Characterization of microstructure and cell wall components of Arabidopsis thaliana overexpressing cyclin-dependent kinase inhibitor 2

Overexpression of a cyclin-dependent kinase inhibitor (KRP2) caused changes in the general morphology in the leaves of Arabidopsis thaliana. The wild type plant had obovate leaves with entire margins whereas the transgenic line had leaves with denticulate margins. The epidermal cells and stomata of the adult transgenic leaves were significantly larger than those of the wild-type plants and the number of stomata was in proportion to the number of epidermal cells. No apparent differences in thickness and structure of cell walls of the mesophyll cells between the two samples were observed. The smaller amount of cell wall material in the transgenic leaves caused by the larger cell size was also apparent in the lower dry weight of the transgenic leaves. The chemical analysis revealed the main differences to be in pectin and neutral sugar contents, and especially in the amounts of glucose, all being higher in the leaves of the KRP2 transgenic plants. p-Coumaric acid content varied more in the transgenic leaf material than in the control one reflecting possibly fewer cross-links in the cell walls of transgenic plants.     

Effects of soil water level on the development of adult plant resistance to powdery mildew in barley

Barley grown in dry soil developed greater adult plant resistance (APR) to powdery mildew (Erysiphe graminis DC. f. sp. hordei Mérat) than barley grown in wet soil. Conidial germination and appressorium formation were less, and fungal development between formation of appressoria and elongating secondary hyphae on upper leaves was inhibited, when adult plants were grown in dry soil. Mildew colonies expanded more slowly on leaves of adult plants than on leaves of seedlings, especially if adult plants had grown in dry soil. APR was reduced if plants, previously grown in dry soil, were well watered more than 32 h before inoculation. Conidia originating from plants grown in dry soil had a lower solute potential and greater ability to infect plants grown in dry but not wet soil than conidia originating from plants grown in wet soil. APR could not be attributed simply to increased cell wall or cuticle thickness, nor to lowered leaf solute potentials, as has sometimes been suggested for powdery mildew diseases. Increasing plant age and water stress induced increases in cell wall and cuticle thickness, but these changes did not always coincide with changes in disease resistance. Increasing plant age and water stress also lowered leaf solute potentials but fungal solute potentials were lower than leaf solute potentials and, more importantly, were lower than leaf water potentials. Thus, fungal growth was not limited by the availability of water from the host during penetration and hyphal establishment. It is suggested that resistance levels may be determined not by the thickness of epidermal structures, nor by lowering of solute potential per se, but by specific substances harmful to the fungus which accumulate in either cell wall, cuticle or sap, and whose concentration is dependent on the age and water stress of leaves.     

Effects of Water Stress and Hardening on the Internal Water Relations and Osmotic Constituents of Cotton Leaves

Experiments were designed to test the hypothesis that the internal water relations of leaves are altered when cotton plants (Gossypium hirsutum L.‘Acala SJ-2′) are conditioned by several cycles of water stress. Preliminary experiments suggested that plants so conditioned are less sensitive to water deficits and that the change might be partly explained by an accumulation of solutes or by structural alterations attendant on development under conditions of water stress. Leaves of preconditioned plants maintained turgor to lower values of water potential than did leaves of well-watered plants. Accompanying this change was a lower osmotic potential at any given leaf water content in preconditioned plants. Tissue analysis of several osmotically active solutes indicated that soluble sugars and malate accumulate to about the same levels (dry-weight basis) in both conditioned and unconditioned plants exposed to stress. These accumulations could not account for the turgor change. Analysis of the data on relative water content indicated that the leaves of conditioned plants had less water per unit dry weight than did leaves of controls. This change accounts for a substantial fraction of the difference between the osmotic potential of conditioned and control plants. The results of a simple model suggest that structural changes may play a significant role in explaining differences in the responses of conditioned and control plants to water stress.     

Cellular Processes Limiting Leaf Growth in Plants under Hypoxic Root Stress

The effects of root hypoxia on leaf growth of a Populus trichocarpa? deltoides hybrid have been assessed. Clonal plants were subjectedto hypoxic root conditions in pot culture by flooding and insolution culture by gassing with nitrogen. The rate of leafexpansion declined within 8 h and was suppressed for the durationof the treatment. Final leaf size was reduced by 35% to 60%compared to aerated plants. Final epidermal cell size and numberdepended both on the developmental stage of the leaf at theonset of stress and on the duration of the treatment. No differencesin bulk leaf water potential were measured between the hypoxicand aerated plants. Cell wall extensibility was lower, leafsolute potential was more negative and turgor potential washigher in leaves of hypoxia-treated plants than of aerated plants.These data suggest that leaf growth of hypoxia-stressed plantsis limited by cell wall extensibility. The mechanism by whichthe root stress induces changes in leaf cell wall characteristicsis not known. Key words: Populus, flooding     

Mikroskopische Beiträge zum Druck-Volumen-Schema des Zellwasserhaushaltes anhand von Einzelzellen und Zellfäden

Summary Microscopic contributions to the pressure-volume-diagram of the cell-water relations as demonstrated with single cells and cell filaments.The diagram described byRichter (1978), which is based on the relationship between osmotic potential and cell water volume, is used for the evaluation of microscopic measurements on algae in osmotically active solutions. Pressure-volume curves forEremosphaera viridis andSpirogyra spp. are very similar to those obtained from organs of higher plants in experiments with a pressure chamber or a thermocouple device. Tissue counterpressure and cell wall water have no influence on data from experiments on single cells with osmotic methods, which therefore give probably less biased values than tissues or organs. Differences in the elasticity of the cell wall, the resistance of the chromatophore against volume changes of the protoplast, and the relative volumes of protoplasm and vacuole show up very distinctly. -values for the elasticity of the cell wall were calculated for the cells analysed. The usefulness of the Richter diagram could fully be confirmed for single cells.

     

Aging in Perennials

Compared with our knowledge of senescence processes in annuals and biennials, relatively little is known about age-related changes in perennials. The study of aging in plants is very complex and there is no consensus in general concepts related to this topic. Furthermore, there is also a problem of scaling up, which makes us wonder whether cells, tissues/organs or whole organisms really age in plants. This is particularly interesting in the case of perennials, which have the ability to make new leaves every year and live for several years or even centuries or millennia. Recent studies indicate that physiological burdens, such as demands on water and nutrient supply, are responsible for reduced growth as plants age. Aside from the extrinsic factors, it is also possible that intrinsic changes in the shoot meristems could occur through repeated cell divisions and could be fixed during plant development, thereby affecting the physiology of leaves that originated from these cells. Additionally, the increased size associated with the aging of woody perennials (trees and shrubs) has also been proposed as a determining factor responsible for the age-related reductions in growth and photosynthetic rates in leaves. This review is aimed at compiling our current understanding of aging in perennials. After defining some fundamental questions and concepts, and introducing the model plants presently used in the study of aging in perennials, the major role meristems play in perenniality and how aging is manifested in the physiology of perennials (changes in phytohormones, water relations, photosynthesis and oxidative stress) are described. Finally, the causes underlying age-related changes in perennials are discussed in detail and a model based on plant plasticity to explain the aging phenomenon in perennials is presented.     

Loss of capacity for acid-induced wall loosening as the principal cause of the cessation of cell enlargement in light-grown bean leaves

Cell enlargement in primary leaves of bean (Phaseolus vulgaris L.) can be induced, free of cell divisions, by exposure of 10-d-old, red-light-grown seedlings to white light. The absolute rate of leaf expansion increases until day 12, then decreases until the leaves reached mature size on day 18. The cause of the reduction in growth rate following day 12 has been investigated. Turgor calculated from measurements of leaf water and osmotic potential fell from 6.5 to 3.5 bar before day 12, but remained constant thereafter. The decline of growth after day 12 is not caused by a decrease in turgor. On the other hand, Instron-measured cell-wall extensibility decreased in parallel with growth rate after day 12. Two parameters influencing extensibility were examined. Light-induced acidification of cell walls, which has been shown to initiate wall extension, remained constant over the growth period (days 10–18). Furthermore, cells of any age could be stimulated to excrete H⁺ by fusicoccin. However, older tissue was not able to grow in response to fusicoccin or light. Measurements of acid-induced extension on preparations of isolated cell walls showed that as cells matured, the cell walls became less able to extend when acidified. These data indicate that it is a decline in the capacity for acid-induced wall loosening that reduces wall extensibility and thus cell enlargement in maturing leaves.Abbreviations and symbols FC fusicoccin - P turgor pressure - RL red light - WEx wall extensibility - WL white light - P _w leaf water potential - P _s osmotic potential     

Water-stressed maize, barley and rice seedlings show species diversity in mechanisms of leaf growth inhibition

The possible occurrence of species diversity in mechanisms underlying leaf-growth inhibition by water stress, was investigated in related cereal plants. Water stress was generated by additions of the osmoticum polyethylene glycol 6000 to the root medium. Effects of external water potentials ranging from 0 to -0.6MPa, on early growth parameters of emerging leaves were measured under controlled environment conditions, using pairs of maize, barley or rice genotypes with differing resistance to water stress under field conditions. Water potentials of -0.4 MPa for 24 h, similarly reduced leaf growth, comparative production rates of leaf epidermal cells and cell size in all genotypes. These reductions did not appear to be caused by reductions in the osmotic potential gradients between the expanding leaf cells and their external water source. However, growth inhibition in maize and barley, was accompanied by significant reductions in comparative leaf and cell wall extensibility. Moreover, regression plots revealed good linear correlations (r=0.83** for maize and r=0.77** for barley) between the reductions in leaf growth induced by a series of water potentials and associated reductions in leaf extensibility. In contrast, the reduction in growth of rice leaves, was not accompanied by any significant changes in leaf or cell wall extensibility. Similarly, regression plots revealed poor correlations between leaf growth and leaf extensibility in both paddy and upland rice (r=0.17 and r=0.07, respectively). Thus, despite numerous inter-species similarities, biophysical changes associated with stress-induced leaf growth inhibition in maize and barley, differed from those in rice.Key words: Cell walls, extensibility, water stress, cereal diversity, leaf growth.      

Leaf growth of white mustard (Sinapis alba) in different environments

Summary The numbers of cells and area of fully expanded leaves were determined on successive leaves of Sinapis alba grown either in 8 hr. photoperiod (vegetative plants) or 16 hr. photoperiod (flowering plants) at a constant temperature of 20°C. In the 8 hr. photoperiod leaf 9 had the greatest area but leaf 12 had most cells. In 16 hr. photperiod leaf 5 had the greatest area but leaf 9 had most cells. The relationship between area and cell number of successive leaves on the main stem fell into 3 distinct phases: in phase (1), cell number increased at a greater rate than leaf area; in phase (2), leaf area decreased while cell number increased; in phase (3), cell number and leaf area decreased proportionally. For an increase in unit area, cell number increased more in 8 hr. than in 16 hr. photoperiod.Using final area and final cell number of successive leaves, by extrapolation the cell number of unit area of primordium has been deduced. Cell number per unit area increased in successive primordia up to a certain node after which it remained constant at succeeding nodes. It was found that in plants grown under different conditions the cell number per unit area in successive primordia increased at a constant logarithmic rate. That is, cells became progressively smaller. It is concluded that changes in cell size of successive primordia are not influenced by the environment but are under internal control.     

Water relations of rough lemon (Citrus jambhiri Lush.) citrus seedlings infected withFusarium solani

Summary Rough lemon citrus seedlings were inoculated withFusarium solani and evaluated for changes in water relations of leaves, stems, and roots. Inoculated seedlings had decreased leaf stomatal conductance, lower leaf water potential, lower water content, and higher leaf osmotic values compared to healthy plants. Visible wilt symptoms occurred as early as 24 h after inoculation. Transpiration and root conductivity were lower in diseased plants but stem conductivity in diseased plants did not differ from the control. Thus, wilting appears to be due to the inability of roots to supply water to the leaves.     

Tropospheric ozone-induced structural changes in leaf mesophyll cell walls in grapevine plants

The structural changes in leaves of grapevine plants (Vitis vinifera L.) exposed to different ozone concentrations were investigated. Ozone fumigations were performed in open-top chambers at four different ozone levels (charcoal-filtered air (F), ambient air (N), ambient air + 25 mm³m⁻³ ozone (O-25) and ambient air + 50 mm³m⁻³ ozone (O-50)). The leaves of plants from chambers with increased ozone concentrations (O-25 and O-50) were significantly thicker than the controls (F), owing to increased thickness of the mesophyll layer. Observing O-50 leaves, it was found that the mesophyll cell wall displayed structural changes. In some places cell wall thickness increased up to 1 μm. We found callose deposits on the inner side of the cell walls of mesophyll cells. These data are in accord with the concept that the mesophyll cell wall acts as a barrier against the penetration of tropospheric ozone into the cells.     

Interrelationships of petiolar air canal architecture,water depth,and convective air flow in Nymphaea odorata (Nymphaeaceae)

• Premise of the study: Nymphaea odorata grows in water up to 2 m deep, producing fewer larger leaves in deeper water. This species has a convective flow system that moves gases from younger leaves through submerged parts to older leaves, aerating submerged parts. Petiolar air canals are the convective flow pathways. This study describes the structure of these canals, how this structure varies with water depth, and models how convective flow varies with depth. • Methods: Nymphaea odorata plants were grown at water depths from 30 to 90 cm. Lamina area, petiolar cross-sectional area, and number and area of air canals were measured. Field-collected leaves and leaves from juvenile plants were analyzed similarly. Using these data and data from the literature, we modeled how convective flow changes with water depth. • Key results: Petioles of N. odorata produce two central pairs of air canals; additional pairs are added peripherally, and succeeding pairs are smaller. The first three pairs account for 96% of air canal area. Air canals form 24% of petiolar cross-sectional area. Petiolar and air canal cross-sectional areas increase with water depth. Petiolar area scales with lamina area, but the slope of this relationship is lower in 90 cm water than at shallower depths. In our model, the rate of convective flow varied with depth and with the balance of influx to efflux leaves. • Conclusions: Air canals in N. odorata petioles increase in size and number in deeper water but at a decreasing amount in relation to lamina area. Convective flow also depends on the number of influx to efflux laminae.     

Changes in the water binding characteristics of the cell walls from transgenic Nicotiana tabacum leaves with enhanced levels of peroxidase activity

Pore size in the cell wall matrix may affect cell wall–water relations, particularly under osmotic stress. Cross linkage of plant cell wall matrix polymers is an important step in the formation of this structure and peroxidases have been proposed to catalyse the cross-linking of phenolic constituents. Transgenic tobacco ( Nicotiana tabacum ) plants expressing a basic tomato peroxidase gene (TPX2) showed increased apoplastic ferulic acid peroxidase activity in mature leaves. This enhanced activity was not associated with a decreased leaf growth. Differential scanning calorimetry (DSC) of control dried cell walls showed a putative glass transition, after Ca²⁺ removal, that was absent in the transgenic line. This would indicate that transgenic walls were more rigid. DSC analysis of water-hydrated cell wall preparations distinguished two pools of water, freezable and non-freezable water. The amount of non-freezable water, which corresponds to strongly bound water, was higher in the transgenic line (64 versus 55%). DSC thermograms of the transgenic cell wall were displaced to lower temperatures, and this may be interpreted as the result of a stronger interaction between this freezable water and this wall. Water sorption and desorption isotherms, obtained at relative humidity ranging from 5 to 93%, demonstrated the presence of very strongly bound water in the transgenic cell walls that was absent in controls. Water sorption–desorption hysteresis of the isotherms was evident in the control wall but not in the transgenic line. These changes in cell wall–water interaction seem to be relevant at the organ level because leaf discs of transgenic plants maintained higher relative water content than control discs, at water potentials between −1.05 and−2.31 MPa.     

Cell growth in expanding primary leaves of Phaseolus

Plants were grown at 25 and 20° C in 6, 12, and 18 h daylengths.Final area of the primary leaf pair ranged from 105 to 209 cm²,and for a given temperature was greatest in the 12 h and leastin 6 h daylength. Cell numbers per leaf were similar for alltreatments. In the 6 h daylength leaves were thinner, containedless chlorophyll and ethanol-insoluble dry matter, and had considerablysmaller cells than leaves on plants in the longer daylengths;final levels of protein and cell-wall material per cell werealso low, although levels of nucleic acid per cell were as highas, or higher than, those for leaves in 12 and 18 h days. Itis concluded that the low levels of protein and cell-wall materialare associated with a low level of photosynthesis, and thatthe small area of these leaves is a result of the reductionin cell size. In the 12- and 18-h daylenghts, protein and cell wall per cellincreased linearly with time, and when expansion of the laminawas completed, values for these parameters were found to besimilar. Cell size, as measured by fresh weight, was also similarat this stage, although small differences in lamina thicknesswere found. Thus the smaller area for leaves in 18-h days wasnot due to a reduction in mean cell size, although differencesin epidermal cell dimensions must be involved. From consideration of simple models it is concluded that increasein cell wall material during lamina expansion is associatedwith increase in wall area, but that the continued formationof wall material after lamina expansion has ceased is accountedfor by deposition on already existing walls. This continuedincrease in wall material occurs at a time when protein andnucleic acid levels per cell are declining.     

Quantitative Untersuchungen zur Genwirkung bei der Blattentwicklung vonOenothera Hookeri de Vries und Einigen Mutanten

The development of the leaves of 8 mutants ofOenothera hookeri is compared with that of the normal type. All mutants show differences from the controls in many characters, which are interconnected by developmental processes in the sense of a relational pleiotropy (Hadorn). In this paper, quantitative observations on growth characteristics are described. Differences between the mutants and the control do not only exist in the size of mature leaves, but also in the changes of size and form of the leaves during the period of growth.Size and form of leaves are described by arithmetic mean and variance for length and width, and by the length to width ratio. Comparison of growth curves and regression-coefficients as a measure of growth rate of the mutants showed significant differences from wild type. The growth in length and width differs not always in the same direction from the normal.In the mutantsSp-1, gi-2 andb differences in growth curves were found even for leaves, which did not show a deviation in size or form from normal ones.In normal leaves the epidermal cells near the tip of the leaves are smaller than those in the middle part. The cells are smaller in the stemleaves compared with the rosettes. All mutants,gi-2 excepted, show differences in cell size compared with the normal form.The relation of cell size to length and width of the leaves showed that growth by cell division is, in all eight mutants investigated, different from normal.     

Cell water potential,osmotic potential,and turgor in the epidermis and mesophyll of transpiring leaves

Water potential, osmotic potential and turgor measurements obtained by using a cell pressure probe together with a nanoliter osmometer were compared with measurements obtained with an isopiestic psychrometer. Both types of measurements were conducted in the mature region of Tradescantia virginiana L. leaves under non-transpiring conditions in the dark, and gave similar values of all potentials. This finding indicates that the pressure probe and the osmometer provide accurate measurements of turgor, osmotic potentials and water potentials. Because the pressure probe does not require long equilibration times and can measure turgor of single cells in intact plants, the pressure probe together with the osmometer was used to determine in-situ cell water potentials, osmotic potentials and turgor of epidermal and mesophyll cells of transpiring leaves as functions of stomatal aperture and xylem water potential. When the xylem water potential was-0.1 MPa, the stomatal aperture was at its maximum, but turgor of both epidermal and mesophyll cells was relatively low. As the xylem water potential decreased, the stomatal aperture became gradually smaller, whereas turgor of both epidermal and mesophyll cells first increased and afterward decreased. Water potentials of the mesophyll cells were always lower than those of the epidermal cells. These findings indicate that evaporation of water is mainly occurring from mesophyll cells and that peristomatal transpiration could be less important than it has been proposed previously, although peristomatal transpiration may be directly related to regulation of turgor in the guard cells.     

Cold hardiness and water relations parameters in Rhododendron cv. Catawbiense Boursault subjected to drought episodes

We determined whether increase in cold hardiness of Rhododendron cv. Catawbiense Boursault induced by water stress was correlated with changes in tissue water relations. Water content of the growing medium was either maintained near field capacity for the duration of the study or plants were subjected to drought episodes at different times between 15 July and 19 February. Watering during a drought episode was delayed until soil water content decreased below 0.4 m³ m⁻³ then watering was resumed at a level to maintain soil water content between 0.3 and 0.4 m³ m⁻³. Cold hardiness was evaluated in the laboratory with freeze tolerance tests on detached leaves. Water relations parameters were determined using pressure-volume analysis. Exposure to drought episodes increased cold hardiness during the cold acclimation stage in late summer and fall but not during the winter. When water-stressed plants were re-watered to field capacity, the previous gain in cold hardiness gradually disappeared. Water relations parameters correlating with seasonal changes of cold hardiness included dry matter content (r =−0.67). apoplastic water content (r =−0.60), and water potential at the turgor loss point (r = 0.40). Changes of cold hardiness in water-stressed plants in reference to well-watered plants were correlated with changes of all water relations parameters, except for osmotic potential at full turgor (r = 0.13). It is proposed that water stress reduced the hydration of cell walls, thereby increasing their rigidity. Increased rigidity of cell walls could result in a development of greater negative turgor pressures at subfreezing temperatures and therefore increased resistance to freeze dehydration.     

Further Studies on Growth and Osmoregulation of Sugar Beet Leaves under Low Salinity Conditions

In previous work, Nunes and Dias (1980) demonstrated that lowsodium concentrations in the root medium of intact or decapitatedyoung sugar beet plants grown under controlled conditions modifiedleaf water relations and increased leaf area and dry weight.The present study confirms these findings and presents furtherresults concerning the effect of salt on the concentrationsof the main osmotic substrates and on the structural and chemicalfractions of the cell dry weight. Increases of water and turgor potentials (0.25 MPa and 0.4 MPa,respectively) and a small decrease in osmotic potential (0.16MPa) were found in the leaves of salt treated plants. In theseplants, osmotic potentials estimated from the concentrationof ions and organic solutes in the leaf sap agree with thosemeasured showing that the observed increase in sodium concentrationmay account for the small decrease in the osmotic potential.No changes were detected in the concentration of orthophosphateor malic acid but total acidity of the leaf sap from salt treatedplants was significantly lower. It was found that all the main components of cell dry matter(total protein, soluble sugars, pigments and crude cell wall)contributed to the dry weight increase in the salt treated plants.Among the polysaccharide fractions of the cell wall, pectinsincreased significantly relative to hemicellulose and cellulose. Key words: Sugar beet, Sodium chloride, Growth, Osmoregulation     

Rolling-leaf14 is a 2OG-Fe (II) oxygenase family protein that modulates rice leaf rolling by affecting secondary cell wall formation in leaves

As an important agronomic trait, leaf rolling in rice (Oryza sativa L.) has attracted much attention from plant biologists and breeders. Moderate leaf rolling increases the amount of photosynthesis in cultivars and hence raises grain yield. Here, we describe the map-based cloning of the gene RL14, which was found to encode a 2OG-Fe (II) oxygenase of unknown function. rl14 mutant plants had incurved leaves because of the shrinkage of bulliform cells on the adaxial side. In addition, rl14 mutant plants displayed smaller stomatal complexes and decreased transpiration rates, as compared with the wild type. Defective development could be rescued functionally by the expression of wild-type RL14. RL14 was transcribed in sclerenchymatous cells in leaves that remained wrapped inside the sheath. In mature leaves, RL14 accumulated mainly in the mesophyll cells that surround the vasculature. Expression of genes related to secondary cell wall formation was affected in rl14-1 mutants, and cellulose and lignin content were altered in rl14-1 leaves. These results reveal that the RL14 gene affects water transport in leaves by affecting the composition of the secondary cell wall. This change in water transport results in water deficiency, which is the major reason for the abnormal shape of the bulliform cells.