving a lower average coefficient of variation (15.2%, 5.8%, and 19.0% for half-time, cell volume [Vo], and hydraulic conductivity [Lp], respectively) than the PC method (25.4%, 22.0%, and 24.2%, respectively). Vo as determined from PC and PR tests was 1.1 to 2.7 nL and in the range of optically estimated Vo values of 1.5 to 4.9 nL. For the same cell, Vo and Lp estimates were significantly lower (about 15% and 30%, respectively) when determined by PC compared with PR. Both methods, however, showed significantly higher LpOUT than LpIN (LpOUT/LpIN ≅ 1.20). Because these results were obtained using small and reversible hydrostatically driven flows in the same cell, the 20% outward biased polarity of water transport is most likely not due to artifacts associated with unstirred layers or to direct effects of externally applied osmotica on the membrane, as has been suggested in previous studies. The rapid reversibility of applied flow direction, particularly for the PR method, and the lack of a clear increase in LpOUT/LpIN over a wide range of Lp values suggest that the observed polarity is an intrinsic biophysical property of the intact membrane/protein complex.