Dynamic Relation between Expansion and Cellular Turgor in Growing Grape (Vitis vinifera L.) Leaves

Measurements of the growth and water relations of expanding grape (Vitis vinifera L.) leaves have been used to determine the relationship between leaf expansion rate and leaf cell turgor. Direct measurement of turgor on the small (approximately 15 micrometer diameter) epidermal cells over the midvein of expanding grape leaves was made possible by improvements in the pressure probe technique. Leaf expansion rate and leaf water status were perturbed by environmentally induced changes in plant transpiration. After establishing a steady state growth rate, a step decrease in plant transpiration resulted in a rapid and large increase in leaf cell turgor (0.25 megapascal in 5 minutes), and leaf expansion rate. Subsequently, leaf expansion rate returned to the original steady state rate with no change in cell turgor. These results indicate that the expansion rate of leaves may not be strongly related to the turgor of the leaf cells, and that substantial control of leaf expansion rate, despite changes in turgor, may be part of normal plant function. It is suggested that a strictly physical interpretation of the parameters most commonly used to describe the relationship between turgor and growth in plant cells (cell wall extensibility and yield threshold) may be inappropriate when considering the process of plant cell expansion.     

Cell wall and protoplast isoperoxidases in relation to injury, indoleacetic Acid, and ethylene effects

A number of plants have been surveyed with respect to isolation by mild grinding in large quantities of leaf cells. The extent of recovery of mesophyll cells per unit leaf area was found to vary with plant species and the method of grinding. Greater than 70% recovery was obtained from the leaves of Canna indica L., Crotalaria Laburnifolia L., and Thunbergia grandiflora Roxb.     

Hpa1 harpin needs nitroxyl terminus to promote vegetative growth and leaf photosynthesis in Arabidopsis

Hpa1 is a harpin protein produced by Xanthomonas oryzae, an important bacterial pathogen of rice, and has the growth-promoting activity in plants. To understand the molecular basis for the function of Hpa1, we generated an inactive variant protein, Hpa1ΔNT, by deleting the nitroxyl-terminal region of the Hpa1 sequence and compared Hpa1ΔNT with the full-length protein in terms of the effects on vegetative growth and related physiological responses in Arabidopsis. When Hpa1 was applied to plants, it acted to enhance the vegetative growth but did not affect the floral development. Enhanced plant growth was accompanied by induced expression of growth-promoting genes in plant leaves. The growth-promoting activity of Hpa1 was further correlated with a physiological consequence shown as promoted leaf photosynthesis as a result of facilitated CO₂ conduction through leaf stomata and mesophyll cells. On the contrary, plant growth, growth-promoting gene expression, and the physiological consequence changed little in response to the Hpa1ΔNT treatment. These analyses suggest that Hpa1 requires the nitroxyl-terminus to facilitate CO₂ transport inside leaf cells and promote leaf photosynthesis and vegetative growth of the plant.     

Developmental and genetic characterization of a short leaf mutant of sorghum (Sorghum bicolor L. (Moench.))

Changes in plant architecture, specifically conversion to compact canopy for cereal crops, have resulted in significant increases in grain yield for wheat (Triticum aestivum) and rice (Oryza sativa). For sorghum (Sorghum bicolor L. Moench.) a versatile crop with an open canopy, plant architecture is an important feature that merits strong consideration for modification. Here, we report the genetic, developmental, and physiological characterization of a sorghum genetic stock, KFS2061, a stable mutant (in the Western Black Hull Kafir background) which exhibit short and erect leaves resulting in compact plant architecture. Genetic study of an F₂ population derived from the cross of KFS2061 to BTx623 showed that the short leaf is recessive and appeared to be controlled by a single gene. The expression of the short leaf trait commenced with the 3rd leaf and is propagated through the entire leaf hierarchy of the canopy. The short leaf mutant exhibited consistent steep leaf angle, 43° (with the main culm as reference), and greener leaves than wild type. Biochemical analyses indicated significantly higher chlorophyll and cellulose content per leaf area in the mutant than wild type. Histological studies revealed reduction in cell length along the longitudinal axis and enlargement of bulliform cells in the adaxial surface of the mutant leaf. Further evaluation of agronomic traits indicated that this mutation could increase harvest index. This study provides information on a short leaf genetic stock that could serve as a vital resource in understanding how to manipulate plant canopy architecture of sorghum.     

Overexpression of OsEXPA8, a Root-Specific Gene,Improves Rice Growth and Root System Architecture by Facilitating Cell Extension

Expansins are unique plant cell wall proteins that are involved in cell wall modifications underlying many plant developmental processes. In this work, we investigated the possible biological role of the root-specific α-expansin gene OsEXPA8 in rice growth and development by generating transgenic plants. Overexpression of OsEXPA8 in rice plants yielded pleiotropic phenotypes of improved root system architecture (longer primary roots, more lateral roots and root hairs), increased plant height, enhanced leaf number and enlarged leaf size. Further study indicated that the average cell length in both leaf and root vascular bundles was enhanced, and the cell growth in suspension cultures was increased, which revealed the cellular basis for OsEXPA8-mediated rice plant growth acceleration. Expansins are thought to be a key factor required for cell enlargement and wall loosening. Atomic force microscopy (AFM) technology revealed that average wall stiffness values for 35S::OsEXPA8 transgenic suspension-cultured cells decreased over six-fold compared to wild-type counterparts during different growth phases. Moreover, a prominent change in the wall polymer composition of suspension cells was observed, and Fourier-transform infrared (FTIR) spectra revealed a relative increase in the ratios of the polysaccharide/lignin content in cell wall compositions of OsEXPA8 overexpressors. These results support a role for expansins in cell expansion and plant growth.     

Effect of Glyphosate on Intact Bean Plants (Phaseolus vulgaris L.) and Isolated Cells

Whole bean (var. “Eastern Butterwax”) plants and isolated cells were used to investigate possible mechanisms of action of glyphosate [N-(phosphonomethyl)glycine]. Results showed that glyphosate was quickly absorbed by the whole plant but not by individual cells and that it caused a rapid reduction in leaf dry matter accumulation, leaf expansion, leaf angle, and stomatal aperture without affecting the water status of the plant. Glyphosate also caused a rapid reduction in cellular uptake of ⁸⁶Rb and ³²P which preceded its detrimental effects on photosynthesis, RNA and protein synthesis, and respiration of isolated cells. This reduction in ion absorption was not due to a loss of membrane integrity, decrease in energy supply or chelation of ions. It was concluded that glyphosate was directly inhibiting the ion absorption process of bean leaf cells.     

Defects in leaf epidermis of Arabidopsis thaliana plants with CDKA;1 activity reduced in the shoot apical meristem

In Arabidopsis thaliana, like in other dicots, the shoot epidermis originates from protodermis, the outermost cell layer of shoot apical meristem. We examined leaf epidermis in transgenic A. thaliana plants in which CDKA;1.N146, a negative dominant allele of A-type cyclin-dependent kinase, was expressed from the SHOOTMERISTEMLESS promoter, i.e., in the shoot apical meristem. Using cleared whole mount preparations of expanding leaves and sequential in vivo replicas of expanding leaf surface, we show that dominant-negative CDKA;1 expression results in defects in epidermis continuity: loss of individual cells and occurrence of gaps between anticlinal walls of neighboring pavement cells. Another striking feature is ingrowth-like invaginations of anticlinal cell walls of pavement cells. Their formation is related to various processes: expansion of cells surrounding the sites of cell loss, defected cytokinesis, and presumably also, the actual ingrowth of an anticlinal cell wall. The mutant exhibits also increased variation in cell size and locally reduced waviness of anticlinal walls of pavement cells. These unusual features of leaf epidermis phenotype may shed a new light on our knowledge on morphogenesis of jigsaw puzzle-shaped pavement cells and on the CDKA;1 role in regulation of plant development via influence on cytoskeleton and plant cell wall.     

A Secreted Effector Protein of Ustilago maydis Guides Maize Leaf Cells to Form Tumors

The biotrophic smut fungus Ustilago maydis infects all aerial organs of maize (Zea mays) and induces tumors in the plant tissues. U. maydis deploys many effector proteins to manipulate its host. Previously, deletion analysis demonstrated that several effectors have important functions in inducing tumor expansion specifically in maize leaves. Here, we present the functional characterization of the effector See1 (Seedling efficient effector1). See1 is required for the reactivation of plant DNA synthesis, which is crucial for tumor progression in leaf cells. By contrast, See1 does not affect tumor formation in immature tassel floral tissues, where maize cell proliferation occurs independent of fungal infection. See1 interacts with a maize homolog of SGT1 (Suppressor of G2 allele of skp1), a factor acting in cell cycle progression in yeast (Saccharomyces cerevisiae) and an important component of plant and human innate immunity. See1 interferes with the MAPK-triggered phosphorylation of maize SGT1 at a monocot-specific phosphorylation site. We propose that See1 interferes with SGT1 activity, resulting in both modulation of immune responses and reactivation of DNA synthesis in leaf cells. This identifies See1 as a fungal effector that directly and specifically contributes to the formation of leaf tumors in maize.     

Effects of osmotic gradients on vacuolar malic Acid storage: a basic principle in oscillatory behavior of crassulacean Acid metabolism

Malate synthesis by CO₂ dark fixation and malate accumulation in the vacuoles of leaf slices of Kalanchoë daigremontiana Hamet et Perrier, a plant performing crassulacean acid metabolism, occurs only in external solutions where the osmotic pressure difference between the cells and the medium is low. Conversely, malate loss from the vacuoles depends on a high osmotic pressure difference between the cells and the medium and is observed in media of low osmotic pressure. This suggests that the diurnal oscillations of malate levels in crassulacean acid metabolism leaf cells are regulated by osmotic gradients. These findings support a model which is introduced to explain how the rhythm of crassulacean acid metabolism may function in the intact plant.     

Size‐dependent and environment‐mediated shifts in leaf traits of a deciduous tree species in a subtropical forest

AimsUnderstanding the joint effects of plant development and environment on shifts of intraspecific leaf traits will advance the understandings of the causes of intraspecific trait variation. We address this question by focusing on a widespread species Clausena dunniana in a subtropical broad‐leaved forest.MethodsWe sampled 262 individuals of C. dunniana at two major topographic habitat types, the slope and hilltop, within the karst forests in Maolan Nature Reserve in southwestern China. We measured individual plant level leaf traits (i.e., specific leaf area (SLA), leaf area, leaf dry‐matter content (LDMC), and leaf thickness) that are associated with plant resource‐use strategies. We adopted a linear mixed‐effects model in which the plant size (i.e., the first principal component of plant basal diameter and plant height) and environmental factors (i.e., topographic habitat, canopy height, and rock‐bareness) were used as independent variables, to estimate their influences on the shifts of leaf traits.Key ResultsWe found that (1) plant size and the environmental factors independently drove the intraspecific leaf trait shifts of C. dunniana, of which plant size explained less variances than environmental factors. (2) With increasing plant size, C. dunniana individuals had increasingly smaller SLA but larger sized leaves. (3) The most influential environmental factor was topographic habitat; it drove the shifts of all the four traits examined. Clausena dunniana individuals on hilltops had leaf traits representing more conservative resource‐use strategies (e.g., smaller SLA, higher LDMC) than individuals on slopes. On top of that, local‐scale environmental factors further modified leaf trait shifts.ConclusionsPlant size and environment independently shaped the variations in intraspecific leaf traits of C. dunniana in the subtropical karst forest of Maolan. Compared with plant size, the environment played a more critical role in shaping intraspecific leaf trait variations, and potentially also the underlying individual‐level plant resource‐use strategies.     

Effects of boron toxicity on root and leaf anatomy in two Citrus species differing in boron tolerance

Key message

Typical toxic symptom only occurred in B-toxic C. grandis leaves. B-toxicity induced PCD of C. grandis leaf phloem tissue. The lower leaf free B might contribute to the higher B-tolerance of C. sinensis.

Abstract

Seedlings of ‘Xuegan’ (Citrus sinensis) and ‘Sour pummelo’ (Citrus grandis) differing in boron (B)-tolerance were irrigated with nutrient solution containing 10 (control) or 400 (B-toxic) μM H₃BO₃ for 15 weeks. Thereafter, the effects of B-toxicity on leaf photosynthesis, chlorophyll, plant B absorption and distribution, root and leaf anatomy were investigated to elucidate the possible B-tolerant mechanisms of Citrus plants. Typical toxic symptom only occurred in B-toxic C. grandis leaves. Similarly, B-toxicity only affected C. grandis photosynthesis and chlorophyll. Although total B concentration in B-toxic roots and leaves was similar between the two species, leaves from B-toxic C. grandis plant middle had higher free B and lower bound B as compared with those from C. sinensis. Effects of B-toxicity on leaf structure were mainly limited to the mesophyll cells and the phloem of leaf veins. Although irregular cell wall thickening was observed in leaf cortex cells and phloem tissue of B-toxic C. grandis and C. sinensis leaves, exocytosis only occurred in the companion cells and the parenchyma cells of B-toxic C. sinensis leaf phloem. Also, B-toxicity induced cell death of phloem tissue through autophagy in C. grandis leaf veins. B-toxicity caused death of root epidermal cells of the two Citrus species. B-toxicity restrained degradation of middle lamella, but did not alter ultrastructure of Golgi apparatus and mitochondria in root elongating zone cells. In conclusion, C. sinensis was more tolerant to B-toxicity than C. grandis. The lower leaf free B and higher bound B might contribute to the higher B-tolerance of C. sinensis.     

Geographic association and temporal variation of chemical and physical defense and leaf damage in Datura stramonium

The evolution of plant defense traits has traditionally been explained trough the “coevolutionary arms race” between plants and herbivores. According to this, specialist herbivores have evolved to cope effectively with the defensive traits of their host plants and may even use them as a cue for host location. We analyzed the geographic association between leaf trichomes, two tropane alkaloids (putative resistance traits), and leaf damage by herbivores in 28 populations of Datura stramonium in central Mexico. Since the specialist leaf beetles Epitrix parvula and Lema trilineata are the main herbivores of D. stramonium in central Mexico, we predicted a positive association between plant defense and leaf damage across populations. Also, if physical environmental conditions (temperature or precipitation) constrain the expression of plant defense, then the geographic variation in leaf damage should be explained partially by the interaction between defensive traits and environmental factors. Furthermore, we studied the temporal and spatial variation in leaf trichome density and leaf damage in five selected populations of D. stramonium sampled in two periods (1997 vs. 2007). We found a positive association between leaf trichomes density and atropine concentration with leaf damage across populations. The interaction between defensive traits and water availability in each locality had a significant effect on the geographic variation in leaf damage. Differences among populations in leaf trichome density are maintained over time. Our results indicate that local plant–herbivore interaction plays an important role in shaping the geographic and temporal variation in plant defense in D. stramonium.     

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.     

A neighboring-inhibition model for stomate patterning

The patternization of stomate distribution was investigated in the first leaf of the sporophyte of the fern Dryopteris thelypteris and in the leaves of the jade plant Crassula argentae. In the fern leaf, stomates arise over a period of 1.1 days (26.4 hr) and attain a frequency of 0.186 of the epidermal cells while in the jade plant the formative period is over only 0.8 days (19.2 hr) and stomates reach a frequency of 0.090 of the epidermal cells. A computer model was devised to simulate the appearance of stomates by an induction process for a new stomate which then inhibits contiguous cells from becoming stomates. The validity of the model was demonstrated in that it gave values for stomate and stomate cluster frequencies upon calculating the frequency of free cells. For the fern leaf, the model required 57 iteration intervals, six iterations for the time from stomate induction until the adjacent cells were inhibited (a period of induction plus inhibitions), and an induction rate of 0.02 cells per time interval. From these theoretical values and the measured period of 26.4 hr when stomates arise during leaf development, a period of induction/inhibitions is calculated as 2.7 hr. In the model for the jade plant, 81 iteration intervals are required with six iterations per period of induction/inhibitions along with an induction rate of 0.002 cells per interation interval. These values give the duration for a period of induction/inhibitions in jade of 1.4 hr. This study describes the patterning process of stomate formation by an explicit, mathematical algorithm, and, from measurements of actual leaves, the various periods in the model can be assigned real time values.     

Subcellular Localization and Functional Analysis of the Arabidopsis GTPase RabE

Membrane trafficking plays a fundamental role in eukaryotic cell biology. Of the numerous known or predicted protein components of the plant cell trafficking system, only a relatively small subset have been characterized with respect to their biological roles in plant growth, development, and response to stresses. In this study, we investigated the subcellular localization and function of an Arabidopsis (Arabidopsis thaliana) small GTPase belonging to the RabE family. RabE proteins are phylogenetically related to well-characterized regulators of polarized vesicle transport from the Golgi apparatus to the plasma membrane in animal and yeast cells. The RabE family of GTPases has also been proposed to be a putative host target of AvrPto, an effector protein produced by the plant pathogen Pseudomonas syringae, based on yeast two-hybrid analysis. We generated transgenic Arabidopsis plants that constitutively expressed one of the five RabE proteins (RabE1d) fused to green fluorescent protein (GFP). GFP-RabE1d and endogenous RabE proteins were found to be associated with the Golgi apparatus and the plasma membrane in Arabidopsis leaf cells. RabE down-regulation, due to cosuppression in transgenic plants, resulted in drastically altered leaf morphology and reduced plant size, providing experimental evidence for an important role of RabE GTPases in regulating plant growth. RabE down-regulation did not affect plant susceptibility to pathogenic P. syringae bacteria; conversely, expression of the constitutively active RabE1d-Q74L enhanced plant defenses, conferring resistance to P. syringae infection.     

Functional identification of AtSKIP as a regulator of the cell cycle signaling pathway in Arabidopsis thaliana

Light is a key environmental cue controlling plant development, which involves meristemic activation by cell proliferation and differentiation. Here, we identify one gene, AtSKIP, associated with cell cycle-regulated root and leaf growth processes in Arabidopsis. The spatial pattern of β-glucuronidase (GUS) activity indicated that AtSKIP is expressed in the leaf primodia, root meristem region and root vascular system, and can be activated by light. Ectopic expression of AtSKIP resulted in enhanced leaf development but suppressed root elongation in Arabidopsis, whereas AtSKIP^DD seedlings displayed retarded leaf growth and normal root growth. Moreover, AtSKIP cells displayed enhanced sensitivity to a cytokinin in a callus induction assay, further demonstrated that AtSKIP expression altered endogenous cell cycle-regulated signaling in plants. Together, these data indicate that AtSKIP participates in cell cycle-mediated growth of leaf and root.     

Rumen bacterial degradation of forage cell walls investigated by electron microscopy

The association of rumen bacteria with specific leaf tissues of the forage grass Kentucky-31 tall fescue (Festuca arundinacea Schreb.) during in vitro degradation was investigated by transmission and scanning electron microscopy. Examination of degraded leaf cross-sections revealed differential rates of tissue degradation in that the cell walls of the mesophyll and pholem were degraded prior to those of the outer bundle sheath and epidermis. Rumen bacteria appeared to degrade the mesophyll, in some cases, and phloem without prior attachment to the plant cell walls. The degradation of bundle sheath and epidermal cell walls appeared to be preceded by attachment of bacteria to the plant cell wall. Ultrastructural features apparently involved in the adhesion of large cocci to plant cells were observed by transmission and scanning electron microscopy. The physical association between plant and rumen bacterial cells during degradation apparently varies with tissue types. Bacterial attachment, by extracellular features in some microorganisms, is required prior to degradation of the more resistant tissues.     

Variation of leaf traits and pigment content in three species of steppe plants depending on the climate aridity

Mesophyll structure and content of photosynthetic pigments in the leaves of three species of steppe plants, Centaurea scabiosa L., Euphorbia virgata Waldst. et Kit., Helichrysum arenarium (L.) Moench, were investigated in four geographical sites of the Volga region and the Urals located in the forest-steppe and steppe zones. Variations of the studied parameters between geographical points depended both on the species and on the structural organization of the leaf. The highest level of variation was observed for leaf area and pigment content per unit leaf area, the size and the number of chloroplasts in the cell changed to a lesser extent. The leaf thickness, leaf area and mesophyll cell sizes mostly depended on the plant species. C. scabiosa had large leaves (40–50 cm²) with large thickness (280–290 μm) and large mesophyll cells (up to 15000 μm³). The leaves of H. arenarium and E. virgata were ten times smaller and characterized by 1.5 times smaller thickness and 2?3 times smaller cell size. Geographical location and climate of the region affected leaf density, proportion of partial tissue volume, and the ratio of the photosynthetic pigments. In the southern point of Volga region with the highest climate aridity, all studied species were characterized by maximum values of volumetric leaf density (LD), due to the high proportion of sclerenchyma and vascular bundles, and specificity of the mesophyll structure. With the decline in latitude, chlorophyll (Chl) and carotenoid (Car) contents in leaf area were reduced, the ratio Chl/Car was increased, and the ratio Chl a/b was declined. The reduction of the pigment content in the leaf in all species was associated with a reduction in the amount of Chl per chloroplast, and for C. scabiosa and H. arenarium it was associated also with the reduction of chloroplast amount in the leaf area. In turn, chloroplast number per leaf area and the total cell area (A_mes/A) depended on the ratio of the number and size of mesophyll cells inherent to this plant species. At the same time, we found a similar mechanism of spatial organization of leaf restructuring for all studied species—decrease in A_mes/A was accompanied by increasing in the proportion of intercellular air spaces in the leaf. It is concluded that variations in structural and functional parameters of the photosynthetic apparatus of steppe plants were associated with plant adaptation to climate features. General direction of the changes of leaf parameters of the studied species with aridity was the increase of LD and the decrease of pigment content per leaf area however the cellular mechanisms of changes in the pigment content and integral parameters of mesophyll were determined by the plant species properties.     

Effects of Alteria alternata f.sp. lycopersici toxins at different levels of tomato plant cell development

Since the host-specific toxins of Alternaria alternata f. sp. lycopersici play an important role in pathogenesis, they potentially could be applied as selective agents in in vitro selection at the cellular level for disease resistance. Prerequisite for this is that sensitivity to the Alternaria alternata f.sp. lycopersici pathotoxins is manifest at the cellular level. To gain insight into cellular effects of AAL-toxins and into the mechanisms of plant insensitivity to AAL-toxins, effects of AAL-toxins on leaves, leaf discs, roots, calli, suspension cells, minicalli and protoplasts of susceptible and resistant tomato genotypes were studied. In leaves of susceptible genotypes, toxins cause severe necrosis, while in leaves of resistant genotypes necrosis was never observed. Inhibition effects of toxins were observed at all other levels in susceptible and resistant genotypes: toxins inhibited shoot induction on leaf discs, root growth and growth of calli, suspension cells and protoplasts. This indicates a cellular site for AAL-toxins. Differences in sensitivity to AAL-toxins between susceptible and resistant genotypes were observed in leaves and roots, but were not observed during shoot induction on leaf discs, in calli, suspension cells and protoplasts. However, differences in sensitivity to AAL-toxins in roots were at least 20 times less than in leaves. Therefore insensitivity seems related to a higher level of tomato plant differentiation and is most pronounced in leaves.