Showing papers in "Planta in 1995"

Organic acid exudation as an aluminum-tolerance mechanism in maize (Zea mays L.)

Abstract: In this study, the role of root organic acid synthesis and exudation in the mechanism of aluminum tolerance was examined in Al-tolerant (South American 3) and Al-sensitive (Tuxpeno and South American 5) maize genotypes. In a growth solution containing 6 μM Al3+, Tuxpeno and South American 5 were found to be two- and threefold more sensitive to Al than South American 3. Root organic acid content and organic acid exudation from the entire root system into the bulk solution were investigated via high-performance liquid chromatographic analysis while exudates collected separately from the root apex or a mature root region (using a dividedroot-chamber technique) were analyzed with a more-sensitive ion chromatography system. In both the Al-tolerant and Al-sensitive lines, Al treatment significantly increased the total root content of organic acids, which was likely the result of Al stress and not the cause of the observed differential Al tolerance. In the absence of Al, small amounts of citrate were exuded into the solution bathing the roots. Aluminum exposure triggered a stimulation of citrate release in the Al-tolerant but not in the Al-sensitive genotypes; this response was localized to the root apex of the Al-tolerant genotype. Additionally, Al exposure triggered the release of phosphate from the root apex of the Al-tolerant genotype. The same solution Al3+ activity that elicited the maximum difference in Al sensitivity between Al-tolerant and Al-sensitive genotypes also triggered maximal citrate release from the root apex of the Al-tolerant line. The significance of citrate as a potential detoxifier for aluminum is discussed. It is concluded that organic acid release by the root apex could be an important aspect of Al tolerance in maize.

Characterisation of Al-stimulated efflux of malate from the apices of Al-tolerant wheat roots

Abstract: Aluminium (Al) stimulates the efflux of malate from the apices of wheat (Triticum aestivum L.) roots (Delhaize et al. 1993, Plant Physiol. 103, 695–702). The response was five to tenfold higher in Al-tolerant seedlings than Al-sensitive seedlings and the capacity for Al-stimulated malate efflux was found to co-segregate with Al tolerance in a pair of near-isogenic wheat lines differing in Al-tolerance at a single dominant locus. We have investigated this response further using excised root apices. Half-maximal efflux of malate from apices of Al-tolerant seedlings was measured with 30 μM Al in 0.2 mM CaCl2, pH 4.2, while saturating rates of 2.0 nmol·apex−1·h−1 occurred with concentrations above 100 μM Al. The stimulation of malate efflux by Al was accompanied by an increase in K+ efflux which appeared to account for electroneutrality. The greater stimulation of malate efflux from Al-tolerant apices compared to Al-sensitive apices could not be explained by differences in the activities of phosphoenolpyruvate carboxylase or NAD-malate dehydrogenase. Several other polyvalent cations, including gallium, indium and the tridecamer Al13, failed to elicit malate efflux. Aluminium-stimulated efflux of malate was correlated with the measured concentration of total monomeric Al present, and with the predicted concentrations of Al3+ and AlOH2+ ions in the solution. Several antagonists of anion channels inhibited Al-stimulated efflux of malate with the following order of effectiveness: niflumic acid≈NPPB>IAA-94≈A-9-C>ethacrynic acid. Lanthanum, chlorate, perchlorate, zinc and α-cyano-4-hydroxycinnamic acid inhibited malate release by less than 30% at 100 μM while 4,4′-diisothiocyanatostilbene-2,2′-disulphonate (DIDS) had no effect. These results suggest that the Al3+ cation stimulates malate efflux via anion channels in apical cells of Al-tolerant wheat roots.

The promoter of the Arabidopsis thaliana SUC2 sucrose-H+ symporter gene directs expression of beta-glucuronidase to the phloem: evidence for phloem loading and unloading by SUC2.

Abstract: The Arabidopsis thaliana (L.) Heynh. SUC2 gene encodes a plasma-membrane sucrose-H+ symporter. The DNA sequence of the SUC2 promoter has been determined. Using a translational fusion of this promoter to the N-terminus of beta-glucuronidase (GUS) and the GUS histochemical assay, the tissue specificity of the SUC2 promoter was studied in Arabidopsis plants transformed with this fusion construct. The SUC2 promoter directed expression of GUS activity with high specificity to the phloem of all green tissues of Arabidopsis such as rosette leaves, stems, and sepals. During leaf development the expression of SUC2-GUS activity was first seen in the tips of young rosette leaves. In older leaves and during their concomitant sink/source transition, expression proceeded from the tips to the bases of the leaves, indicating that expression of the SUC2 sucrose-H+ symporter is tightly coupled to the source-strength of Arabidopsis leaves. Expression of SUC2-GUS activity was also seen, however, in sink tissues such as roots and developing Arabidopsis pods, suggesting that the product of the SUC2 gene might not only be important for phloem loading, but also for phloem unloading. A possible regulatory effect of carbohydrates (glucose and sucrose) on the activity of the SUC2 promoter was studied and excluded, both in excised leaves and young seedlings of transgenic Arabidopsis plants. The overall pattern of SUC2-GUS expression correlated well with that of the Arabidopsis thaliana AHA3 plasma-membrane H(+)-ATPase which is also expressed in the phloem and most likely represents the primary pump generating the energy for secondary active transporters such as SUC2.

Influence of leaf water status on stomatal response to humidity, hydraulic conductance, and soil drought in Betula occidentalis

Abstract: Whole-canopy measurements of water flux were used to calculate stomatal conductance (g s ) and transpiration (E) for seedlings of western water birch (Betula occidentalis Hook.) under various soil-plant hydraulic conductances (k), evaporative driving forces (ΔN; difference in leaf-to-air molar fraction of water vapor), and soil water potentials (Ψs). As expected, g s dropped in response to decreased k or ΨS, or increased ΔN(> 0.025). Field data showed a decrease in mid-day g s with decreasing k from soil-to-petiole, with sapling and adult plants having lower values of both parameters than juveniles. Stomatal closure prevented E and Ψ from inducing xylem cavitation except during extreme soil drought when cavitation occurred in the main stem and probably roots as well. Although all decreases in g s were associated with approximately constant bulk leaf water potential (ψl), this does not logically exclude a feedback response between ΨL and g s . To test the influence of leaf versus root water status on g s , we manipulated water status of the leaf independently of the root by using a pressure chamber enclosing the seedling root system; pressurizing the chamber alters cell turgor and volume only in the shoot cells outside the chamber. Stomatal closure in response to increased ΔN, decreased k, and decreased ΨS was fully or partially reversed within 5 min of pressurizing the soil. Bulk ΨL remained constant before and after soil pressurizing because of the increase in E associated with stomatal opening. When ΔN was low (i.e., < 0.025), pressurizing the soil either had no effect on g s , or caused it to decline; and bulk ΨL increased. Increased Ψl may have caused stomatal closure via increased backpressure on the stomatal apparatus from elevated epidermal turgor. The stomatal response to soil pressurizing indicated a central role of leaf cells in sensing water stress caused by high ΔN, low k, and low ΨS. Invoking a prominent role for feedforward signalling in short-term stomatal control may be premature.

An evaluation of direct and indirect mechanisms for the "sink-regulation" of photosynthesis in spinach: Changes in gas exchange, carbohydrates, metabolites, enzyme activities and steady-state transcript levels after cold-girdling source leaves

Abstract: Mature source leaves of spinach (Spinacia oleracea L.) plants growing hydroponically in a 9 h light (350 μmol photons·m−2 · s−1)/15 h dark cycle at 20° C in a climate chamber were fitted with a cold girdle around the petiole, 2 h into the light period. Samples were taken 1, 3 and 7 h later, and at the end of the photoperiod for the following 4 d. Control samples were taken from ungirdled leaves. In the first 7 h after fitting the cold girdle there was (compared to the control leaves) a two to five-fold accumulation of sucrose, glucose, fructose and starch, a 40–50% increase of hexose-phosphates and ribulose-1,5-bisphosphate, a decrease of glycerate-3-phosphate, a small decrease in sucrose-phosphate synthase activation, an increase of fructose-2,6-bisphosphate, increased activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), but no significant change in photosynthetic rate or stomatal conductance. Steady-state transcript levels for rbcS (small subunit of Rubisco) and atp-D (D-subunit of the thylakoid ATP synthase) decreased 30%, cab (chlorophyll-a-binding protein) decreased by 15% and agp-S (S-isoenzyme of ADP-glucose pyrophosphorylase) and nra (nitrate reductase) rose twofold. On the following days, levels of carbohydrates continued to rise and the changes of metabolites were maintained. Transcripts for rbcS, cab and atpD declined to 20, 70 and 25% of the control values. From day 3 onward the maximum activity of Rubisco declined. This was accompanied by a further increase of Rubisco activation to over 90% and, from day 4 onwards, an inhibition of photosynthesis which was associated with high internal CO2 concentration (ci), high ribulose-1,5-bisphosphate, and low glycerate-3-phosphate. When the cold-girdle was removed on day 5 there was a gradual recovery of photosynthesis and decline of ci over the next 2 d. Hexose-phosphates levels and transcripts for rbcS, cab and atp-D completely recovered within 2 d, even though the levels of carbohydrates had not fully recovered. Activity of Rubisco only reverted partly after 2 d, and Rubisco activation state and the ribulose-1,5-bisphosphate/glycerate-3-phosphate ratio were still higher than in control leaves. Transcripts for nra and agp-S were also still higher than in control leaves. It is concluded (i) that a reversible modulation of gene expression in response to the export rate plays a central role in the mid-term feedback “sink” regulation of photosynthesis, and (ii) that feedback regulation of CO2 fixation by changes of Pi are of little importance in spinach under these conditions. Further (iii) the rapid and reciprocal changes in nra and agpS transcripts, compared to rbcS, provide evidence that gene expression could also contribute to the modulation of nitrate assimilation and carbohydrate storage in conditions of decreased sink demand.

Seasonal changes in photosystem II organisation and pigment composition in Pinus sylvestris

Abstract: Conifers of the boreal zone encounter considerable combined stress of low temperature and high light during winter, when photosynthetic consumption of excitation energy is blocked. In the evergreen Pinus sylvestris L. these stresses coincided with major seasonal changes in photosystem II (PSII) organisation and pigment composition. The earliest changes occurred in September, before any freezing stress, with initial losses of chlorophyll, the D1-protein of the PSII reaction centre and of PSII light-harvesting-complex (LHC II) proteins. In October there was a transient increase in F0, resulting from detachment of the light-harvesting antennae as reaction centres lost D1. The D1-protein content eventually decreased to 90%, reaching a minimum by December, but PSII photochemical efficiency [variable fluorescence (Fv)/maximum fluorescence (Fm)] did not reach the winter minimum until mid-February. The carotenoid composition varied seasonally with a twofold increase in lutein and the carotenoids of the xanthophyll cycle during winter, while the epoxidation state of the xanthophylls decreased from 0.9 to 0.1 from October to January. The loss of chlorophyll was complete by October and during winter much of the remaining chlorophyll was reorganised in aggregates of specific polypeptide composition, which apparently efficiently quench excitation energy through non-radiative dissipation. The timing of the autumn and winter changes indicated that xanthophyll de-epoxidation correlates with winter quenching of chlorophyll fluorescence while the drop in photochemical efficiency relates more to loss of D1-protein. In April and May recovery of the photochemistry of PSII, protein synthesis, pigment rearrangements and zeaxanthin epoxidation occurred concomitantly. Indoor recovery of photosynthesis in winter-stressed branches under favourable conditions was completed within 3 d, with rapid increases in F0, the epoxidation state of the xanthophylls and in light-harvesting polypeptides, followed by recovery of D1-protein content and Fv/Fm, all without net increase in chlorophyll. The fall and winter reorganisation allow Pinus sylvestris to maintain a large stock of chlorophyll in a quenched, photoprotected state, allowing rapid recovery of photosynthesis in spring.

The cellular pathway of postphloem sugar transport in developing tomato fruit

Abstract: The cellular pathway of postphloem sugar transport was elucidated in the outer pericarp of tomato (Lycopersicon esculentum Mill cv. Floradade) fruit at 13–14 and 23–25 days after anthesis (DAA). These developmental stages are characterized by phloem-imported sugars being accumulated as starch and hexose, respectively. The symplasmic tracer, 5(6)-carboxyfluorescein, loaded into the storage parenchyma cells of pericarp discs, moved readily in the younger fruit but was immobile in fruit at 23–25 DAA. Symplasmic mobility of [14C]glucose was found to be identical to 5(6)-carboxyfluorescein. For the older fruit, the pericarp apoplasm was shown to be freely permeable to the apoplasmic tracer, trisodium 3-hydroxy-5,8,10-pyrenetrisulfonate. Indeed, the transport capacity of the pericarp apoplasm was such that the steady-state rate of in-vitro glucose uptake by pericarp discs accounted fully for the estimated rate of in-vivo glucose accumulation. For fruit at 23–25 DAA, the inhibitory effects of the sulfhydryl group modifier, p-chloromer-curibenzenesulfonic acid (PCMBS), on [14C]glucose and [14C]fructose uptake by the pericarp discs depended on the osmolality of the external solution. The inhibition was most pronounced for pericarp discs enriched in storage parenchyma. Consistent with the PCMBS study, strong fluorescent signals were exhibited by the storage parenchyma cells of pericarp discs exposed to the membrane-impermeable thiol-binding fluorochrome, mono-bromotrimethylammoniobimane. The fluorescent weak acid, sulphorhodamine G, was accumulated preferentially by the storage parenchyma cells. Accumulation of sulphorhodamine G was halted by the ATPase inhibitor erythrosin B, suggesting the presence of a plasma-membrane-bound H+-ATPase. A linkage between the putative H+-ATPase activity and hexose transport was demonstrated by an erythrosin-B inhibition of [14C]glucose and [14C]fructose uptake. In contrast, comparable evidence for an energy-coupled hexose porter could not be found in the pericarp of younger fruit at 13–14 DAA. Overall, the data are interpreted to indicate that: (i) The postphloem cellular pathway in the outer fruit pericarp shifts from the symplasm during starch accumulation (13–14 DAA) to the apoplasm for rapid hexose accumulation (23–25 DAA). (ii) An energy-coupled plasma-membrane hexose carrier is expressed specifically in storage parenchyma cells at the latter stage of fruit development.

Thioredoxin h is one of the major proteins in rice phloem sap

Abstract: Sieve tubes play important roles in the transfer of nutrients as well as signals. Hundreds of proteins were found in pure phloem sap collected from rice (Oryza sativa L. cv. Kantou) plants through the cut ends of insect stylets. These proteins may be involved in nutrient transfer and signal transduction. To characterize the nature of these proteins, the partial amino-acid sequence of a 13-kDa protein, named RPP13-1, that was abundant in the pure phleom sap was determined. A cDNA clone of 687 bp, containing an open reading frame of 122 amino acids, was isolated using corresponding oligonucleotides as a probe. The deduced amino-acid sequence was very similar to that of the ubiquitous thiol redox protein, thioredoxin. The consensus sequences of thioredoxins are highly conserved. No putative signal peptide was identified. Antiserum against wheat thioredoxin h cross-reacted with RPP13-1 in the phloem sap of rice plants. RPP13-1 produced in Escherichia coli was reactive to antiserum against wheat thioredoxin h. Both E. coli-produced RPP13-1 and the phloem sap proteins catalyzed the reduction of the disulfide bonds of insulin in the presence of dithiothreitol. These results indicate that an active thioredoxin is a major protein translocating in rice sieve tubes.

High susceptibility to photoinhibition of young leaves of tropical forest trees

Abstract: Photoinhibition of photosynthesis was studied in young (but almost fully expanded) and mature canopy sun leaves of several tropical forest tree species, both under controlled conditions (exposure of detached leaves to about 1.8 mmol photons·m-2·s-1) and in the field. The degree of photoinhibition was determined by means of the ratio of variable to maximum chlorophyll (Chl) fluorescence emission (FV/FM) and also by gas-exchange measurements. For investigations in situ, young and mature leaves with similar exposure to sunlight were compared. The results show a consistently higher degree of photoinhibition in the young leaves. In low light, fast recovery was observed in both types of leaves in situ, as well as in the laboratory. The fluorescence parameter 1 — FS/F′M (where FS = stationary fluorescence and f′M = maximum fluorescence during illumination) was followed in situ during the course of the day in order to test its suitability as a measure of the photosynthetic yield of photosystem II (PSII). Electron-transport rates were calculated from these fluorescence signals and compared with rates of net CO2 assimilation. Measurements of diurnal changes in PSII ‘yield’ confirmed the increased susceptibility of young leaves to photoinhibition. Calculated electron transport qualitatively reflected net CO2 uptake in situ during the course of the day. Photosynthetic pigments were analyzed in darkened and illuminated leaves. Young and mature leaves showed the same Chl a/b ratio, but young leaves contained about 50% less Chl a + b per unit leaf area. The capacity of photosynthetic O2 evolution per unit leaf area was decreased to a similar extent in young leaves. On a Chl basis, young leaves contained more α-carotene, more xanthophyll cycle pigments and, under strong illumination, more zeaxanthin than mature leaves. The high degree of reversible photoinhibition observed in these young sun leaves probably represents a dynamic regulatory process protecting the photosynthetic apparatus from severe damage by excess light.

Accumulation of jasmonate, abscisic acid, specific transcripts and proteins in osmotically stressed barley leaf segments

Abstract: The accumulation of abundant proteins and their respective transcripts, induced by 10−4 M cisabscisic acid or 10−5 M jasmonic acid methyl ester, was studied in barley (Hordeum vulgare L.) leaf segments and compared to that resulting from osmotic stress caused by floating the segments on solutions of sorbitol, glucose, polyethyleneglycol (PEG)-6000 or NaCl. Osmotic stress or treatment with abscisic acid led to the synthesis of novel proteins which were identical to jasmonateinduced proteins (JIPs) with respect to immunological properties and molecular masses. The most prominent polypeptides were characterized by molecular masses of 66, 37 and 23 kDa and were newly synthesized. Whereas sorbitol, mannitol, sucrose, glucose and PEG provoked the synthesis of JIPs, 2deoxyglucose and NaCl did not. We provide evidence that the synthesis of JIPs induced by osmotic stress is directly correlated with a preceding rise in endogenous jasmonates. These jasmonates, quantified by an enzyme immunoassay specific for (−)jasmonic acid and its aminoacid conjugates, increased remarkably in leaf segments treated with sorbitol, glucose or other sugars. In contrast, no increase in jasmonates could be observed in tissues exposed to salts (NaCl). The results strengthen the hypothesis that the accumulation of jasmonates, probably by de-novo synthesis, is an intermediate and essential step in a signalling pathway between (osmotic) stress and activation of genes coding for polypeptides of high abundance.

Enzymes regulating the accumulation of active oxygen species during the hypersensitive reaction of bean to Pseudomonas syringae pv. phaseolicola

Abstract: Changes in the activities of superoxide dismutase (SOD; EC 1.15.1.1), peroxidase (POD; EC 1.11.1.7) and catalase (CAT; EC 1.11.1.6) which regulate the persistence of active oxygen species (AOS) were examined in leaves of bean (Phaseolus vulgaris L. cv. Tendergreen) undergoing compatible and incompatible interactions to race 6 and race 3 strains, respectively, of the halo-blight bacterium Pseudomonas syringae pv. phaseolicola. Resistance of cv. Tendergreen to race 3 is determined by the R3 gene and was expressed by a hypersensitive reaction (HR) which was associated with a rapid increase in lipid peroxidation between 8 and 12 h after inoculation. Five main isoforms of SOD were resolved by native polyacrylamidegel electrophoresis (PAGE). Major changes were found in the activities of the cytosolic Cu, Zn-SOD3 and Cu, ZnSOD5 isoforms, which increased by 6 h after inoculation with race 3, and the possibly peroxisomal MnSOD2 isoform, which decreased rapidly in tissue undergoing the HR. Three further minor isoforms of SOD showed a strong increase in activity during the HR. A low level of extracellular SOD activity was also resolved; two isoforms, one of which increased dramatically in activity during the HR, were detected within intercellular fluids recovered from inoculation sites. Fewer changes in SOD activities were found during the compatible interaction to race 6, and they did not occur until 16 h after inoculation. In tissue around infiltration sites, no decrease in the activity of Mn-SOD2 was observed but slight increases in some other isoforms were found. Four groups of POD isoforms were detected in both 3,3-diaminobenzidine/H2O2-and o-dianisidine/H2O2-stained PAGE gels. Significant changes in activity were again associated with development of the HR. In particular, by 2 h after inoculation, increases in POD3a, b and c isoforms were detected within total soluble extracts and also in POD3c within intercellular fluids (no other isoform was found in the apoplasm). By contrast, POD1 and POD2 activities generally declined following inoculation. The principal change in activity in tissues surrounding infiltration sites was an increase in POD3 isoforms following inoculation with race 3. Measurements of total activity showed a decrease in CAT activity as early as 2 h after inoculation, followed by a recovery after 8 h and a further decrease as infiltrated tissue collapsed during the HR. A more-gradual decline in CAT activity was observed at sites undergoing the compatible interaction and also in tissue surrounding inoculation sites. The spatial and temporal changes detected in activities of CAT and isoforms of SOD and POD clearly demonstrate the complexity and potential subtlety of control of the production and persistence of AOS in bean following microbial challenge. The generation of AOS through HR-specific, early increases in extra-cellular POD and SOD isoforms is discussed.

Monoterpene and sesquiterpene biosynthesis in glandular trichomes of peppermint (mentha piperita) rely exclusively on plastid-derived isopentenyl diphosphate

Abstract: The subcellular compartmentation of isopentenyl diphosphate (IPP) synthesis was examined in secretory cells isolated from glandular trichomes of peppermint (Mentha x piperita L. cv. Black Mitcham). As a consequence of their anatomy and the conditions of their isolation, the isolated secretory cells are non-specifically permeable to low-molecular-weight water-soluble metabolites. Thus, the cytoplasm is readily accessible to the exogenous buffer whereas the selective permeability of subcellular organelles is maintained. With the appropriate choice of exogenous substrates, this feature allows the assessment of cytoplasmic and organellar (e.g. plastidic) metabolism in situ. Glycolytic substrates such as [14C]glucose-6-phosphate and [14C]pyruvic acid are incorporated into both monoterpenes and sesquiterpenes with a monoterpene:sesquiterpene ratio that closely mimics that observed in vivo, indicating that the correct subcellular partitioning of these substrates is maintained in this model system. Additionally, exogenous [14C]mevalonic acid and [14C]IPP, which are both intitially metabolized in the cytoplasm, produce an abnormally high proportion of sesquiterpenes. In contrast, incubation with either [14C]citrate or [14C]acetyl-CoA results in the accumulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) with no detectable isoprenoids formed. Taken together, these results indicate that the cytoplasmic mevalonic acid pathway is blocked at HMG-CoA reductase and that the IPP utilized for both monoterpene and sesquiterpene biosynthesis is synthesized exclusively in the plastids.

Light inactivation of functional photosystem II in leaves of peas grown in moderate light depends on photon exposure

Abstract: To determine the dependence of in vivo photosystem (PS) II function on photon exposure and to assign the relative importance of some photoprotective strategies of PSII against excess light, the maximal photochemical efficiency of PSII (Fv/Fm) and the content of functional PSII complexes (measured by repetitive flash yield of oxygen evolution) were determined in leaves of pea (Pisum satlvum L.) grown in moderate light. The modulation of PSII functionality in vivo was induced by varying either the duration (from 0 to 3 h) of light treatment (fixed at 1200 or 1800 μmol photons · m-2 · s-1) or irradiance (from 0 to 3000 μmol photons · m-2 · s-1) at a fixed duration (1 h) after infiltration of leaves with water (control), lincomycin (an inhibitor of chloroplast-encoded protein synthesis), nigericin (an uncoupler), or dithiothreitol (an inhibitor of the xanthophyll cycle) through the cut petioles of leaves of 22 to 24-day-old plants. We observed a reciprocity of irradiance and duration of illumination for PSII function, demonstrating that inactivation of functional PSII depends on the total number of photons absorbed, not on the rate of photon absorption. The Fv/Fm ratios from photoinhibitory light-treated leaves, with or without inhibitors, declined pseudo-linearly with photon exposure. The number of functional PSII complexes declined multiphasically with increasing photon exposure, in the following decreasing order of inhibitor effect: lincomycin > nigericin > DTT, indicating the central role of D1 protein turnover. While functional PSII and Fv/Fm ratio showed a linear relationship under high photon exposure conditions, in inhibitor-treated leaves the Fv/Fm ratio failed to reveal the loss of up to 25% of the total functional PSII under low photon exposure. The loss of this 25% of less-stable functional PSII was accompanied by a decrease of excitation-energy trapping capacity at the reaction centre of PSII (revealed by the fluorescence parameter, 1/Fo-1/Fm, where Fo and Fm stand for chlorophyll fluorescence when PSII reaction centres are open and closed, respectively), but not by a loss of excitation energy at the antenna (revealed by the fluorescence parameter, 1/Fm). We conclude that (i) PSII is an intrinsic photon counter under photoinhibitory conditions, (ii) PSII functionality is mainly regulated by D1 protein turnover, and to a lesser extent, by events mediated via the transthylakoid pH gradient, and (iii) peas exhibit PSII heterogeneity in terms of functional stability during photon exposure.

Does photorespiration protect the photosynthetic apparatus in French bean leaves from photoinhibition during drought stress

Abstract: Ten days after withholding water from bean (Phaseolus vulgaris L. cv. Contender) plants net photosynthetic CO2 uptake by leaves declined and no net CO2 uptake occurred after 15d. A similar decline in stomatal conductance also occurred over this 15-d period. Leaf relative water content and tugor pressure remained very similar to that measured on control plants during most of the experimental period. The decline of net CO2 uptake by leaves during dehydration is attributed to stomatal closure since CO2-dependent O2 evolution measured on the same leaves at saturating light and CO2 concentration always remained identical to that of control plants. Dehydration of the leaves had no effect on the quantum yield of CO2-dependent O2 evolution during the experimental period. Leaves from dehydrating plants maintained in normal (21% O2 + 350 μmol·mol-1 CO2) air exhibited a substantial rate of photosynthetic activity (about 38% of that measured at saturation light and CO2 concentration) associated with O2 reduction. Decreasing the oxygen content of the air from 21% to 2% in order to inhibit photorespiration produced a decrease in the linear electron transport rate by ca. 65% However, inhibiting photosynthetic O2 reduction during high-light treatment did not increase the extent of photoinhibition of photosystem II photochemistry and did not change the amount of violaxanthin converted to zeaxanthin. It is concluded that photorespiration does not protect the photosynthetic apparatus against high-light damage during drought conditions. Thermal deactivation of energy in the photosystem II antennae appears to be the main protective mechanism against deleterious effects of high light.

Identification of multiple isoforms of soluble and granule-bound starch synthase in developing wheat endosperm

Abstract: We have investigated the nature and locations of isoforms of starch synthase in the developing endosperm of wheat (Triticum aestivum L.). There are three distinct granule-bound isoforms of 60 kDa (the Waxy gene product), 77 kDa and 100–105 kDa. One of these isoforms, the 77-kDa protein, is also present in the soluble fraction of the endosperm but it contributes only a small proportion of the total soluble activity. Most of the soluble activity is contributed by isoforms which are apparently not also granule-bound. The 60-kDa and 77kDa isoforms of wheat are antigenically related to isoforms of very similar size in the developing pea embryo, but the other isoforms in the endosperm appear to have no counterparts in the pea embryo. The significance of these results in terms of the diversity of isoforms of starch synthase and their locations is discussed.

Biosynthesis and metabolism of abscisic acid in tomato leaves infected with Botrytis cinerea

Abstract: Two virulent strains ofBotrytis cinerea Pers., one of them (Bc 6) producing abscisic acid (ABA) via 1′,4′-trans-diol-ABA in defined liquid culture, and a second strain (Bc 9) without the ability to form ABA or its fungal precursor, and two near-isogenic lines of tomato were used to study the biosynthesis and metabolism of ABA in infected isolated leaves. The tomato plants used wereLycopersicon esculentum Mill. cv. Ailsa Craig (wild type) and the ABA-deficient mutantflacca. The level of 1′,4′-trans-diol-ABA increased in Ailsa Craig andflacca leaves in a similar pattern to about 4 μg·(gDW)−1 after conidiospore infection with Bc 6, but not after infection with Bc 9. Pulse-feeding experiments showed that [214-C]-1′,4′-trans-diol-ABA was metabolised to ABA and to further plant metabolites of ABA (phaseic acid, dihydrophaseic acid and polar compounds) in both uninfected and infected leaves. Following infection, the turnover of 1′,4′-trans-diol-ABA was reduced. The level of endogenous ABA in leaves infected with the ABA-producing strain Bc 6 rose more than tenfold in Ailsa Craig and twofold inflacca, respectively. Infection of Ailsa Craig leaves with Bc 9 caused a fivefold increase in ABA, and no increase of ABA inflacca. It is concluded that at least four processes control the level of ABA in wild-type tomato leaves infected withBotrytis cinerea: stimulation of fungal ABA biosynthesis by the host; release of ABA or its precursor by the fungus; stimulation of biosynthesis of plant ABA by the fungus; inhibition of its metabolism by the fungus. Application of ABA together with fungal spores to tomato leaves caused a faster development of necrotic leaf area than spore inoculation only.

Altered photosynthesis, flowering, and fruiting in transgenic tomato plants that have an increased capacity for sucrose synthesis

Abstract: Photosynthesis, leaf assimilate partitioning, flowering, and fruiting were examined in two lines of Lycopersicon esculentum Mill. transformed with a gene coding for sucrose-phosphate synthase (SPS) (EC 2.3.1.14) from Zea mays L. expressed from a tobacco ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit promoter. Plants were grown at either 35 or 65 Pa CO2 and high light (1000 μmol photons·m−2·s−1). Limiting and maximum SPS activities were significantly greater (up to 12 times) in the leaves of SPS-transformed lines for all treatments. Partitioning of carbon into sucrose increased 50% for the SPS transformants. Intact leaves of the control lines exhibited CO2-insensitivity of photosynthesis at high CO2 levels, whereas the SPS transformants did not exhibit CO2-insensitivity. The O2-sensitivity of photosynthesis was also greater for the SPS-transformed lines compared to the untransformed control when measured at 65 Pa CO2. These data indicate that the SPS transformants had a reduced limitation on photosynthesis imposed by end-product synthesis. Growth at 65 Pa CO2 resulted in reduced photosynthetic capacity for control lines but not for SPS-transformed lines. When grown at 65 Pa CO2, SPS transformed lines had a 20% greater photosynthetic rate than controls when measured at 65 Pa CO2 and a 35% greater rate when measured at 105 Pa CO2. Photosynthetic rates were not different between lines when grown at 35 Pa CO2. The time to 50% blossoming was reduced and the total number of inflorescences was significantly greater for the SPS transformants when grown at either 35 or 65 Pa CO2. At 35 Pa CO2, the total fruit number of the SPS transformants was up to 1.5 times that of the controls, the fruit matured earlier, and there was up to a 32% increase in total fruit dry weight. Fruit yield was not significantly different between the lines when grown at 65 Pa CO2. Therefore, there was not a strict relationship between yield and leaf photosynthesis rate. Flowering and fruit development of the SPS-transformed lines grown at 35 Pa CO2 showed similar trends to the controls grown at 65 Pa CO2. Incidences of blossom-end rot were also reduced in the SPS-transformed lines. These data indicate that altering starch/sucrose partitioning by increasing the capacity for sucrose synthesis can affect acclimation to elevated CO2 partial pressure and flowering and fruiting in tomato.

Contrasting roles in ion transport of two K(+)-channel types in root cells of Arabidopsis thaliana

Abstract: Plant roots accumulate K+ over a range of external concentrations. Root cells have evolved at least two parallel plasma-membrane K+ transporters which operate at millimolar and micromolar external [K+]: high-affinity K+ uptake is energised by symport with H+, while low-affinity uptake is assumed to occur via ion channels. To determine the role of ion channels in low-affinity K+ uptake, a characterisation of the principal K+-selective ion channels in the plasma membrane of Arabidopsis thaliana (L.) Heynh. cv. Columbia roots was undertaken. Two classes of K+-selective channels were frequently observed: one inward (IRC) and one outward (ORC) rectifying with unitary conductances of 5 pS, 20 pS (IRCs) and 15 pS (ORC), measured in symmetrical 10 mM KCl. The dominant IRC (5 pS) and ORC (15 pS) were highly cation-selective (PCl PK < 0.025) but less selective amongst monovalent cations (PNa∶PK≈0.17–0.3). Both the IRC and the ORC were blocked by Ba2+, Cs+ and tetra-ethyl-ammonium, whereas 4-aminopyridine and quinidine selectively inhibited the ORC. The ORC open probability was steeply voltage-dependent and ORC activation potentials were close to the potassium equilibrium potential (EK+), enabling ORCs to conduct mainly outward, but occasionally inward, K+ current. By contrast, gating of the 5-pS IRC was weakly voltageependent and IRC gating was invariably restricted to membrane potentials more negative than EK+, ensuring K+ transport was always inwardly directed. Studies on channel activity were conducted for a large number of root cells grown at two levels of external [K+], one where K+ uptake is likely to be principally through channels (6 mM K+) and one where it must be energised (100 μM K+). Shifting growth conditions from high to low K+ did not affect single-channel properties such as conductance and selectivity, nor the manifestation of the ORC and 20-pS IRC, but led to enhanced activity of the 5-pS IRC. The enhanced activity of the 5-pS IRC was mirrored by a parallel increase in unidirectional 86Rb+ influx after low-K+ growth, clearly indicating a dominant role of this particular channel in K+ uptake at supra millimolar external [K+].

Characterization of a voltage-dependent Ca2+-selective channel from wheat roots

Abstract: A new mechanism for calcium flux in wheat (Triticum aestivum L.) root cells has been characterized. Membrane vesicles were enriched in plasma membrane using aqueous-polymer two-phase partitioning and incorporated into artificial lipid bilayers, allowing characterization of single channels under voltage-clamp conditions. Membrane marker activities showed 74% and 83% purity in plasma membrane when expressed in terms of membrane area and activity, respectively. Since membrane vesicles obtained by aqueous-polymer two-phase partitioning yield a population of membrane vesicles of regular orientation, and vesicle fusion into planar lipid bilayers occurs in a defined manner, the orientation of the channel upon vesicle incorporation could be determined. Thus ionic activities and potentials could be controlled appropriately on what we propose to be the cytosolic (trans) and extracellular (cis) faces of the channel. The unitary conductance in symmetrical 1 mM CaCl2 was 27±0.4 (pS). The correlation between the theoretical and observed reversal potentials in asymmetrical conditions showed that the channel was highly selective for Ca2+ over Cl−. Experiments simulating physiological ionic conditions showed a PCa 2+/PK + of 17–26, decreasing in this range as the extracellular CaCl2 concentration increased from 0.1 to 1 mM. The channel was also permeable to the essential nutrient ions, Mg2+ and Mn2+. The open probability of the channel was strongly dependent on the membrane potential. Inactivation with time was observed at more negative membrane potentials, and was immediately reversed as soon as the membrane potential was decreased. At membrane potentials more negative than -130mV, the channel remained mainly in the closed state, suggesting that in vivo the channel would remain largely closed and would open only upon membrane depolarization. The channel was blocked by micromolar concentrations of extracellular verapamil and trivalent cations, Al3+ being the most effective of those tested. Exposure of the cytosolic and extracellular sides of the channel to inositol 1,4,5-trisphosphate had no effect on the channel activity. We suggest a plasma-membrane origin for the channel as shown by biochemical and electrophysiological evidence, and discuss possible physiological roles of this channel, both in Ca2+ uptake into roots and in signal transduction.

Protons and calcium modulate SV-type channels in the vacuolar-lysosomal compartment - channel interaction with calmodulin inhibitors

Abstract: Slowly activating vacuolar (SV-type; Hedrich and Neher 1987, Nature 329: 833–835) ion channels provide the predominant membrane conductance of the vacuolar-lysosomal compartment of Vicia faba L. guard cells and sugar beet (Beta vulgaris L.) taproots. Applying the patch-clamp technique to isolated vacuoles of both tissues, the electrical and pharmacological properties of guard-cell SV-type currents were studied and compared to the sugar beet channel with regard to its modulation by cytoplasmic Ca2+ and pH. This outward rectifier of V. faba guard cells showed a half-maximum activation at 55–60 mV with an apparent gating charge equivalent of z ≈ 4. Studies on the single-channel and whole-vacuole level revealed an extremely high conductance of 280 pS for the guard-cell channels at a mean density of 0.37 μm-2 compared to taproots (120–140 pS at about 0.16 channels per μm2). Guard-cell SV-type channels are weakly selective for cations over anions and lack saturation at KC1 concentrations of up to 1 M. Since in the absence of physiological K+ concentrations, Ca2+ is the major permeable ion, relative changes in the amounts of the two ions might control the permeation process. In spite of their different origins and physiological functions, in guard cells and beet taproot cells, cytoplasmic Ca2+ and protons, both considered as candidates for intracellular signalling in plants, modulate the voltage dependence of SV-type channels. While the two effectors do not alter the single-channel conductance, they strongly interact with the voltage sensor. The calmodulin (CaM) antagonists N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide hydrochloride (W-7), trifluoperazine (TFP) and calmidazolium hydrochloride (R 24571) effectively blocked the channel in an antagonist-specific manner. In agreement with the properties of a Ca2+-permeable channel, CaM could be involved in the modulation of the activation threshold of the SV-type channel. We therefore conclude that guard-cell SV-type channels, which might be responsible for the release of K+, Cl- and to a smaller extent Ca2+ during stomatal closure, could serve as an intracellular sensor for changes in cytosolic calcium (calcium-CaM) and pH.

An epitope of rice threonine- and hydroxyproline-rich glycoprotein is common to cell wall and hydrophobic plasma-membrane glycoproteins.

Abstract: A monoclonal antibody, LM1, has been derived that has a high affinity for an epitope of hydroxyproline-rich glycoproteins (HRGPs). In suspension-cultured rice (Oryza sativa L.) cells the epitope is carried by three major proteins with different biochemical properties. The most abundant is the 95-kDa extracellular rice extensin, a threonine- and hydroxyproline-rich glycoprotein (THRGP) occurring in the cell wall and secreted into the medium. This THRGP can be selectively oxidatively cross-linked in the presence of hydrogen peroxide and an endogenous peroxidase with the result that it does not enter a protein gel. A second polypeptide with the LM1 epitope (180 kDa), also occurring in the suspension-cultured cells and medium, is not oxidatively cross-linked. Three further polypeptides (52, 65 and 110 kDa) with the characteristics of hydrophobic proteins of the plasma-membrane also carry the LM1 epitope as determined by immuno-blotting of detergent/aqueous partitions of a plasma-membrane preparation and immuno-fluorescence studies with rice protoplasts. At the rice root apex the LM1 epitope is carried by four glycoproteins and is developmentally regulated. The major locations of the epitope are at the surface of cells associated with the developing protoxylem and metaxylem in the stele, the longitudinal radial walls of epidermal cells and a sheath-like structure at the surface of the root apex.

Specific reduction of chloroplast glyceraldehyde-3-phosphate dehydrogenase activity by antisense RNA reduces CO2 assimilation via a reduction in ribulose bisphosphate regeneration in transgenic tobacco plants

Abstract: The reduction of 3-phosphoglycerate (PGA) to triose phosphate is a key step in photosynthesis linking the photochemical events of the thylakoid membranes with the carbon metabolism of the photosynthetic carbon-reduction (PCR) cycle in the stroma. Glyceraldehyde-3-phosphate dehydrogenase: NADP oxidoreductase (GAPDH) is one of the two chloroplast enzymes which catalyse this reversible conversion. We report on the engineering of an antisense RNA construct directed against the tobacco (Nicotiana tabacum L.) chloroplastlocated GAPDH (A subunit). The construct was integrated into the tobacco genome by Agrobacterium-mediated transformation of leaf discs. Of the resulting transformants, five plants were recovered with reduced GAPDH activities ranging from 11 to 24% of wild-type (WT) activities. Segregation analysis of the kanamycin-resistance character in self-pollinated T1 seed from each of the five transformants revealed that one plant (GAP-R) had two active DNA inserts and the others had one insert. T1 progeny from GAP-R was used to generate plants with GAPDH activities ranging from WT levels to around 7% of WT levels. These were used to study the effect of variable GAPDH activities on metabolite pools for ribulose1,5-bisphosphate (RuBP) and PGA, and the accompanying effects on the rate of CO2 assimilation and other gasexchange parameters. The RuBP pool size was linearly related to GAPDH activity once GAPDH activity dropped below the range for WT plants, but the rate of CO2 assimilation was not affected until RuBP levels dropped to 30–40% of WT levels. That is, the CO2 assimilation rate fell when RuBP per ribulose-1,5-biphosphate carboxylase-oxygenase (Rubisco) site fell below 2 mol·(mol site)−1 while the ratio for WT plants was 4–5 mol·m(mol site)−1. Leaf conductance was not reduced in leaves with reduced GAPDH activities, resulting in an increase in the ratio of intercellular to ambient CO2 partial pressure. Conductance in plants with reduced GAPDH activities was still sensitive to CO2 and showed a normal decline with increases in CO2 partial pressure. Although PGA levels did not fluctuate greatly, the effect of reduced GAPDH activity on RuBP-pool size and assimilation rate can be interpreted as being due to a blockage in the regeneration of RuBP. Concomitant gas-ex change and chlorophyll a fluorescence measurements indicated that photosynthesis changed from being Rubisco-limited to being RuBP-regeneration-limited at a lower CO2 partial pressure in the antisense plants than in WT plants. Photosynthetic electron transport was down-regulated by the build-up of a large proton gradient and the electron-transport chain did not become over-reduced due to a shortage of NADP. Plants with severely reduced GAPDH activity were not photoinhibited despite the continuous presence of a large thylakoid proton gradient in the light. Along with plant size, Rubisco activity, leaf soluble protein and chlorophyll content were reduced in plants with the lowest GAPDH activities. We conclude that chloroplastic GAPDH activity does not appear to limit steady-state photosynthetic CO2 assimilation at ambient CO2. This is because WT leaves maintain the ratio of RuBP per Rubisco site about twofold higher than the level required to achieve a maximal rate of CO2 assimilation.

Modulation of germination of embryos isolated from dormant and nondormant barley grains by manipulation of endogenous abscisic acid

Abstract: Dormant and non-dormant barley (Hordeum distichum L.) grains with identical genetic backgrounds were obtained by maturing grains under different climate conditions. When isolated embryos from dormant grains were incubated in a well containing a fixed volume of water (300 μl), the germination rate and percentage were dependent on the embryo number per well. A higher embryo number per well was correlated with a lower germination rate and percentage. However, this was not the case for the embryos isolated from nondormant grains. During germination, the endogenous cis-abscisic acid (ABA) in isolated embryos from both dormant and nondormant grains was analyzed. The inhibitory effect on germination of a higher number per well of isolated dormant embryos was due to diffusion of endogenous ABA out of the embryos and accumulation of ABA in the incubation medium. Moreover, there was de-novo synthesis of ABA in embryos isolated from dormant grains during incubation but not in embryos isolated from nondormant grains. The inhibitory effect of ABA on germination of embryos isolated from dormant grains could be mimicked by addition of ABA or the medium in which dormant embryos had been placed. Embryos isolated from nondormant grains were insensitive to addition of ABA and medium from dormant embryos. Our results demonstrate that diffusion of endogenous ABA, de-novo ABA synthesis and ABA sensitivity play a role in the control of germination. It is proposed that dormancy-breaking treatments act via changes to these processes.

Assimilation images of leaves of Glechoma hederacea: Analysis of non-synchronous stomata related oscillations

Abstract: An experimental system is described to analyze the spacial distribution of leaf assimilation from chlorophyll-a fluorescence images. Fluorescence parameters were determined with a digitized camera system pixel-by-pixel and calibrated against the assimilation rate from integrated gas exchange. The approach is an extension of the work by P.F. Daley, et al. (1989, Plant Physiol. 90, 1233–1238) and allows the visualisation of assimilation images of leaves with high resolution. With a leaf of Glechoma hederacea L., assimilation images were taken at short time intervals during stomata-related oscillations of the assimilation rate (period about 13 min) over a time period of more than 3 h. Mathematical pixel-by-pixel analysis of images shows nearly Gaussian distribution of assimilation values around a minimum (5 μmol CO2 · m−2 · s−1) and maximum (13 μmol CO2 · m−2 · s−1) peak value during oscillations. Synchronous oscillations, initiated by rapid changes in gas composition, changed into non-synchronous oscillations, due to slight local variations in the period. As a consequence, the oscillation of gas exchange (H2O and CO2) died out whilst leaf assimilation continued to oscillate non-synchronously and patchily. Patchy distribution of frequencies largely followed gas-exchange compartmentation (‘alveoli’), whilst non-homogeneous distribution of the amplitude was on a larger scale, with a decrease in amplitudes from the leaf base towards distal areas. The data are discussed considering the framework of physiological processes involved in stomatal control.

Arabinogalactan-protein epitopes in somatic embryogenesis of Daucus carota L.

Abstract: Two monoclonal antibodies (ZUM 15 and ZUM 18) directed against carrot (Daucus carota L.) seed arabinogalactan proteins (AGPs) were used to isolate specific AGP fractions. For both carrot and tomato (Lycopersicon esculentum Mill.) seed AGPs analyzed by crossedelectrophoresis, the ZUM 15 and ZUM 18 AGP fractions showed one identical peak. However, the Rf values for the two species were different: 0.82 for carrot seed AGPs and 0.52 for tomato seed AGPs. When the fractionated AGPs (carrot or tomato) were added to carrot cell lines they had a dramatic effect on the culture. One AGP fraction (ZUM 15 AGPs) was able to induce vacuolation of embryogenic cells. Those cells failed to produce embryos. The other AGP fraction (ZUM 18 AGPs) increased the percentage of embryognic cells from about 40% up to 80% within one week and this subsequently resulted in the formation of more embryos on hormone-free medium. This activity was higher than that of unfractionated carrot seed AGPs, while the optimum concentration was 50-fold lower. Since both ZUM 18 AGPs (carrot or tomato) yielded identical responses it can be concluded that neither the Rf value nor the source are essential for biological activity. The dose-response curve of ZUM 18 AGPs showed a sharp optimum. When the AGPs that also bound to the antibody ZUM 15 were removed, the dose-response curve of the remaining AGPs (containing only the ZUM 18 epitope, not the ZUM 15 epitope) resembled a saturation curve. Regardless of its concentration, the fraction in which AGP molecules contained both epitopes showed no appreciable embryogenesis-promoting activity. The biological activity of AGPs was therefore determined by the presence of embryogenesis-enhancing and-inhibiting epitopes. The inhibiting and enhancing epitopes can be located on separate molecules or one single AGP molecule.

Autoradiographic and biochemical evidence for the systemic translocation of systemin in tomato plants

Abstract: The movement of systemin, the 18-amino-acid polypeptide inducer of proteinase inhibitors in tomato (Lycopersicon esculentum L.) plants, was investigated in young tomato plants following the application of [14C]systemin to wounds on the surface of leaves. Wholeleaf autoradiographic analyses revealed that [14C]systemin was distributed throughout the wounded leaf within 30 min, and then during the next several hours was transported to the petiole, to the main stem, and to the upper leaves. The movement of [14C]systemin was similar to the movement of [14C]sucrose when applied to leaf wounds, except that sucrose was slightly more mobile than systemin. Analyses of the radioactivity in the petiole phloem exudates at intervals over a 5-h period following the application of [14C]systemin to a wound demonstrated that intact [14C]systemin was present in the phloem over the entire time, indicating that the polypeptide was either stable for long periods in the phloem or was being continually loaded into the phloem from the source leaf. The translocation pathway of systemin was also investigated at the cellular level, using light microscopy and autoradiography. Within 15 min after application of [3H]systemin to a wound on a terminal leaflet, it was found distributed throughout the wounded leaf and was primarily concentrated in the xylem and phloem tissues within the leaf veins. After 30 min, the radioactivity was found mainly associated with vascular strands of phloem tissue in the petiole and, at 90 min, label was found in the phloem of the main stem. Altogether, these and previous results support a role for systemin as a systemic wound signal in tomato plants.

Effect of water deficit on photosynthetic oxygen exchange measured using 18O2 and mass spectrometry in Solanum tuberosum L. leaf discs

Abstract: The effect of leaf dehydration on photosynthetic O2 exchange of potato (Solanum tuberosum L., cv. Haig) leaf discs was examined using 18O2 as a tracer and mass spectrometry. In normal air (350 μl·l−1CO2) and under an irradiance of 390 μmol photons·m−2·s1, a relative water deficit (RWD) of about 30% severely decreased net O2 evolution and increased O2 uptake by about 50%, thus indicating an enhancement of photorespiration. Increasing CO2 concentrations diminished O2 uptake and stimulated net O2 evolution both in well-hydrated and in dehydrated (RWD of about 30%) leaves. Much higher CO2 concentrations (up to 4%) were required to observe a complete effect of CO2 in dehydrated leaves. The chloroplastic CO2 concentration at the ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) level (Cc) was calculated from O2-exchange data in both well-hydrated and dehydrated leaves, assuming that the specificity factor of Rubisco was unaffected by desiccation. When plotting net O2 photosynthesis as a function of Cc, a similar relationship was obtained for well-hydrated and waterstressed leaf discs, thus showing that the main effect of water deficit is a decrease of the chloroplastic CO2 concentration. At saturating CO2 levels, the non-cyclic electron-transport rate, measured either as gross O2 photosynthesis or as the chlorophyll fluorescence ratio (Fm -Fs)/Fm, was insensitive to water deficit, provided RWD was below 40%. In this range of RWD, the decrease in gross O2 photosynthesis observed in normal air was attributed to the inability of oxidative processes to sustain the maximal electron-flow rate at low chloroplastic CO2 concentration. The maximal efficiency of photosystem II, estimated as the chlorophyll fluorescence ratio (Fm -F0)/Fm measured in dark-adapted leaves, was not affected by water deficits up to 60%.

Whole -root iron(III)-reductase activity throughout the life cycle of iron-grown Pisum sativum L. (Fabaceae): relevance to the iron nutrition of developing seeds

Abstract: To understand the whole-plant processes which influence the Fe nutrition of developing seeds, we have characterized root Fe(III)-reductase activity and quantified whole-plant Fe balance throughout the complete 10-week (10-wk) life cycle of pea (Pisum sativum L., cv. Sparkle). Plants were grown hydroponically in complete nutrient solution with a continuous supply of chelated Fe; all side shoots were removed at first appearance to yield plants with one main shoot. Root Fe(III)-reductase activity was assayed with Fe(III)-EDTA. Flowering of the experimental plants began on wk 4 and continued until wk 6; seed growth and active seed import occurred during wks 5–10. Vegetative growth terminated at wk 6. Iron(III) reduction in whole-root systems was found to be dynamically modulated throughout the plant's life cycle, even though the plants were maintained on an Fe source. Iron(III)-reductase activity ranged from 1–3 μmol Fe reduced · g −1 DW · h−1 at early and late stages of the life cycle to 9.5 μmol Fe reduced · g−1 DW · h−1 at wk 6. Visual assays demonstrated that Fe(III)-reductase activity was localized to extensive regions of secondary and tertiary lateral roots during this peak activity. At midstages of growth (wks 6–7), root Fe(III)-reductase activity could be altered by changes in internal shoot Fe demand or external root Fe supply: removal of all pods or interruption of phloem transport from the reproductive portion of the shoot (to the roots) resulted in lowered root Fe(III)-reductase activity, while removal of Fe from the nutrient solution resulted in a stimulation of this activity. Total shoot Fe content increased throughout the 10-wk growth period, with Fe content in the non-seed tissues of the shoot declining by 50% of their maximal level and accounting for 35% of final seed Fe content. At maturity, total seed Fe represented 74% of total shoot Fe; total Fe in the roots (apoplasmic and symplasmic Fe combined) was minimal. These studies demonstrate that the root Fe(III)-reductase system responds to Fe status and/or Fe requirements of the shoot, apparently through shoot-to-root communication involving a phloem-mobile signal. During active seed-fill, enhanced root Fe(III)-reductase activity is necessary to generate sufficient Fe2+ for continued root Fe acquisition. This continuing Fe supply to the shoot is essential for the developing seeds to attain their Fe-content potential. Increased rates of root Fe(III) reduction would be necessary for seed Fe content to be enhanced in Pisum sativum.

Cell-type specific, coordinate expression of two ADP-glucose pyrophosphorylase genes in relation to starch biosynthesis during seed development of Vicia faba L.

Abstract: Several cDNA clones encoding two different ADP-glucose pyrophosphorylase (AGPase, EC 2.7.7.27) polypeptides denoted VfAGPC and VfAGPP were isolated from a cotyledonary library of Vicia faba L. Both sequences are closely related to AGPase small-subunit sequences from other plants. Whereas mRNA levels of VfAGPP were equally high in developing cotyledons and leaves, the mRNA of VfAGPC was present in considerable amounts only in cotyledons. During development of cotyledons, both mRNAs accumulated until the beginning of the desiccation phase and disappeared afterwards. The increase of AGPase activity in cotyledons during the phase of storage-product synthesis was closely followed by the accumulation of starch. The AGPase activity in crude extracts of cotyledons was insensitive to 3-phosphoglycerate whereas the activity from leaves could be activated more than five-fold. Inorganic phosphate inhibited the enzyme from both tissues but was slightly more effective on the leaf enzyme. There was a correlation at the cellular level between the distribution of VfAGPP and VfAGPC mRNAs and the accumulation of starch, as studied by in-situ hybridisation and by histochemical staining in parallel tissue sections of developing seeds, respectively. During the early phase of seed development (12–15 days after fertilization) VfAGPase mRNA and accumulation of starch were detected transiently in the hypodermal, chlorenchymal and outer parenchymal cell layers of the seed coat but not in the embryo. At 25 days after fertilization both synthesis of VfAGPase mRNA and biosynthesis of starch had started in parenchyma cells of the inner adaxial zone of the cotyledons. During later stages, the expression of VfAGPase and synthesis of starch extended over most of the cotyledons but were absent from peripheral cells of the abaxial zone, provascular and procalyptral cells.

The effect of auxin on cytokinin levels and metabolism in transgenic tobacco tissue expressing an ipt gene

Abstract: The ipt gene from the T-DNA of Agrobacterium tumefaciens was transferred to tobacco (Nicotiana tabacum L.) in order to study the control which auxin appears to exert over levels of cytokinin generated by expression of this gene. The transgenic tissues contained elevated levels of cytokinins, exhibited cytokinin and auxin autonomy and grew as shooty calli on hormone-free media. Addition of 1-naphthylacetic acid to this culture medium reduced the total level of cytokinins by 84% while 6-benzylaminopurine elevated the cytokinin level when added to media containing auxin. The cytokinins in the transgenic tissue were labelled with 3H and auxin was found to promote conversion of zeatin-type cytokinins to 3H-labelled adenine derivatives. When the very rapid metabolism of exogenous [3H]zeatin riboside was suppressed by a phenylurea derivative, a noncompetitive inhibitor of cytokinin oxidase, auxin promoted metabolism to adenine-type compounds. Since these results indicated that auxin promoted cytokinin oxidase activity in the transformed tissue, this enzyme was purified from the tobacco tissue cultures. Auxin did not increase the level of the enzyme per unit tissue protein, but did enhance the activity of the enzyme in vitro and promoted the activity of both glycosylated and non-glycosylated forms. This enhancement could contribute to the decrease in cytokinin level induced by auxin. Studies of cytokinin biosynthesis in the transgenic tissues indicated that trans-hydroxylation of isopentenyladenine-type cytokinins to yield zeatin-type cytokinins occurred principally at the nucleotide level.