Wolfram Hartung

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.

The Stomatal Response to Reduced Relative Humidity Requires Guard Cell-Autonomous ABA Synthesis

Stomata are pores on the leaf surface, bounded by two guard cells, which control the uptake of CO(2) for photosynthesis and the concomitant loss of water vapor. In 1898, Francis Darwin showed that stomata close in response to reduced atmospheric relative humidity (rh); however, our understanding of the signaling pathway responsible for coupling changes in rh to alterations in stomatal aperture is fragmentary. The results presented here highlight the primacy of abscisic acid (ABA) in the stomatal response to drying air. We show that guard cells possess the entire ABA biosynthesis pathway and that it appears upregulated by positive feedback by ABA. When wild-type Arabidopsis and the ABA-deficient mutant aba3-1 were exposed to reductions in rh, the aba3-1 mutant wilted, whereas the wild-type did not. However, when aba3-1 plants, in which ABA synthesis had been specifically rescued in guard cells, were challenged with dry air, they did not wilt. These data indicate that guard cell-autonomous ABA synthesis is required for and is sufficient for stomatal closure in response to low rh. Guard cell-autonomous ABA synthesis allows the plant to tailor leaf gas exchange exquisitely to suit the prevailing environmental conditions.

Abscisic acid and hydraulic conductivity of maize roots: a study using cell- and root-pressure probes.

Abstract. Using root- and cell-pressure probes, the effects of the stress hormone abscisic acid (ABA) on the water-transport properties of maize roots (Zea mays L.) were examined in order to work out dose and time responses for root hydraulic conductivity. Abscisic acid applied at concentrations of 100–1,000 nM increased the hydraulic conductivity of excised maize roots both at the organ (root Lpr: factor of 3–4) and the root cell level (cell Lp: factor of 7–27). Effects on the root cortical cells were more pronounced than at the organ level. From the results it was concluded that ABA acts at the plasmalemma, presumably by an interaction with water channels. Abscisic acid therefore facilitated the cell-to-cell component of transport of water across the root cylinder. Effects on cell Lp were transient and highly specific for the undissociated (+)-cis-trans-ABA. The stress hormone ABA facilitates water uptake into roots as soils start drying, especially under non-transpiring conditions, when the apoplastic path of water transport is largely excluded.

An abscisic acid-related reduced transpiration promotes gradual embolism repair when grapevines are rehydrated after drought

* Proposed mechanisms of embolism recovery are controversial for plants that are transpiring while undergoing cycles of dehydration and rehydration. * Here, water stress was imposed on grapevines (Vitis vinifera), and the course of embolism recovery, leaf water potential (Psi(leaf)), transpiration (E) and abscisic acid (ABA) concentration followed during the rehydration process. * As expected, Psi(leaf) and E decreased upon water stress, whereas xylem embolism and leaf ABA concentration increased. Upon rehydration, Psi(leaf) recovered in 5 h, whereas E fully recovered only after an additional 48 h. The ABA content of recovering leaves was higher than in droughted controls, both on the day of rewatering and the day after, suggesting that ABA accumulated in roots during drought was delivered to the rehydrated leaves. In recovering plants, xylem embolism in petioles, shoots, and roots decreased during the 24 h following rehydration. * A model is proposed to describe plant recovery after rehydration based on three main points: embolism repair occurs progressively in shoots and further in roots and in petioles, following an almost full recovery of Psi(leaf); hydraulic conductance recovers during diurnal transpiring hours, when formation and repair of embolisms occurs in all plant organs; an ABA residual signal in rehydrated leaves hinders stomatal opening even when water relations have recovered, suggesting that an ABA-induced transpiration control promotes gradual embolism repair in rehydrated grapevines.

Cadmium leads to stimulated expression of the lipid transfer protein genes in barley: implications for the involvement of lipid transfer proteins in wax assembly.

Abstract. In order to investigate the nature of genes expressed in leaf epidermal cells of higher plants, we have identified the nucleotide sequence of a cDNA designated ltp 7a2b encoding a novel nonspecific lipid transfer protein of barley (Hordeum vulgare L. cv. Gerbel). The cDNA of 755 basepairs contains an open reading frame of 366 nucleotides coding for a 12.3-kDa polypeptide. The first 29 amino acids constitute the putative signal peptide, characteristic for targeting to the secretory pathway. Analysis of mRNA levels by Northern blotting indicated that ltp 7a2b is preferentially expressed in the leaf epidermis. Levels of mRNA decreased during ageing of leaf tissue. Expression of ltp 7a2b was stimulated by a factor of 2–3 when the seedlings were grown in the presence of cadmium (10–1600 μM). Concomitantly, the primary leaves of Cd-exposed seedlings contained elevated levels of abscisic acid and a thicker wax layer of the cuticle. At 100 μM Cd in the hydroponic medium, the wax cover was increased by 50%. The increase in abscisic acid content, ltp 7a2b mRNA and wax coverage was either not seen, or seen much less, in Ni- and Zn-stressed seedlings. The data add circumstantial evidence to the recently proposed hypothesis that nonspecific lipid transfer proteins function in transfer of cutin and/or wax monomers from the site of synthesis in the cell to the cuticle.

Regulation of the ABA-sensitive Arabidopsis potassium channel gene GORK in response to water stress.

The phytohormone abscisic acid (ABA) regulates many stress‐related processes in plants. In this context ABA mediates the responsiveness of plants to environmental stresses such as drought, cold or salt. In response to water stress, ABA induces stomatal closure by activating Ca2+, K+ and anion channels in guard cells. To understand the signalling pathways that regulate these turgor control elements, we studied the transcriptional control of the K+ release channel gene GORK that is expressed in guard cells, roots and vascular tissue. GORK transcription was up‐regulated upon onset of drought, salt stress and cold. The wilting hormone ABA that integrates responses to these stimuli induced GORK expression in seedlings in a time‐ and concentration‐dependent manner and this induction was dependent on extracellular Ca2+. ABA‐responsive expression of GORK was impaired in the ABA‐insensitive mutants abi1‐1 and abi2‐1, indicating that these protein phosphatases are regulators of GORK expression. Application of ABA to suspension‐cultured cells for 2 min followed by a 4 h chase was sufficient to manifest transcriptional activation of the K+ channel gene. As predicted for a process involved in drought adaptation, only 12–24 h after the release of the stress hormone, GORK mRNA slowly decreased. In contrast to other tissues, GORK expression as well as K+ out channel activity in guard cells is ABA insensitive, allowing the plant to adjust stomatal movement and water status control separately.

Whole-plant hydraulic conductance and root-to-shoot flow of abscisic acid are independently affected by water stress in grapevines

In order to investigate whether plant hydraulic conductance (gplant) is reduced under drought conditions via an ABA-related mechanism, a water-stress experiment was carried out using split-rooted grapevines. In addition, inversion of shoot growth orientation was imposed to reduce gplant independently of soil water availability, and thus of the putative ABA root-generated stress message. As expected, water stress imposed on split-roots affected ABA accumulation. ABA drought-stress message negatively affected stomatal conductance (gs) and transpiration (E), but modified neither leaf or stem water potentials (Ψleaf and Ψstem, respectively), nor gplant. When gplant was reduced in split-rooted, shoot-inverted (s-r/s-i) grapevines, Ψleaf and Ψstem decreased, without changes in ABA accumulation, gs and E. The ABA drought-stress message did not modify gplant, nor did gplant (impaired by shoot-growth inversion) decrease ABA delivery to the leaves. However, leaf growth was depressed in s-r/s-i grapevines. The fact that no interaction between ABA stress messages (caused by split-root technique) and hydraulic constraints to sap flow (caused by shoot inversion) was necessary to impair leaf growth suggests that the targets of ABA and hydraulic-limitation effects on leaf expansion are not the same.

Compartmental distribution and redistribution of abscisic acid in intact leaves : II. Model analysis.

A computer model written for whole leaves and described in the preceding publication (Slovik et al. 1992, this volume) has been developed for calculating the distribution and fluxes of weak acids or bases amongst different leaf tissues and their compartments, considering membrane transport, transpiration-driven mass transport, symplasmic and apoplasmic diffusion, and metabolic turnover rates in specified compartments. The model is used to analyse flux equilibria and the transport behaviour of the phytohormone abscisic acid (ABA) in unstressed and stressed leaves. We compare experimental data of unstressed Valerianella locusta L. leaves and expectations based on the detailed analysis of the data. (i) The mean daily influx of ABA into the leaf lamina via the xylem sap is about 10 nmol · m−2 · day−1. It is balanced by the sum of an export of ABA via the phloem sap (0.7%), possibly also by a basipetal ABA transport in the petiole parenchyma of young leaves (up to 18%), by an irreversible conjugation of ABA (0.4–4%) and by net degradation of ABA in the leaf lamina (80–95%). (ii) The estimated kinetic parameters of this net degradation are for the mesophyll apoplasm: apparent Km = 3.7 nM and Vmax = 12.9 nmol · m−3 · s−1, or for the mesophyll cytosol: apparent Km = 8.1 nM and Vmax = 32.3 nmol · m−3 · s−1. (iii) The dynamic ABA concentration in the phloem sap of Valerianella is 2.8 nM. This is only 5.5% of the static ABA equilibrium concentration in excised leaves or 70% of the ABA concentration in the mesophyll apoplasm, and it equilibrates within a few hours after source concentrations in the mesophyll apoplasm are changed under stress. Thus, the phloem sap is a flexible medium for transporting ‘new phytohormone information’ from the lamina to the shoot and roots, (iv) Measured compartmental ABA concentrations are close to calculated equilibrium concentrations in unstressed leaves. We conclude that model calculations are close to reality, (v) pH gradients within the apoplasm influence the apoplasmic distribution of ABA. Its concentration is maximally about twofold higher in guard-cell walls relative to the mesophyll apoplasm. (vi) Unexpectedly, all compartmental equilibrium concentrations of ABA in the leaf lamina depend on plasmalemma conductances for undissociated ABA and on the transport properties of the plasmodesmata. This is a consequence of the cyclic diffusion pathway: mesophyll cytosol — mesophyll plasmalemma — mesophyll apoplasm — epidermal apoplasm — epidermal plasmalemma — epidermal cytosol — plasmodesmata — mesophyll cytosol (in this direction), if there are different apoplasmic or cytosolic pH values in both tissues. The cyclisation rate is 42 fmol · s−1 · m−2 leaf area, which corresponds to a turnover time = 11.0 h for the total ABA content within the leaf lamina. A decrease of the epidermal plasmalemma conductance by 90% yields a threefold ABA concentration in the guard-cell free space, (vii) Compartmental relaxation-time coefficients are estimated and summarised for all leaf tissues and its major compartments. They range from 1.5 min for chloroplasts up to 3.3 d for mesophyll vacuoles, (viii) The highest ABA concentration, which can be expected in any leaf compartment, is 7 mM in the guard-cell cytoplasm of certain plant species, (ix) We employed circadian changes (equal day + night, 12 h each = equinoctium) of the stromal pH ± 0.3 in C3 plants, and for Crassulacean acid metabolism (CAM) plants, additionally, vacuolar pH ± 2.5 changes, and calculated the consequences for ABA redistribution within the lamina. In plants of both photosynthesis types, the ABA concentration in guard-cell walls is only 1.5 times higher in the night relative to the day. We conclude that stomata may not be regulated by ABA in a night-day regime. The influence of the extreme vacuolar pH changes on ABA distribution is small in CAM plants for two reasons: the ABA content in CAM mesophyll vacuoles is low (maximum 2.7% of the total ABA mass per unit leaf area) and there is only a 6.5-fold increase of the mole fraction of undissociated ABA when the the vacuolar pH is lowered from 5.5 to 3.0 (importance of the absolute pKa = 4.75 of ABA).

Identification of a novel E3 ubiquitin ligase that is required for suppression of premature senescence in Arabidopsis

During leaf senescence, resources are recycled by redistribution to younger leaves and reproductive organs. Candidate pathways for the regulation of onset and progression of leaf senescence include ubiquitin-dependent turnover of key proteins. Here, we identified a novel plant U-box E3 ubiquitin ligase that prevents premature senescence in Arabidopsis plants, and named it SENESCENCE-ASSOCIATED E3 UBIQUITIN LIGASE 1 (SAUL1). Using in vitro ubiquitination assays, we show that SAUL1 has E3 ubiquitin ligase activity. We isolated two alleles of saul1 mutants that show premature senescence under low light conditions. The visible yellowing of leaves is accompanied by reduced chlorophyll content, decreased photochemical efficiency of photosystem II and increased expression of senescence genes. In addition, saul1 mutants exhibit enhanced abscisic acid (ABA) biosynthesis. We show that application of ABA to Arabidopsis is sufficient to trigger leaf senescence, and that this response is abolished in the ABA-insensitive mutants abi1-1 and abi2-1, but enhanced in the ABA-hypersensitive mutant era1-3. We found that increased ABA levels coincide with enhanced activity of Arabidopsis aldehyde oxidase 3 (AAO3) and accumulation of AAO3 protein in saul1 mutants. Using label transfer experiments, we showed that interactions between SAUL1 and AAO3 occur. This suggests that SAUL1 participates in targeting AAO3 for ubiquitin-dependent degradation via the 26S proteasome to prevent premature senescence.

Apoplastic transport of abscisic acid through roots of maize: effect of the exodermis

Abstract. The exodermal layers that are formed in maize roots during aeroponic culture were investigated with respect to the radial transport of cis-abscisic acid (ABA). The decrease in root hydraulic conductivity (Lpr) of aeroponically grown roots was stimulated 1.5-fold by ABA (500 nM), reaching Lpr values of roots lacking an exodermis. Similar to water, the radial flow of ABA through roots (JABA) and ABA uptake into root tissue were reduced by a factor of about three as a result of the existence of an exodermis. Thus, due to the cooperation between water and solute transport the development of the ABA signal in the xylem was not affected. This resulted in unchanged reflection coeffcients for roots grown hydroponically and aeroponically. Despite the well-accepted barrier properties of exodermal layers, it is concluded that the endodermis was the more effective filter for ABA. Owing to concentration polarisation effects, ABA may accumulate in front of the endodermal layer, a process which, for both roots possessing and lacking an exodermis, would tend to increase solvent drag and hence ABA movement into the xylem sap at increased water flow (JVr). This may account for the higher ABA concentrations found in the xylem at greater pressure difference.