Wieland Fricke

The Biophysics of Leaf Growth in Salt-Stressed Barley. A Study at the Cell Level

Biophysical parameters potentially involved in growth regulation were studied at the single-cell level in the third leaf of barley (Hordeum vulgare) after exposure to various degrees of NaCl stress for 3 to 5 d. Gradients of elongation growth were measured, and turgor pressure, osmolality, and water potentials (ψ) were determined (pressure probe and picoliter osmometry) in epidermal cells of the elongation zone and the mature blade. Cells in the elongation zone adjusted to decreasing external ψ through increases in cell osmolality that were accomplished by increased solute loads and reduced water contents. Cell turgor changed only slightly. In contrast, decreases in turgor also contributed significantly to ψ adjustment in the mature blade. Solute deposition rates in the elongation zone increased at moderate stress levels as compared with control conditions, but decreased again at more severe NaCl exposure. Growth-associated ψ gradients between expanding epidermal cells and the xylem were significant under control and moderate stress conditions (75 mm NaCl) but seemed negligible at severe stress (120 mm NaCl). We conclude that leaf cell elongation in NaCl-treated barley is probably limited by the rate at which solutes can be taken up to generate turgor, particularly at high NaCl levels.

Aquaporin-facilitated water uptake in barley (Hordeum vulgare L.) roots

It is not known to what degree aquaporin-facilitated water uptake differs between root developmental regions and types of root. The aim of this study was to measure aquaporin-dependent water flow in the main types of root and root developmental regions of 14- to 17-d-old barley plants and to identify candidate aquaporins which mediate this flow. Water flow at root level was related to flow at cell and plant level. Plants were grown hydroponically. Hydraulic conductivity of cells and roots was determined with a pressure probe and through exudation, respectively, and whole-plant water flow (transpiration) determined gravimetrically in response to the commonly used aquaporin inhibitor HgCl2. Expression of aquaporins was analysed by real-time PCR and in situ hybridization. Hydraulic conductivity of cortical cells in seminal roots was largest in lateral roots; it was smallest in the fully mature zone and intermediate in the not fully mature ‘transition’ zone along the main root axis. Adventitious roots displayed an even higher (3- to 4-fold) cortical cell hydraulic conductivity in the transition zone. This coincided with 3- to 4-fold higher expression of three aquaporins (HvPIP2;2, HvPIP2;5, HvTIP1:1). These were expressed (also) in cortical tissue. The largest inhibition of water flow (83–95%) in response to HgCl2 was observed in cortical cells. Water flow through roots and plants was reduced less (40–74%). It is concluded that aquaporins contribute substantially to root water uptake in 14- to 17-d-old barley plants. Most water uptake occurs through lateral roots. HvPIP2;5, HvPIP2;2, and HvTIP1;1 are prime candidates to mediate water flow in cortical tissue.

Root pressure and a solute reflection coefficient close to unity exclude a purely apoplastic pathway of radial water transport in barley (Hordeum vulgare)

*Aquaporins can contribute to the control of root water uptake, provided that at least one membrane is crossed between the root medium and the xylem and that water does not move only along the apoplast. In the present study we have critically assessed the possibility of such a purely apoplastic pathway. *A range of methods were used to analyse root water flow (root pressure probe, root exudation, vacuum perfusion and cell pressure probe) and associated driving forces (gradients in hydrostatic pressure and osmolality). These methods were complemented by theoretical approaches in which we predicted, for a particular main pathway of water movement, values for root pressure and osmolality and compared these with measured values. *The mere existence of root pressure excludes the possibility of a purely apoplastic pathway of radial water uptake. A membrane(s) must be crossed. Equilibrium measurements of gradients in hydrostatic and osmotic pressure between the root medium and the xylem point to a root reflection coefficient for solutes close to 1.0. *The generally accepted composite model of water transport across roots should be revised. Barley (Hordeum vulgare) roots behave as perfect osmometers. Root reflection coefficients significantly smaller than unity may be experimental artefacts.

Water uptake by seminal and adventitious roots in relation to whole-plant water flow in barley (Hordeum vulgare L.)

Prior to an assessment of the role of aquaporins in root water uptake, the main path of water movement in different types of root and driving forces during day and night need to be known. In the present study on hydroponically grown barley (Hordeum vulgare L.) the two main root types of 14- to 17-d-old plants were analysed for hydraulic conductivity in dependence of the main driving force (hydrostatic, osmotic). Seminal roots contributed 92% and adventitious roots 8% to plant water uptake. The lower contribution of adventitious compared with seminal roots was associated with a smaller surface area and number of roots per plant and a lower axial hydraulic conductance, and occurred despite a less-developed endodermis. The radial hydraulic conductivity of the two types of root was similar and depended little on the prevailing driving force, suggesting that water uptake occurred along a pathway that involved crossing of membrane(s). Exudation experiments showed that osmotic forces were sufficient to support night-time transpiration, yet transpiration experiments and cuticle permeance data questioned the significance of osmotic forces. During the day, 90% of water uptake was driven by a tension of about –0.15 MPa.

Salinity and the growth of non-halophytic grass leaves: the role of mineral nutrient distribution

Salinity is increasingly limiting the production of graminaceous crops constituting the main sources of staple food (rice, wheat, barley, maize and sorghum), primarily through reductions in the expansion and photosynthetic yield of the leaves. In the present review, we summarise current knowledge of the characteristics of the spatial distribution patterns of the mineral elements along the growing grass leaf and of the impact of salinity on these patterns. Although mineral nutrients have a wide range of functions in plant tissues, their functions may differ between growing and non-growing parts of the grass leaf. To identify the physiological processes by which salinity affects leaf elongation in non-halophytic grasses, patterns of mineral nutrient deposition related to developmental and anatomical gradients along the growing grass leaf are discussed. The hypothesis that a causal link exists between ion deficiency and / or toxicity and the inhibition of leaf growth of grasses in a saline environment is tested.

Developmental pattern of aquaporin expression in barley (Hordeum vulgare L.) leaves

Aquaporins are multifunctional membrane channels which belong to the family of major intrinsic proteins (MIPs) and are best known for their ability to facilitate the movement of water. In the present study, earlier results from microarray experiments were followed up. These experiments had suggested that, in barley (Hordeum vulgare L.), aquaporin family members are expressed in distinct patterns during leaf development. Real-time PCR and in situ hybridization were used to analyse the level and tissue-distribution of expression of candidate aquaporins, focusing on plasma membrane and tonoplast intrinsic proteins (PIPs, TIPs). Water channel function of seven aquaporins, whose transcripts were the most abundant and the most variable, was tested through expression in yeast and, in part, through expression in oocytes. All PIP1 and PIP2 subfamily members changed in expression during leaf development, with expression being much higher or lower in growing compared with mature tissue. The same applied to those TIPs which were expressed at detectable levels. Specific roles during leaf development are proposed for particular aquaporins.

In planta function of compatible solute transporters of the AtProT family

The three proline transporters of Arabidopsis thaliana (AtProTs) transport the compatible solutes proline and glycine betaine and the stress-induced compound γ-aminobutyric acid when expressed in heterologous systems. The aim of the present study was to show transport and physiological relevance of these three AtProTs in planta. Using single, double, and triple knockout mutants and AtProT-overexpressing lines, proline content, growth on proline, transport of radiolabelled betaine, and expression of AtProT genes and enzymes of proline metabolism were analysed. AtProT2 was shown to facilitate uptake of L- and D-proline as well as [14C]glycine betaine in planta, indicating a role in the import of compatible solutes into the root. Toxic concentrations of L- and D-proline resulted in a drastic growth retardation of AtProT-overexpressing plants, demonstrating the need for a precise regulation of proline uptake and/or distribution. Furthermore evidence is provided that AtProT genes are highly expressed in tissues with elevated proline content—that is, pollen and leaf epidermis.

Potassium channels in barley: cloning, functional characterization and expression analyses in relation to leaf growth and development

It is not known how the uptake and retention of the key osmolyte K(+) in cells are mediated in growing leaf tissue. In the present study on the growing leaf 3 of barley, we have cloned the full-length coding sequence of three genes which encode putative K(+) channels (HvAKT1, HvAKT2, HvKCO1/HvTPK1), and of one gene which encodes a putative K(+) transporter (HvHAK4). The functionality of the gene products of HvAKT1 and HvAKT2 was tested through expression in Xenopus laevis oocytes. Both are inward-rectifying K(+) channels which are inhibited by Cs(+). Function of HvAKT1 in oocytes requires co-expression of a calcineurin-interacting protein kinase (AtCIPK23) and a calcineurin B-like protein (AtCBL9) from Arabidopsis, showing cross-species complementation of function. In planta, HvAKT1 is expressed primarily in roots, but is also expressed in leaf tissue. HvAKT2 is expressed particularly in leaf tissue, and HvHAK4 is expressed particularly in growing leaf tissue. Within leaves, HvAKT1 and HvAKT2 are expressed predominantly in mesophyll. Expression of genes changes little in response to low external K(+) or salinity, despite major changes in K(+) concentrations and osmolality of cells. Possible contributions of HvAKT1, HvAKT2, HvKCO1 and HvHAK4 to regulation of K(+) relations of growing barley leaf cells are discussed.

Cloning and expression analysis of candidate genes involved in wax deposition along the growing barley (Hordeum vulgare) leaf

The aim of the present study was to isolate clones of genes which are likely to be involved in wax deposition on barley leaves. Of particular interest were those genes which encode proteins that take part in the synthesis and further modification of very long chain fatty acids (VLCFAs), the precursors of waxes. Previously, it had been shown that wax deposition commences within a spatially well-defined developmental zone along the growing barley leaf (Richardson et al. in Planta 222:472–483, 2005). In the present study, a barley microarray approach was used to screen for candidate contig-sequences (www.barleybase.org) that are expressed particularly in those leaf zones where wax deposition occurs and which are expressed specifically within the epidermis, the site of wax synthesis. Candidate contigs were used to screen an established in-house cDNA library of barley. Six full-length coding sequences clones were isolated. Based on sequence homologies, three clones were related to Arabidopsis CER6/CUT1, and these clones were termed HvCUT1;1, HvCUT1;2 and HvCUT1;3. A fourth clone, which was related to Arabidopsis Fiddlehead (FDH), was termed HvFDH1;1. These clones are likely to be involved in synthesis of VLCFAs. A fifth and sixth clone were related to Arabidopsis CER1, and were termed HvCER1;1 and HvCER1;2. These clones are likely to be involved in the decarbonylation pathway of VLCFAs. Semi-quantitative RT-PCR confirmed microarray expression data. In addition, expression analyses at 10-mm resolution along the blade suggest that HvCUT1;1 (and possibly HvCUT1;2) and HvCER1;1 are involved in commencement of wax deposition during barley leaf epidermal cell development.

Do root hydraulic properties change during the early vegetative stage of plant development in barley (Hordeum vulgare)

BACKGROUND AND AIMS As annual crops develop, transpirational water loss increases substantially. This increase has to be matched by an increase in water uptake through the root system. The aim of this study was to assess the contributions of changes in intrinsic root hydraulic conductivity (Lp, water uptake per unit root surface area, driving force and time), driving force and root surface area to developmental increases in root water uptake. METHODS Hydroponically grown barley plants were analysed during four windows of their vegetative stage of development, when they were 9-13, 14-18, 19-23 and 24-28 d old. Hydraulic conductivity was determined for individual roots (Lp) and for entire root systems (Lp(r)). Osmotic Lp of individual seminal and adventitious roots and osmotic Lp(r) of the root system were determined in exudation experiments. Hydrostatic Lp of individual roots was determined by root pressure probe analyses, and hydrostatic Lp(r) of the root system was derived from analyses of transpiring plants. KEY RESULTS Although osmotic and hydrostatic Lp and Lp(r) values increased initially during development and were correlated positively with plant transpiration rate, their overall developmental increases (about 2-fold) were small compared with increases in transpirational water loss and root surface area (about 10- to 40-fold). The water potential gradient driving water uptake in transpiring plants more than doubled during development, and potentially contributed to the increases in plant water flow. Osmotic Lp(r) of entire root systems and hydrostatic Lp(r) of transpiring plants were similar, suggesting that the main radial transport path in roots was the cell-to-cell path at all developmental stages. CONCLUSIONS Increase in the surface area of root system, and not changes in intrinsic root hydraulic properties, is the main means through which barley plants grown hydroponically sustain an increase in transpirational water loss during their vegetative development.