Electrogenic proton/L-glutamate symport in isolated asparagus sprengeri mesophyll cell

Medium' alkaliniiation occurred -lipon the addition of L-Glu to mechanically isolated Asparagus sprenger-i mesophyll cells suspended in 1 mM CaS04. Alkalinization resulted from the coupled entry of H+ and L-Glu anion into the cells. This H+ IL-Glu symport did not stimulate K+ efflux. K+ efflux has been observed during H~ lamino acid symport in other systems. The stimulation of K+ efflux by proton coupled symport is regarded as an indicator of a plasma membrane depolarizing electrogenic symport process. H+ IL-Glu symport in Asparagus sprengerimesophyl1 cells was investigated to determine whether or not the process was electrogenic. The rate of uptake of 0.25 11M 3H-MTPP+ ( Methyltriphenylphosphonium, methyl-3H ) is a probe for monitoring changes in the membrane potential. 3HMTPP+ uptake was reduced by K+ or CCCP, agents known to depolarize the membrane potential. Uptake of 3H-MTPP+ was also inhibited by L-Glu but not by D-Glu. Conversely, 10 mM external MTPP+ inhibited the uptake of 14C-U-LGlu. Simultaneous measurements of the rates of 14C-U-L-Glu uptake and L-Glu dependent H+ influx showed that the molar stoichiometry of H+ IL-Glu symport was 2 to 1. K+ or Na+ stimulated H+ efflux was completely inhibited by DCCD, DES, oligomycin and antimycin reagents which inhibit ATP driven H+ efflux. The H+ efflux \Vas also stimulate.d by the weak acids, butyric acid and acetic acid, which are known fo-aCidify the cytoplasm. This weak acid stimulated H+ efflux was also completely inhibited by oligomycin. It was calculated that net L-Glu dependent H+ influx increased by 100% in the presence of oligomycin and that despite net medium alkalinization H+ IL-Glu symport stimulates ATP dependent H+ efflux. 11 The data presented in this study indicate that H+ IL-Glu symport is electrogenic. The data also show that ATP dependent Ht efflux rather than K+ efflux is the- process compensating for thi~ electrogenic H+ IL-Glu symport.

Natriuretic peptides and immunoreactants modify osmoticum-dependent volume changes in Solanum tuberosum L. mesophyll cell protoplasts

In plants, as in vertebrates, natriuretic peptide (NP) hormones can influence water and solute homeostasis. Here we demonstrate that a synthetic peptide identical to the C-terminus (amino acids 99–126) of the rat atrial natriuretic peptide (rANP) modulates osmotically induced swelling of mesophyll cell protoplasts (MCPs) in a concentration and time-dependent manner. Osmotically-induced volume changes in MCPs are enhanced by plant extracts with NP immunoreactivity and this effect is concentration-dependent. In contrast, pre-treatment of the plant extracts with rabbit anti-human ANP (99–126) antiserum suppresses enhanced osmoticum-induced swelling. Isolated plant peptides (irPNP) that have been immunoaffinity purified with rabbit anti-human ANP (99–126) antiserum also enhance osmotically-induced swelling. While rANP and irPNP cause increases in cGMP levels in MCPs, elevated cGMP levels do not cause increases in osmoticum-dependent swelling but exert an inhibitory effect. These findings are consistent with a NP-dependent, cGMP-independent effect on plant cell volume regulation and a role in homeostasis for peptides that are recognized by antibodies directed against the C-terminus of vertebrate ANPs.

Proteasome Inhibitors Prevent Tracheary Element Differentiation in Zinnia Mesophyll Cell Cultures1

To determine whether proteasome activity is required for tracheary element (TE) differentiation, the proteasome inhibitors clasto-lactacystin β-lactone and carbobenzoxy-leucinyl-leucinyl-leucinal (LLL) were used in a zinnia (Zinnia elegans) mesophyll cell culture system. The addition of proteasome inhibitors at the time of culture initiation prevented differentiation otherwise detectable at 96 h. Inhibition of the proteasome at 48 h, after cellular commitment to differentiation, did not alter the final percentage of TEs compared with controls. However, proteasome inhibition at 48 h delayed the differentiation process by approximately 24 h, as indicated by examination of both morphological markers and the expression of putative autolytic proteases. These results indicate that proteasome function is required both for induction of TE differentiation and for progression of the TE program in committed cells. Treatment at 48 h with LLL but not clasto-lactacystin β-lactone resulted in partial uncoupling of autolysis from differentiation. Results from gel analysis of protease activity suggested that the observed incomplete autolysis was due to the ability of LLL to inhibit TE cysteine proteases.

Effect of Water Stress on Cell Division and Cdc2-Like Cell Cycle Kinase Activity in Wheat Leaves1

In wheat (Triticum aestivum) seedlings subjected to a mild water stress (water potential of −0.3 MPa), the leaf-elongation rate was reduced by one-half and the mitotic activity of mesophyll cells was reduced to 42% of well-watered controls within 1 d. There was also a reduction in the length of the zone of mesophyll cell division to within 4 mm from the base compared with 8 mm in control leaves. However, the period of division continued longer in the stressed than in the control leaves, and the final cell number in the stressed leaves reached 85% of controls. Cyclin-dependent protein kinase enzymes that are required in vivo for DNA replication and mitosis were recovered from the meristematic zone of leaves by affinity for p13suc1. Water stress caused a reduction in H1 histone kinase activity to one-half of the control level, although amounts of the enzyme were unaffected. Reduced activity was correlated with an increased proportion of the 34-kD Cdc2-like kinase (an enzyme sharing with the Cdc2 protein of other eukaryotes the same size, antigenic sites, affinity for p13suc1, and H1 histone kinase catalytic activity) deactivated by tyrosine phosphorylation. Deactivation to 50% occurred within 3 h of stress imposition in cells at the base of the meristematic zone and was therefore too fast to be explained by a reduction in the rate at which cells reached mitosis because of slowing of growth; rather, stress must have acted more immediately on the enzyme. The operation of controls slowing the exit from the G1 and G2 phases is discussed. We suggest that a water-stress signal acts on Cdc2 kinase by increasing phosphorylation of tyrosine, causing a shift to the inhibited form and slowing cell production.

高产小麦旗叶和芒细胞结构及其光合性能的研究

小麦旗叶的光合产物是其籽粒碳水化合物的主要来源，因此如何提高旗叶的光合能力从而提高小麦产量一直是小麦研究的热点。但是以往对高产小麦旗叶的研究主要集中在光合功能和生理生化指标等方面，很少涉及其结构与功能的联系，以及对亲本与子代的旗叶进行对比观察。本文以冬小麦亲本小偃54 、8602及其子代小偃81（高产品种）灌浆期的旗叶为材料，应用细胞离析法、组织切片和荧光显微技术等，对旗叶中叶肉细胞形态、叶绿体数目、叶片厚度、维管束数目和面积等进行了比较观察和测定，旨在探讨小麦旗叶结构与其光合效率的关系。研究结果表明，与亲本小偃54、8602相比，子代小偃81的叶片较厚，横切面内中央大维管束的周长与面积较大;高环数叶肉细胞所占比例、叶肉细胞的周长及其平面面积和细胞内叶绿体的数目等均大于亲本。由此可见，通过小麦品种的改良确实能使其旗叶的结构与光合细胞发生了明显的变化，从而为植物细胞结构和功能的密切关系提供了有力的证据;同时也为作物改良育种提供了又一种新的育种目标。 　　小麦非叶器官之一的芒，对其结构与光合特性关系的研究尚不够深入和广泛。本实验以具芒小麦高产耐旱品种京411籽粒不同发育时期的芒及旗叶为材料，对其叶绿体结构、放氧速率和磷酸烯醇式丙酮酸羧化酶(PEPCase EC 4.1.1.31)的活性进行了比较观察和测定。超微结构显示，从抽穗期开始，芒和旗叶中的叶绿体基粒及其垛叠度均有增加，之后由灌浆末期开始，叶绿体的膜系统开始逐渐解体。通过放氧速率的测定表明，在芒和旗叶中，光合速率在前几个时期呈上升趋势，随着器官的衰老逐渐下降，但是，旗叶放氧速率的下降比芒中更早。另外，芒的PEPCase活性在籽粒发育的整个过程中均高于旗叶，其中以籽粒干物质形成末期尤为显著。因此，芒对高产小麦籽粒的形成，特别是在干物质形成的后期起着更为重要的作用。 　　

Rubisco small subunit, chlorophyll a/b-binding protein and sucrose : fructan-6-fructosyl transferase gene expression and sugar status in single barley leaf cells in situ. Cell type specificity and induction by light

Chungui Lu, Olga A. Koroleva, John F. Farrar, Joe Gallagher, Chris J. Pollock, and A. Deri Tomos (2002). Rubisco small subunit, chlorophyll a/b-binding protein and sucrose : fructan-6-fructosyl transferase gene expression and sugar status in single barley leaf cells in situ. Cell type specificity and induction by light. Plant Physiology, 130 (3) pp.1335-1348 Sponsorship: BBSRC RAE2008

Amino acid transport : the special case of a H/L-glutamate cotransport system in Asparagus sprengeri mesophyll cells /

The addition of L-Glutamate (L-GLU) and L-Hethionine ~ulfoximine (L-HSO) to mechanically isolated. photosynthetically competent, Asparagus sprengeri mesophyll cells ~u~pended in 1mM CaS04 cau~ed an immediate transient alkalinization of the cell su~pension medium in both the light and dark. The alkalinization response was specific and stereospecific as none of the L-isomers of the other 19 protein amino acids tested or D-GLU gave this response. Uptake of 14C-L-GLU was stimulated by the light. The addition of non-radioactive L-GLU. or L-GLU analogs together with 14C-L-GLU showed that only L-GLU and L-HSO stimulated alkalinization whilst inhibiting the uptake of 14C-L-GLU. Both the L-GLU dependent alkalinization and the upt~ke of 14C-L-GLU were stimulated when the external pH was decreased from 6.5 to 5.5. Increasing external K+ concentrations inhibited the uptake of 14C-L-GLU. Fusicoccin (FC) stimulated uptake. The L-GLU dependent alkalinization re~ponse exhibited monophasic saturation kinetics while the uptake of 14C-L-GLU exhibited biphasic saturation kinetics. In addition to a saturable component. the uptake kinetics also showed a linear component of uptake. Addition of L-GLU and L-MSO caused internal acidification of the cell as measured by a change in the distribution of 14C-DMO. There was no change in K+ efflux when L-GLU was added. A H+ to L-GLUinflux stoichiometry of 3:1 wa~ mea~ured at an external I.-GLU concentration of O.5mM and increased with increasing external 13 L-QLU concentration. Metabolism of L-GLU was detected manometrlcally by observing an increase in COa evolution upon the addition of L-QLU and by detection of i*C02 evolution upon the addition of »*C-L-GLU. »*C02 evolution was higher in the dark than in the light. The data are consistent with the operation of a H+/L-QLO cotransport system. The data also show that attempts to quantify the stoichlometry of the process were complicated by the metabolism of L-GLU.

GABA accumulation and the hypersensitive response in isolated mesophyll cells treated with the G protein activator mastoparan

GABA (y-amino butyric acid) is a non-protein amino acid synthesized through the a-decarboxylation of L-glutamate. This reaction is catalyzed by L-glutamate decarboxylase (EC 4.1.1.15), a cytosolic Ca2+/calmodulin-stimulated enzyme. The purpose of this study is to determine whether or not GABA accumulation is associated with the hypersensitive response of isolated Asparagus sprengeri mesophyll cells. The addition of 25 J.lM mastoparan, a G protein activator, to suspensions of isolated asparagus mesophyll cells significantly increased GABA synthesis and cell death. Cell death was assessed using Evan's blue dye and fluorescein diacetate tests for cell viability. In addition, mastoparan stimulated pH-dependent alkalinization of the external medium, and a rapid and large 02 consumption followed by a loss of photosynthetic activity. The rate of 02 consumption and the net decrease in 02 in the dark was enhanced by light. The inactive mastoparan analogue Mas17 was ineffective in stimulating GABA accumulation, medium alkalinization, 02 uptake and cell death. Accumulation of H202 in response tomastoparan was not detected, however, mastoparan caused the cell-dependent degradation of added H202. The pH dependence of mastoparan-stimulated alkalinization suggests cellular electrolyte leakage, while the consumption of 02 corresponds to the oxidative burst in which 02 at the cell surface is reduced to form various active oxygen species. The results are indicative of the "hypersensitive response" of plants to pathogen attack, namely, the death of cells in the locality of pathogen invasion. The data are compatible with a model in which mastoparan triggers G protein activity, subsequent intracellular signal transduction pathway/s, and the hypersensitive response. It is postulated that the physiological elicitation of the hypersensitive response involves G protein signal transduction. The synthesis of GABA during the hypersensitive response has not been documented previously; however the role/s of GABA synthesis in the hypersensitive response, if any, remain unclear.

Plant natriuretic peptide active site determination and effects on cGMP and cell volume regulation

Natriuretic peptides (NP) were first identified in animals where they play a role in the regulation of salt and water balance. This regulation is partly mediated by intracellular changes in cyclic GMP (cGMP). NP immunoanalogues occur in many plants and have been isolated, with two NP encoding genes characterised in Arabidopsis thaliana L. (AtPNP-A and AtPNP-B). Part of AtPNP-A contains the region with homology to human atrial (A)NP. We report here on the effects of recombinant AtPNP-A and smaller synthetic peptides within the ANP-homologous region with a view to identifying the biologically active domain of the molecule. Furthermore, we investigated interactions between AtPNP-A and the hormone, abscisic acid (ABA). ABA does not significantly affect Arabidopsis mesophyll protoplast volume regulation, whereas AtPNP-A and synthetic peptides promote water uptake into the protoplasts causing swelling. This effect is promoted by the membrane permeable cGMP analogue, 8-Br-cGMP, and inhibited by guanylate cyclase inhibitors indicating that increases in cGMP are an essential component of the plant natriuretic peptides (PNP) signalling cascade. ABA does not induce cGMP transients and does not affect AtPNP-A dependent cGMP increases, hence the two regulators differ in their second messenger signatures. Interestingly, AtPNP-A significantly delays and reduces the extent of ABA stimulated stomatal closure that is also based on cell volume regulation. We conclude that a complex interplay between observed PNP effects (stomatal opening and protoplast swelling) and ABA is likely to be cell type specific.

Implications of the mesophyll conductance to CO2 for photosynthesis and water-use efficiency during long-term water stress and recovery in two contrasting Eucalyptu species

Water stress (WS) slows growth and photosynthesis (An), but most knowledge comes from short-time studies that do not account for longer term acclimation processes that are especially relevant in tree species. Using two Eucalyptus species that contrast in drought tolerance, we induced moderate and severe water deficits by withholding water until stomatal conductance (gsw) decreased to two pre-defined values for 24 d, WS was maintained at the target gsw for 29 d and then plants were re-watered. Additionally, we developed new equations to simulate the effect on mesophyll conductance (gm) of accounting for the resistance to refixation of CO2. The diffusive limitations to CO2, dominated by the stomata, were the most important constraints to An. Full recovery of An was reached after re-watering, characterized by quick recovery of gm and even higher biochemical capacity, in contrast to the slower recovery of gsw. The acclimation to long-term WS led to decreased mesophyll and biochemical limitations, in contrast to studies in which stress was imposed more rapidly. Finally, we provide evidence that higher gm under WS contributes to higher intrinsic water-use efficiency (iWUE) and reduces the leaf oxidative stress, highlighting the importance of gm as a target for breeding/genetic engineering.

Ozone inhibits guard cell K+ channels implicated in stomatal opening

Ozone (O3) deleteriously affects organisms ranging from humans to crop plants, yet little is understood regarding the underlying mechanisms. In plants, O3 decreases CO2 assimilation, but whether this could result from direct O3 action on guard cells remained unknown. Potassium flux causes osmotically driven changes in guard cell volume that regulate apertures of associated microscopic pores through which CO2 is supplied to the photosynthetic mesophyll tissue. We show in Vicia faba that O3 inhibits (i) guard cell K+ channels that mediate K+ uptake that drives stomatal opening; (ii) stomatal opening in isolated epidermes; and (iii) stomatal opening in leaves, such that CO2 assimilation is reduced without direct effects of O3 on photosynthetic capacity. Direct O3 effects on guard cells may have ecological and agronomic implications for plant productivity and for response to other environmental stressors including drought.

Phloem sap proteins from Cucurbita maxima and Ricinus communis have the capacity to traffic cell to cell through plasmodesmata

In angiosperms, the functional enucleate sieve tube system of the phloem appears to be maintained by the surrounding companion cells. In this study, we tested the hypothesis that polypeptides present within the phloem sap traffic cell to cell from the companion cells, where they are synthesized, into the sieve tube via plasmodesmata. Coinjection of fluorescently labeled dextrans along with size-fractionated Cucurbita maxima phloem proteins, ranging in size from 10 to 200 kDa, as well as injection of individual fluorescently labeled phloem proteins, provided unambiguous evidence that these proteins have the capacity to interact with mesophyll plasmodesmata in cucurbit cotyledons to induce an increase in size exclusion limit and traffic cell to cell. Plasmodesmal size exclusion limit increased to greater than 20 kDa, but less than 40 kDa, irrespective of the size of the injected protein, indicating that partial protein unfolding may be a requirement for transport. A threshold concentration in the 20–100 nM range was required for cell-to-cell transport indicating that phloem proteins have a high affinity for the mesophyll plasmodesmal binding site(s). Parallel experiments with glutaredoxin and cystatin, phloem sap proteins from Ricinus communis, established that these proteins can also traffic through cucurbit mesophyll plasmodesmata. These results are discussed in terms of the requirements for regulated protein trafficking between companion cells and the sieve tube system. As the threshold value for plasmodesmal transport of phloem sap proteins falls within the same range as many plant hormones, the possibility is discussed that some of these proteins may act as long-distance signaling molecules.

The Relationship between Photosynthesis and a Mastoparan-Induced Hypersensitive Response in Isolated Mesophyll Cells1

The G-protein activator mastoparan (MP) was found to elicit the hypersensitive response (HR) in isolated Asparagus sprengeri mesophyll cells at micromolar concentrations. The HR was characterized by cell death, extracellular alkalinization, and an oxidative burst, indicated by the reduction of molecular O2 to O2⋅−. To our knowledge, this study was the first to monitor photosynthesis during the HR. MP had rapid and dramatic effects on photosynthetic electron transport and excitation energy transfer as determined by variable chlorophyll a fluorescence measurements. A large increase in nonphotochemical quenching of chlorophyll a fluorescence accompanied the initial stages of the oxidative burst. The minimal level of fluorescence was also quenched, which suggests the origin of this nonphotochemical quenching to be a decrease in the antenna size of photosystem II. In contrast, photochemical quenching of fluorescence decreased dramatically during the latter stages of the oxidative burst, indicating a somewhat slower inhibition of photosystem II electron transport. The net consumption of O2 and the initial rate of O2 uptake, elicited by MP, were higher in the light than in the dark. These data indicate that light enhances the oxidative burst and suggest a complex relationship between photosynthesis and the HR.

Turgor Regulation via Cell Wall Adjustment in White Spruce1

Turgor regulation at reduced water contents was closely associated with changes in the elastic quality of the cell walls of individual needles and shoots of naturally drought-resistant seedlings of white spruce (Picea glauca [Moench] Voss.) and of seedlings of intermediate resistance that had been pretreated with paclobutrazol, a stress-protecting, synthetic plant-growth regulator. Paclobutrazol-treated seedlings showed marked increases in drought resistance, and pressure-volume analysis combined with Chardakov measurements confirmed observations that water stress was ameliorated during prolonged drought. Turgor was maintained in the paclobutrazol-treated and in the naturally resistant drought-stressed seedlings despite water contents near or below the turgor-loss volumes of well-watered controls. The maintenance of turgor in these seedlings was in large part a function of the dynamic process of cell wall adjustment, as reflected by marked reductions in both the saturated and turgor-loss volumes and by large increases in the elastic coefficients of the tissues. Shear and Young's moduli, calculated from pressure-volume curves and the radii and wall thicknesses of mesophyll cells, also confirmed observed changes in the elastic qualities of the cell walls. Elastic coefficients of well-watered, paclobutrazol-treated seedlings were consistently larger than those in well-watered controls and several times larger than the values in untreated plants, which succumbed rapidly to drought. In contrast, untreated seedlings that withstood prolonged drought without wilting displayed elastic coefficients similar to those in seedlings that had been treated with paclobutrazol but that had not been exposed to drought.