An in Vivo Imaging Assay Detects Spatial Variability in Glucose Release from Plant Roots.

Plants secrete a plethora of metabolites into the rhizosphere that allow them to obtain nutrients necessary for growth and modify microbial communities around the roots. Plants release considerable amounts of photosynthetically fixed carbon into the rhizosphere; hence, it is important to understand how carbon moves from the roots into the rhizosphere. Approaches used previously to address this question involved radioactive tracers, fluorescent probes, and biosensors to study sugar movement in the roots and into the rhizosphere. Although quite effective for studying sugar movement, it has been challenging to obtain data on spatial and temporal variability in sugar exudation using these techniques. In this study, we developed a gel-based enzyme-coupled colorimetric and fluorometric assay to image glucose (Glc) in vivo and used this assay to show that there is spatial variability in Glc release from plant roots. We found that the primary roots of maize (Zea mays) released more Glc from the base of the root than from the root tip and that the Glc release rate is reduced in response to water stress. These findings were confirmed independently by quantifying Glc release in well-watered and water-stressed maize primary roots using high-performance anion-exchange chromatography. Additionally, we demonstrated differential patterns of Glc exudation in different monocot and eudicot plant species. These findings and their implications on root-rhizosphere interactions are discussed.

Enzyme-Less Growth in Chara and Terrestrial Plants.

Enzyme-less chemistry appears to control the growth rate of the green alga Chara corallina. The chemistry occurs in the wall where a calcium pectate cycle determines both the rate of wall enlargement and the rate of pectate deposition into the wall. The process is the first to indicate that a wall polymer can control how a plant cell enlarges after exocytosis releases the polymer to the wall. This raises the question of whether other species use a similar mechanism. Chara is one of the closest relatives of the progenitors of terrestrial plants and during the course of evolution, new wall features evolved while pectate remained one of the most conserved components. In addition, charophytes contain auxin which affects Chara in ways resembling its action in terrestrial plants. Therefore, this review considers whether more recently acquired wall features require different mechanisms to explain cell expansion.

Why small fluxes matter: the case and approaches for improving measurements of photosynthesis and (photo)respiration.

Since its inception, the Farquhar et al. (1980) model of photosynthesis has been a mainstay for relating biochemistry to environmental conditions from chloroplast to global levels in terrestrial plants. Many variables could be assigned from basic enzyme kinetics, but the model also required measurements of maximum rates of photosynthetic electron transport (J max ), carbon assimilation (Vcmax ), conductance of CO2 into (g s ) and through (g m ) the leaf, and the rate of respiration during the day (R d ). This review focuses on improving the accuracy of these measurements, especially fluxes from photorespiratory CO2, CO2 in the transpiration stream, and through the leaf epidermis and cuticle. These fluxes, though small, affect the accuracy of all methods of estimating mesophyll conductance and several other photosynthetic parameters because they all require knowledge of CO2 concentrations in the intercellular spaces. This review highlights modified methods that may help to reduce some of the uncertainties. The approaches are increasingly important when leaves are stressed or when fluxes are inferred at scales larger than the leaf.

Impact of cuticle on calculations of the CO2 concentration inside leaves.

MAIN CONCLUSION: Water vapor over-estimates the CO 2 entering leaves during photosynthesis because the cuticle and epidermis transmit more water vapor than CO 2 . Direct measurements of internal CO 2 concentrations may be preferred. The CO2 concentration inside leaves (c i) is typically calculated from the relationship between water vapor diffusing out while CO2 diffuses in. Diffusion through the cuticle/epidermis is usually not considered. This study was undertaken to determine how much the calculations would be affected by including cuticle properties. Previous studies indicate that measurable amounts of CO2 and water vapor move through the cuticle, although much less CO2 than water vapor. The present experiments were conducted with sunflower (Helianthus annuus L) leaves in a gas exchange apparatus designed to directly measure c i, while simultaneously calculating c i. Results showed that, in normal air, calculated c i were always higher than directly measured ones, especially when abscisic acid was fed to the leaves to close the stomata and cause gas exchange to be dominated by the cuticle. The effect was attributed mostly to the reliance on the gas phase for the calculations without taking cuticle properties into account. Because cuticle properties are usually unknown and vary with the turgor of the leaf, which can stretch the waxes, it is difficult to include cuticle properties in the calculation. It was concluded that direct measurement of c i may be preferable to the calculations.

Turgor and the transport of CO2 and water across the cuticle (epidermis) of leaves.

Leaf photosynthesis relies on CO2 diffusing in while water vapour diffuses out. When stomata close, cuticle waxes on the epidermal tissues increasingly affect this diffusion. Also, changes in turgor can shrink or swell a leaf, varying the cuticle size. In this study, the properties of the cuticle were investigated while turgor varied in intact leaves of hypo stomatous grape (Vitis vinifera L.) or amphistomatous sunflower (Helianthus annuus L.). For grape, stomata on the abaxial surface were sealed and high CO2 concentrations outside the leaf were used to maximize diffusion through the adaxial, stoma-free cuticle. For sunflower, stomata were closed in the dark or with abscisic acid to maximize the cuticle contribution to the path. In both species, the internal CO2 concentration was measured directly and continuously while other variables were determined to establish the cuticle properties. The results indicated that stomatal closure diminished the diffusion of both gases in both species, but for CO2 more than for water vapour. Decreasing the turgor diminished the movement of both gases through the cuticle of both species. Because this turgor effect was observed in the adaxial surface of grape, which had no stomata, it could only be attributed to cuticle tightening. Comparing calculated and measured concentrations of CO2 in leaves revealed differences that became large as stomata began to close. These differences in transport, together with turgor effects, suggest calculations of the CO2 concentration inside leaves need to be viewed with caution when stomata begin to close.

Differences in membrane selectivity drive phloem transport to the apoplast from which maize florets develop.

BACKGROUND AND AIMS: Floral development depends on photosynthetic products delivered by the phloem. Previous work suggested the path to the flower involved either the apoplast or the symplast. The objective of the present work was to determine the path and its mechanism of operation. METHODS: Maize (Zea mays) plants were grown until pollination. For simplicity, florets were harvested before fertilization to ensure that all tissues were of maternal origin. Because sucrose from phloem is hydrolysed to glucose on its way to the floret, the tissues were imaged and analysed for glucose using an enzyme-based assay. Also, carboxyfluorescein diacetate was fed to the stems and similarly imaged and analysed. KEY RESULTS: The images of live sections revealed that phloem contents were released to the pedicel apoplast below the nucellus of the florets. Glucose or carboxyfluorescein were detected and could be washed out. For carboxyfluorescein, the plasma membranes of the phloem parenchyma appeared to control the release. After release, the nucellus absorbed apoplast glucose selectively, rejecting carboxyfluorescein. CONCLUSIONS: Despite the absence of an embryo, the apoplast below the nucellus was a depot for phloem contents, and the strictly symplast path is rejected. Because glucose and carboxyfluorescein were released non-selectively, the path to the floret resembled the one later when an embryo is present. The non-selective release indicates that turgor at phloem termini cannot balance the full osmotic potential of the phloem contents and would create a downward pressure gradient driving bulk flow toward the sink. Such a gradient was previously measured by Fisher and Cash-Clark in wheat. At the same time, selective absorption from the apoplast by the nucellar membranes would support full turgor in this tissue, isolating the embryo sac from the maternal plant. The isolation should continue later when an embryo develops.

Pectate chemistry links cell expansion to wall deposition in Chara corallina.

Pectate (polygalacturonic acid) acts as a chelator to bind calcium and form cross-links that hold adjacent pectate polymers and thus plant cell walls together. When under tension from turgor pressure in the cell, the cross-links appear to distort and weaken. New pectate supplied by the cytoplasm is undistorted and removes wall calcium preferentially from the weakened bonds, loosening the wall and accelerating cell expansion. The new pectate now containing the removed calcium can bind to the wall, strengthening it and linking expansion to wall deposition. But new calcium needs to be added as well to replenish the calcium lost from the vacated wall pectate.  A recent report demonstrated that growth was disrupted if new calcium was unavailable.  The present addendum highlights this conclusion by reviewing an experiment from before the chelation chemistry was understood. Using cell wall labeling, a direct link appeared between wall expansion and wall deposition. Together, these experiments support the concept that newly supplied pectate has growth activity on its way to deposition in the wall. Growth rate is thus controlled by signals affecting the rate of pectate release. After release, the coordination of expansion and deposition arises naturally from chelation chemistry when polymers are under tension from turgor pressure. 

Calcium deprivation disrupts enlargement of Chara corallina cells: further evidence for the calcium pectate cycle.

Pectin is a normal constituent of cell walls of green plants. When supplied externally to live cells or walls isolated from the large-celled green alga Chara corallina, pectin removes calcium from load-bearing cross-links in the wall, loosening the structure and allowing it to deform more rapidly under the action of turgor pressure. New Ca(2+) enters the vacated positions in the wall and the externally supplied pectin binds to the wall, depositing new wall material that strengthens the wall. A calcium pectate cycle has been proposed for these sub-reactions. In the present work, the cycle was tested in C. corallina by depriving the wall of external Ca(2+) while allowing the cycle to run. The prediction is that growth would eventually be disrupted by a lack of adequate deposition of new wall. The test involved adding pectate or the calcium chelator EGTA to the Ca(2+)-containing culture medium to bind the calcium while the cycle ran in live cells. After growth accelerated, turgor and growth eventually decreased, followed by an abrupt turgor loss and growth cessation. The same experiment with isolated walls suggested the walls of live cells became unable to support the plasma membrane. If instead the pectate or EGTA was replaced with fresh Ca(2+)-containing culture medium during the initial acceleration in live cells, growth was not disrupted and returned to the original rates. The operation of the cycle was thus confirmed, providing further evidence that growth rates and wall biosynthesis are controlled by these sub-reactions in plant cell walls.

Sugar input, metabolism, and signaling mediated by invertase: roles in development, yield potential, and response to drought and heat.

Invertase (INV) hydrolyzes sucrose into glucose and fructose, thereby playing key roles in primary metabolism and plant development. Based on their pH optima and sub-cellular locations, INVs are categorized into cell wall, cytoplasmic, and vacuolar subgroups, abbreviated as CWIN, CIN, and VIN, respectively. The broad importance and implications of INVs in plant development and crop productivity have attracted enormous interest to examine INV function and regulation from multiple perspectives. Here, we review some exciting advances in this area over the last two decades, focusing on (1) new or emerging roles of INV in plant development and regulation at the post-translational level through interaction with inhibitors, (2) cross-talk between INV-mediated sugar signaling and hormonal control of development, and (3) sugar- and INV-mediated responses to drought and heat stresses and their impact on seed and fruit set. Finally, we discuss major questions arising from this new progress and outline future directions for unraveling mechanisms underlying INV-mediated plant development and their potential applications in plant biotechnology and agriculture.

Sucrose feeding reverses shade-induced kernel losses in maize.

BACKGROUND AND AIMS: Water limitations can inhibit photosynthesis and change gene expression in ways that diminish or prevent reproductive development in plants. Sucrose fed to the plants can reverse the effects. To test whether the reversal acts generally by replacing the losses from photosynthesis, sucrose was fed to the stems of shaded maize plants (Zea mays) during reproductive development. METHODS: Shading was adjusted to mimic the inhibition of photosynthesis around the time of pollination in water-limited plants. Glucose and starch were imaged and quantified in the female florets. Sucrose was infused into the stems to vary the sugar flux to the ovaries. KEY RESULTS: Ovaries normally grew rapidly and contained large amounts of glucose and starch, with a glucose gradient favouring glucose movement into the developing ovary. Shade inhibited photosynthesis and diminished ovary and kernel size, weight, and glucose and starch contents compared with controls. The glucose gradient became small. Sucrose fed to the stem reversed these losses, and kernels were as large as the controls. CONCLUSIONS: Despite similar inhibition of photosynthesis, the depletion of ovary glucose and starch was not as severe in shade as during a comparable water deficit. Ovary abortion prevalent during water deficits did not occur in the shade. It is suggested that this difference may have been caused by more translocation in shade than during the water deficit, which prevented low sugar contents necessary to trigger an up-regulation of senescence genes known to be involved in abortion. Nevertheless, sucrose feeding reversed kernel size losses and it is concluded that feeding acted generally to replace diminished photosynthetic activity.

Cell wall biosynthesis and the molecular mechanism of plant enlargement.

Recently discovered reactions allow the green alga Chara corallina (Klien ex. Willd., em. R.D.W.) to grow well without the benefit of xyloglucan or rhamnogalactan II in its cell wall. Growth rates are controlled by polygalacturonic acid (pectate) bound with calcium in the primary wall, and the reactions remove calcium from these bonds when new pectate is supplied. The removal appears to occur preferentially in bonds distorted by wall tension produced by the turgor pressure (P). The loss of calcium accelerates irreversible wall extension if P is above a critical level. The new pectate (now calcium pectate) then binds to the wall and decelerates wall extension, depositing new wall material on and within the old wall. Together, these reactions create a non-enzymatic but stoichiometric link between wall growth and wall deposition. In green plants, pectate is one of the most conserved components of the primary wall, and it is therefore proposed that the acceleration-deceleration-wall deposition reactions are of wide occurrence likely to underlie growth in virtually all green plants. C. corallina is one of the closest relatives of the progenitors of terrestrial plants, and this review focuses on the pectate reactions and how they may fit existing theories of plant growth.

Calcium pectate chemistry causes growth to be stored in Chara corallina: a test of the pectate cycle.

Calcium pectate chemistry was reported to control the growth rate of cells of Chara corallina, and required turgor pressure (P) to do so. Accordingly, this chemistry should account for other aspects of growth, particularly the ability of plants to compensate for brief exposure to low P, that is, to 'store' growth. Live Chara cells or isolated walls were attached to a pressure probe, and P was varied. Low P caused growth to be inhibited in live cells, but when P returned to normal (0.5 MPa), a flush of growth completely compensated for that lost at low P for as long as 23-53 min. This growth storage was absent in isolated walls, mature cells and live cells exposed to cold, indicating that the cytoplasm delivered a metabolically derived growth factor needing P for its action. Because the cytoplasm delivered pectate needing P for its action, pectate was supplied to isolated walls at low P as though the cytoplasm had done so. Growth was stored while otherwise none occurred. It was concluded that a P-dependent cycle of calcium pectate chemistry not only controlled growth rate and new wall deposition, but also accounted for stored growth.

Xylem tension affects growth-induced water potential and daily elongation of maize leaves.

Diurnal rates of leaf elongation vary in maize (Zea mays L.) and are characterized by a decline each afternoon. The cause of the afternoon decline was investigated. When the atmospheric environment was held constant in a controlled environment, and water and nutrients were adequately supplied to the soil or the roots in solution, the decline persisted and indicated that the cause was internal. Inside the plants, xylem fluxes of water and solutes were essentially constant during the day. However, the forces moving these components changed. Tensions rose in the xylem, and gradients of growth-induced water potentials decreased in the surrounding growing tissues of the leaf. These potentials, measured with isopiestic thermocouple psychrometry, changed because the roots became less conductive to water as the day progressed. The increased tensions were reversed by applying pressure to the soil/root system, which rehydrated the leaf. Afternoon elongation immediately recovered to rapid morning rates. The rapid morning rates did not respond to soil/root pressurization. It was concluded that increased xylem tension in the afternoon diminished the gradients in growth-induced water potential and thus inhibited elongation. Because increased tensions cause a similar but larger inhibition of elongation if maize dehydrates, these hydraulics are crucial for shaping the growth-induced water potential and thus the rates of leaf elongation in maize over the entire spectrum of water availability.

Osmotic adjustment leads to anomalously low estimates of relative water content in wheat and barley.

Relative water content (RWC) is used extensively to determine the water status of plants relative to their fully turgid condition. However, plants often adjust osmotically to salinity or water deficit, which maintains turgor pressure and obscures the definition of 'full turgidity'. To explore this problem, turgor was measured by isopiestic psychrometry in mature leaf blades of barley (Hordeum vulgare) and durum wheat (Triticum turgidum ssp. durum) salinised to 150 mm NaCl, or bread wheat (Triticum aestivum) grown in soil dehydrated to varying degrees. Osmotic adjustment maintained turgor in all the plants but despite full maintenance in some of the salinised plants, their leaf RWC decreased substantially. This occurred because excess water was absorbed while the samples were floated on water as part of the RWC measurement. The absorption falsely increased the weight of the 'fully turgid' condition, causing RWC to be anomalously low by 10-15%. Cell solution was secreted into intercellular spaces and was seen under a microscope, which is a test encouraged for all RWC measurements. Several alternate methods are suggested for rehydrating tissues while minimising excess water absorption, but no simple definition of 'full turgidity' seems possible. In general, direct measurements of osmotic adjustment and turgor are preferred.

Tension required for pectate chemistry to control growth in Chara corallina.

Recent work showed that polygalacturonate (pectate) chemistry controlled the growth rate of the large-celled alga Chara corallina when turgor pressure (P) was normal (about 0.5 MPa). The mechanism involved calcium withdrawal from the wall by newly supplied pectate acting as a chelator. But P itself can affect growth rate. Therefore, pectate chemistry was investigated at various P. A pressure probe varied P in isolated walls, varying the tension on the calcium pectate cross-links bearing the load of P. When soluble pectate was newly supplied, the wall grew irreversibly but the pectate was inactive below a P of 0.2 MPa, indicating that tension was required in the existing wall before new pectate acted. It was suggested that the tension distorted some of the wall pectate (the dominant pectin), weakening its calcium cross-links and causing the calcium to be preferentially lost to the new pectate, which was not distorted. The preferential loss provided a molecular mechanism for loosening the wall structure, resulting in faster growth. However, the resulting relaxation of the vacated wall pectate would cause calcium to be exchanged with load-bearing calcium pectate nearby, auto-propagating throughout the wall for long periods. There is evidence for this effect in isolated walls. In live cells, there is also evidence that auto-propagation is controlled by binding the newly supplied pectate (now calcium pectate) to the wall and/or by additional Ca(2+) entering the wall structure. A tension-dependent cycle of pectate chemistry thus appeared to control growth while new wall was deposited as a consequence.

Functional reversion to identify controlling genes in multigenic responses: analysis of floral abortion.

In many situations, organisms respond to stimuli by altering the activity of large numbers of genes. Among these, certain ones are likely to control the phenotype while others play a secondary role or are passively altered without directly affecting the phenotype. Identifying the controlling genes has proven difficult. However, in a few instances, it has been possible to reverse the phenotype by physiological or biochemical means without altering the genetics of the organism. During this functional reversion, only a few genes may respond, thus identifying those likely to be controlling the phenotype. Floral abortion during a water shortage in maize is an example because the response is inherently multigenic, and the phenotype can be reversed by physiological/biochemical means. A recent analysis used this reversal to reveal that only a few genes are likely to control the abortion phenotype. In maize, these genes coded for a cell wall invertase (Incw2), a soluble invertase (Ivr2), a ribosome-inactivating protein (RIP2), and phospholipase D (PLD1). The invertases appeared to control the normal sugar uptake by the ovaries. Their down-regulation depleted ovary sugar pools and resulted in an up-regulation of the genes for ribosome-inactivating protein and for phospholipase. The latter changes appeared to initiate senescence that degraded cell membranes, thus causing irreversible abortion. With these findings, these genes have become targets for preventing abortion. This approach might have value in other contexts with some additional methods.

Leaf shrinkage decreases porosity at low water potentials in sunflower.

Leaves often shrink significantly when soil water is limited. For gas exchange measurmements, the shrinkage can require correction for changing amounts of tissue in the apparatus. In sunflower plants (Helianthus annuus L.), a comparison was made between mathematically-corrected transpiration and clamping leaves at their original turgid size without mathematical correction. These methods should give the same result, but transpiration was substantially greater in the clamped leaves than in the shrunken and mathematically-corrected ones. Because the clamped leaves remained at their original turgid area, wounding was not a factor. If shrunken leaves were stretched to their original area, transpiration increased immediately and was traced to increased leaf conductance to water vapor and greater porosity for bulk air movement through the leaf, implicating the stomata. Releasing the leaf caused each of these properties to return to the tightened condition. When all the leaves were held at their original size during a soil water deficit, whole-plant water use was greater than when the leaves shrank naturally. It was concluded that shrinkage decreases the porosity of sunflower leaves. This natural tightening can be disrupted by stretching leaves during gas exchange measurements. However, stretching provides a useful means of changing leaf porosity for experimental purposes.

Calcium pectate chemistry controls growth rate of Chara corallina.

Pectin, a normal constituent of cell walls, caused growth rates to accelerate to the rates in living cells when supplied externally to isolated cell walls of Chara corallina. Because this activity was not reported previously, the activity was investigated. Turgor pressure (P) was maintained in isolated walls or living cells using a pressure probe in culture medium. Pectin from various sources was supplied to the medium. Ca and Mg were the dominant inorganic elements in the wall. EGTA or pectin in the culture medium extracted moderate amounts of wall Ca and essentially all the wall Mg, and wall growth accelerated. Removing the external EGTA or pectin and replacing with fresh medium returned growth to the original rate. A high concentration of Ca2+ quenched the accelerating activity of EGTA or pectin and caused gelling of the pectin, physically inhibiting wall growth. Low pH had little effect. After the Mg had been removed, Ca-pectate in the wall bore the longitudinal load imposed by P. Removal of this Ca caused the wall to burst. Live cells and isolated walls reacted similarly. It was concluded that Ca cross-links between neighbouring pectin molecules were strong wall bonds that controlled wall growth rates. The central role of Ca-pectate chemistry was illustrated by removing Ca cross-links with new pectin (wall "loosening"), replacing vacated cross-links with new Ca2+ ("Ca2+-tightening"), or adding new cross-links with new Ca-pectate that gelled ("gel tightening"). These findings establish a molecular model for growth that includes wall deposition and assembly for sustained growth activity.

Identifying cytoplasmic input to the cell wall of growing Chara corallina.

Plants enlarge mostly because the walls of certain cells enlarge, with accompanying input of wall constituents and other factors from the cytoplasm. However, the enlargement can occur without input, suggesting an uncertain relationship between cytoplasmic input and plant growth. Therefore, the role of the input was investigated by quantitatively comparing growth in isolated walls (no input) with that in living cells (input occurring). Cell walls were isolated from growing internodes of Chara corallina and filled with pressurized oil to control turgor pressure while elongation was monitored. Turgor pressure in living cells was similarly controlled and monitored by adding/removing cell solution. Temperature was varied in some experiments. At all pressures and temperatures, isolated walls displayed turgor-driven growth indistinguishable in every respect from that in living cells, except the rate decelerated in the isolated walls while the living cells grew rapidly. The growth in the isolated walls was highly responsive to temperature, in contrast to the elastic extension that has been shown to be insensitive to similar temperatures. Consequently, strong intermolecular bonds were responsible for growth and weak bonds for elastic extension. Boiling the walls gave the same results, indicating that enzyme activities were not controlling these bonds. However, pectin added to isolated walls reversed their growth deceleration and returned the rate to that in the living cells. The pectin was similar to that normally produced by the cytoplasm and deposited in the wall, suggesting that continued cytoplasmic input of pectin may play a role in sustaining turgor-driven growth in Chara.