Showing papers in "Planta in 2000"

Cellular compartmentation of cadmium and zinc in relation to other elements in the hyperaccumulator Arabidopsis halleri.

Abstract: The cellular compartmentation of elements was analysed in the Zn hyperaccumulator Arabidopsis halleri (L.) O'Kane & Al-Shehbaz (=Cardaminopsis halleri) using energy-dispersive X-ray microanalysis of frozen-hydrated tissues. Quantitative data were obtained using oxygen as an internal standard in the analyses of vacuoles, whereas a peak/background ratio method was used for quantification of elements in pollen and dehydrated trichomes. Arabidopsis halleri was found to hyperaccumulate not only Zn but also Cd in the shoot biomass. While large concentrations of Zn and Cd were found in the leaves and roots, flowers contained very little. In roots grown hydroponically, Zn and Cd accumulated in the cell wall of the rhizodermis (root epidermis), mainly due to precipitation of Zn/Cd phosphates. In leaves, the trichomes had by far the largest concentrations of Zn and Cd. Inside the trichomes there was a striking sub-cellular compartmentation, with almost all the Zn and Cd being accumulated in a narrow ring in the trichome base. This distribution pattern was very different from that for Ca and P. The epidermal cells other than trichomes were very small and contained lower concentrations of Zn and Cd than mesophyll cells. In particular, the concentrations of Cd and Zn in the mesophyll cells increased markedly in response to increasing Zn and Cd concentrations in the nutrient solution. This indicates that the mesophyll cells in the leaves of A. halleri are the major storage site for Zn and Cd, and play an important role in their hyperaccumulation.

Nitric oxide stimulates seed germination and de-etiolation, and inhibits hypocotyl elongation, three light-inducible responses in plants

Abstract: Seed germination, greening of etiolated plants and inhibition of hypocotyl elongation are stimulated by light, which is sensed by various types of photoreceptor. Nitric oxide (NO) has proven to be a bioactive molecule, especially in mammalian cells and, most recently, in plants. Like some phytochrome-dependent processes, many NO-mediated ones are accomplished through increases in cGMP levels. Given these similarities, we proposed that NO could take part in light-mediated events in plants. Here we show that NO promotes seed germination and de-etiolation, and inhibits hypocotyl and internode elongation, processes mediated by light. Two NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine induced germination of lettuce (Lactuca sativa L. cv. Grand Rapids) seeds in conditions in which this process is dependent on light (e.g. 26 °C). This was a dose-dependent response and was arrested by addition of an NO scavenger, carboxy-PTIO. In addition, nitrite and nitrate, two NO-decomposition products were ineffective in stimulating germination. Wheat seedlings sprayed with SNP and grown in darkness contained 30–40% more chlorophyll than control seedlings. Nitric-oxide-mediated partial greening was increased by light pulses, wounding and biotic stress. Arabidopsis thaliana (L.) Heynh. (ecotype Columbia) and lettuce seedlings grown in the dark had 20%-shorter hypocotyls in NO treatments than in control ones. On the other hand, internode lengths of potato plants growing under low light intensity and sprayed with 100 μM SNP were also 20% shorter than control ones. These results implicate NO as a stimulator molecule in plant photomorphogenesis, either dependent on or independent of plant photoreceptors.

Characterization of the sink/source transition in tobacco ( Nicotiana tabacum L.) shoots in relation to nitrogen management and leaf senescence.

Abstract: The metabolic, biochemical and molecular events occurring during tobacco (Nicotiana tabacum) leaf ageing are presented, with a particular emphasis on nitrogen metabolism. An integrated model describing the source/sink relationship existing between leaves of different developmental stages along the main plant axis is proposed. The results of our study show that a tobacco plant can be divided into two main sections with regards to sink/source relationships. Sink-to-source transition occurs at a particular leaf stage in which a breakpoint corresponding to an accumulation of carbohydrates and a depletion of both organic and inorganic nitrogen is observed. The sink/source transition is also marked by the appearence of endoproteolytic activities and the induction of both cytosolic glutamine synthetase and NAD(H)-dependent glutamate dehydrogenase transcripts, proteins and activities. The role of the newly induced enzymes and the nature of the potential metabolic and developmental signals involved in the regulation of their expression during leaf senescence are discussed.

Changes in carotenoids, tocopherols and diterpenes during drought and recovery, and the biological significance of chlorophyll loss in Rosmarinus officinalis plants.

Abstract: Two-year-old rosemary (Rosmarinus officinalis L.) plants were subjected to severe stress by exposure to prolonged drought during a Mediterranean summer. Severely stressed plants recovered completely after the autumn rainfalls although the relative water content remained below 35% for 3 months and the chlorophyll content of leaves was reduced by up to 85% during the drought. In severe stress: (i) α-tocopherol increased 9-fold per g dry weight and 20-fold per unit of chlorophyll; (ii) lutein and β-carotene contents decreased on a dry-weight basis, but an 80% increase in lutein and constant levels of β-carotene were observed on a chlorophyll basis; (iii) there were transient and sustained increases in the de-epoxidation state of the xanthophyll cycle; and (iv) the highly oxidised abietane diterpene isorosmanol increased 8-fold as a result of the oxidation of carnosic acid. With the autumn rainfalls, water status, α-tocopherol and violaxanthin recovered first and the levels of photosynthetic pigments and abietane diterpenes increased later. The photoprotection conferred by the xanthophyll cycle and the antioxidant function of tocopherols, lutein and diterpenes may help to avoid irreversible damage in severe drought, making possible the recovery of functional membranes after the autumn rainfalls. Besides, chlorophyll loss reduces the amount of photons absorbed by leaves, which enhances the photoprotective and antioxidant capacity of leaves per amount of photons absorbed, since the ratios of xanthophylls, α-tocopherol and abietane diterpenes to chlorophyll increase.

12-oxophytodienoate reductase 3 (OPR3) is the isoenzyme involved in jasmonate biosynthesis

Abstract: In addition to OPR1 and OPR2, two isoenzymes of 12-oxophytodienoate reductase, a third isoform (OPR3) has recently been identified in Arabidopsis thaliana (L) Heynh The expression of the OPR3 gene is induced not only by a variety of stimuli, such as touch, wind, wounding, UV-light and application of detergent, but also by brassinosteroids The three enzymes were expressed in a functional form in Escherichia coli, and OPR2 was additionally expressed in insect cell cultures and overexpressed in A thaliana Substrate conversion was analyzed using a stereospecific assay The results show that OPR3 effectively converts the natural (9S,13S)-12-oxophytodienoic acid [K m = 35 μM, V max 537 nkat (mg protein)−1] to the corresponding 3-2(2′(Z)-pentenyl) cyclopentane-1-octanoic acid (OPC-8:0) stereoisomer while OPR1 and OPR2 convert (9S,13S)-12-oxophytodienoic acid with greatly reduced efficiency compared to OPR3 Thus, OPR3 is the isoenzyme relevant for jasmonate biosynthesis

Herbivore-induced ethylene suppresses a direct defense but not a putative indirect defense against an adapted herbivore.

Abstract: Herbivory induces both direct and indirect defenses in plants; however, some combinations of these defenses may not be compatible. The jasmonate signal cascade activated both direct (nicotine accumulations) and indirect (mono- and sesquiterpene emissions) whole-plant defense responses in the native tobacco Nicotiana attenuata Torr. Ex Wats. Nicotine accumulations were proportional to the amount of leaf wounding and the resulting increases in jasmonic acid (JA) concentrations. However, when larvae of the nicotine-tolerant herbivore, Manduca sexta, fed on plants or their oral secretions were applied to leaf punctures, the normal wound response was dramatically altered, as evidenced by large (4- to 10-fold) increases in the release of (i) volatile terpenoids and (ii) ethylene, (iii) increased (4- to 30-fold) accumulations of endogenous JA pools, but (iv) decreased or unchanged nicotine accumulations. The ethylene release, which was insensitive to inhibitors of induced JA accumulation, was sufficient to account for the attenuated nicotine response. Applications of ethylene and ethephon suppressed the induced nicotine response and pre-treatment of plants with a competitive inhibitor of ethylene receptors, 1-methylcyclopropene, restored the full nicotine response. This ethylene burst, however, did not inhibit the release of volatile terpenoids. Because parasitoids of Manduca larvae are sensitive to the dietary intake of nicotine by their hosts, this ethylene-mediated switching from direct to a putative indirect defense may represent an adaptive tailoring of a plant's defense response.

Regulation of auxin transport by aminopeptidases and endogenous flavonoids.

Abstract: The 1-N-naphthylphthalamic acid (NPA)-binding protein is a putative negative regulator of polar auxin transport that has been shown to block auxin efflux from both whole plant tissues and microsomal membrane vesicles. We previously showed that NPA is hydrolyzed by plasma-membrane amidohydrolases that co-localize with tyrosine, proline, and tryptophan-specific aminopeptidases (APs) in the cotyledonary node, hypocotyl-root transition zone and root distal elongation zone of Arabidopsisthaliana (L.) Heynh. seedlings. Moreover, amino acyl-β-naphthylamide (aa-NA) conjugates resembling NPA in structure have NPA-like inhibitory activity on growth, suggesting a possible role of APs in NPA action. Here we report that the same aa-NA conjugates and the AP inhibitor bestatin also block auxin efflux from seedling tissue. Bestatin and, to a lesser extent, some aa-NA conjugates were more effective inhibitors of low-affinity specific [3H]NPA-binding than were the flavonoids quercetin and kaempferol but had no effect on high-affinity binding. Since the APs are inhibited by flavonoids, we compared the localization of endogenous flavonoids and APs in seedling tissue. A correlation between AP and flavonoid localization was found in 5- to 6-d-old seedlings. Evidence that these flavonoids regulate auxin accumulation in vivo was obtained using the flavonoid-deficient mutant, tt4. In whole-seedling [14C]indole-3-acetic acid transport studies, the pattern of auxin distribution in the tt4 mutant was shown to be altered. The defect appeared to be in auxin accumulation, as a considerable amount of auxin escaped from the roots. Treatment of the tt4 mutant with the missing intermediate naringenin restored normal auxin distribution and accumulation by the root. These results implicate APs and endogenous flavonoids in the regulation of auxin efflux.

Abscisic acid and hydraulic conductivity of maize roots: a study using cell- and root-pressure probes.

Abstract: Using root- and cell-pressure probes, the effects of the stress hormone abscisic acid (ABA) on the water-transport properties of maize roots (Zea mays L.) were examined in order to work out dose and time responses for root hydraulic conductivity. Abscisic acid applied at concentrations of 100–1,000 nM increased the hydraulic conductivity of excised maize roots both at the organ (root Lpr: factor of 3–4) and the root cell level (cell Lp: factor of 7–27). Effects on the root cortical cells were more pronounced than at the organ level. From the results it was concluded that ABA acts at the plasmalemma, presumably by an interaction with water channels. Abscisic acid therefore facilitated the cell-to-cell component of transport of water across the root cylinder. Effects on cell Lp were transient and highly specific for the undissociated (+)-cis-trans-ABA. The stress hormone ABA facilitates water uptake into roots as soils start drying, especially under non-transpiring conditions, when the apoplastic path of water transport is largely excluded.

Control of seed dormancy in Nicotiana plumbaginifolia: post-imbibition abscisic acid synthesis imposes dormancy maintenance

Abstract: The physiological characteristics of seed dormancy in Nicotiana plumbaginifolia Viv are described The level of seed dormancy is defined by the delay in seed germination (ie the time required prior to germination) under favourable environmental conditions A wild-type line shows a clear primary dormancy, which is suppressed by afterripening, whereas an abscisic acid (ABA)-deficient mutant shows a non-dormant phenotype We have investigated the role of ABA and gibberellic acid (GA(3)) in the control of dormancy maintenance or breakage during imbibition in suitable conditions It was found that fluridone, a carotenoid biosynthesis inhibitor, is almost as efficient as GA(3) in breaking dormancy Dry dormant seeds contained more ABA than dry afterripened seeds and, during early imbibition, there was an accumulation of ABA in dormant seeds, but not in afterripened seeds In addition, fluridone and exogenous GA(3) inhibited the accumulation of ABA in imbibed dormant seeds This reveals an important role for ABA synthesis in dormancy maintenance in imbibed seeds

The hypersensitive response is associated with host and nonhost resistance to Phytophthora infestans.

Abstract: The interaction between Phytophthora infestans (Mont.) de Bary and Solanum was examined cytologically using a diverse set of wild Solanum species and potato (S. tuberosum L.) cultivars with various levels of resistance to late blight. In wild Solanum species, in potato cultivars carrying known resistance (R) genes and in nonhosts the major defense reaction appeared to be the hypersensitive response (HR). In fully resistant Solanum species and nonhosts, the HR was fast and occurred within 22 h. This resulted in the death of one to three cells. In partially resistant clones, the HR was induced between 16 and 46 h, and resulted in HR lesions consisting of five or more dead cells, from which hyphae were occasionally able to escape to establish a biotrophic interaction. These results demonstrate the quantitative nature of the resistance to P. infestans. The effectiveness of the HR in restricting growth of the pathogen differed considerably between clones and correlated with resistance levels. Other responses associated with the defense reaction were deposition of callose and extracellular globules containing phenolic compounds. These globules were deposited near cells showing the HR, and may function in cell wall strengthening.

Fractionation of carbohydrates in Arabidopsis root cell walls shows that three radial swelling loci are specifically involved in cellulose production.

Abstract: Three non-allelic radial swelling mutants (rsw1, rsw2 and rsw3) of Arabidopsisthaliana L. Heynh. were shown to be specifically impaired in cellulose production. Fractionation methods that identify, characterise and quantify some of the major cell wall polysaccharides in small quantities of seedlings demonstrated that changes in the production of cellulose are much more pronounced than changes in the production of non-cellulosic polysaccharides. A crude cell wall pellet was sequentially extracted with chloroform methanol (to recover lipids), dimethyl sulphoxide (starch), ammonium oxalate (pectins) and alkali (hemicelluloses). Crystalline cellulose remained insoluble through subsequent treatments with an acetic/nitric acid mixture and with trifluoroacetic acid. Cetyltrimethylammonium bromide precipitation resolved neutral and acidic polymers in the fractions, and precipitation behaviour, monosaccharide composition and glycosidic linkage patterns identified the major polysaccharides. The deduced composition of the walls of wild-type seedlings and the structure and solubility properties of the major polymers were broadly typical of other dicots. The three temperature-sensitive, radial swelling mutants produced less cellulose in their roots than the wild type when grown at their restrictive temperature (31 °C). There were no significant differences at 21 °C where no radial swelling occurs. The limited changes seen in the monosaccharide compositions, glycosidic linkage patterns and quantities of non-cellulosic polysaccharides support the view that the RSW1, RSW2 and RSW3 genes are specifically involved in cellulose synthesis. Reduced deposition of cellulose was accompanied by increased accumulation of starch.

Minor vein structure and sugar transport in Arabidopsis thaliana.

Abstract: Leaf and minor vein structure were studied in Arabidopsis thaliana (L) Heynh to gain insight into the mechanism(s) of phloem loading Vein density (length of veins per unit leaf area) is extremely low Almost all veins are intimately associated with the mesophyll and are probably involved in loading In transverse sections of veins there are, on average, two companion cells for each sieve element Phloem parenchyma cells appear to be specialized for delivery of photoassimilate from the bundle sheath to sieve element-companion cell complexes: they make numerous contacts with the bundle sheath and with companion cells and they have transfer cell wall ingrowths where they are in contact with sieve elements Plasmodesmatal frequencies are high at interfaces involving phloem parenchyma cells The plasmodesmata between phloem parenchyma cells and companion cells are structurally distinct in that there are several branches on the phloem parenchyma cell side of the wall and only one branch on the companion cell side Most of the translocated sugar in A thaliana is sucrose, but raffinose is also transported Based on structural evidence, the most likely route of sucrose transport is from bundle sheath to phloem parenchyma cells through plasmodesmata, followed by efflux into the apoplasm across wall ingrowths and carrier-mediated uptake into the sieve element-companion cell complex

Form of aluminium for uptake and translocation in buckwheat (Fagopyrum esculentum Moench)

Abstract: The forms of Al for uptake by the roots and translocation from the root to the shoot were investigated in a buckwheat (Fagopyrum esculentum Moench, cv. Jianxi) that accumulates Al in its leaves. The Al concentration in the xylem sap was 15-fold higher in the plants exposed to AlCl3 than in those exposed to an Al-oxalate (1:3) complex, suggesting that the roots take up Al in the ionic form. The Al concentration in the xylem sap was 4-fold higher than that in the external solution after a 1-h exposure to AICl3 solution and 10-fold higher after a 2-h exposure. The Al concentration in the xylem sap increased with increasing Al concentration in the external solution. The Al uptake was not affected by a respiratory inhibitor, hydroxylamine, but significantly inhibited by the addition of La. These results suggest that Al uptake by the root is a passive process, and La3+ competes for the binding sites for Al3+ on the plasma membrane. The form of Al in the xylem sap was identified by 27Al-nuclear magnetic resonance analysis. The chemical shift of 27Al in the xylem sap was around 10.9 ppm, which is consistent with that of the Al-citrate complex. Furthermore, the dominant organic acid in the xylem sap was citric acid, indicating that Al was translocated in the form of Al-citrate complex. Because Al is present as Al-oxalate (1:3) in the root, the present data show that ligand exchange from oxalate to citrate occurs before Al is released to xylem.

Regulation and activation of phytoene synthase, a key enzyme in carotenoid biosynthesis, during photomorphogenesis

Abstract: During photomorphogenesis in higher plants, a coordinated increase occurs in the chlorophyll and carotenoid contents. The carotenoid level is under phytochrome control, as reflected by the light regulation of the mRNA level of phytoene synthase (PSY), the first enzyme in the carotenoid biosynthetic pathway. We investigated PSY protein levels, enzymatic activity and topological localization during photomorphogenesis. The results revealed that PSY protein levels and enzymatic activity increase during de-etiolation and that the enzyme is localized at thylakoid membranes in mature chloroplasts. However, under certain light conditions (e.g., far-red light) the increases in PSY mRNA and protein levels are not accompanied by an increase in enzymatic activity. Under those conditions, PSY is localized in the prolamellar body fraction in a mostly enzymatically inactive form. Subsequent illumination of dark-grown and/or in far-red light grown seedlings with white light causes the decay of these structures and a topological relocalization of PSY to developing thylakolds which results in its enzymatic activation. This light-dependent mechanism of enzymatic activation of PSY in carotenoid biosynthesis shares common features with the regulation of the NADPH:protochlorophyllide oxidoreductase, the first light-regulated enzyme in chlorophyll biosynthesis. The mechanism of regulation described here may contribute to ensuring a spatially and temporally coordinated increase in both carotenoid and chlorophyll contents.

Transgenic Arabidopsis plants can accumulate polyhydroxybutyrate to up to 4% of their fresh weight

Abstract: Transgenic Arabidopsis thaliana (L.) Heynh. plants expressing the three enzymes encoding the biosynthetic route to polyhydroxybutyrate (PHB) are described. These plants accumulated more than 4% of their fresh weight (≈40% of their dry weight) in the form of PHB in leaf chloroplasts. These very high producers were obtained and identified following a novel strategy consisting of a rapid GC-MS analysis of a large number of transgenic Arabidopsis plants generated using a triple construct, thus allowing the parallel transfer of all three genes necessary for PHB synthesis in a single transformation event. The level of PHB produced was 4-fold greater than previously published values, thus demonstrating the large potential of plants to produce this renewable resource. However, the high levels of the polymer produced had severe effects on both plant development and metabolism. Stunted growth and a loss of fertility were observed in the high-producing lines. Analysis of the metabolite composition of these lines using a GC-MS method that we have newly developed showed that the accumulation of high levels of PHB was not accompanied by an appreciable change in either the composition or the amount of fatty acids. Substantial changes were, however, observed in the levels of various organic acids, amino acids, sugars and sugar alcohols.

Hormonal regulation of fruitlet abscission induced by carbohydrate shortage in citrus.

Abstract: The hormonal signals controlling fruitlet abscission induced by sugar shortage in citrus were identified in Satsuma mandarin, Citrus unshiu (Mak.) Marc, cv. Clausellina and cv. Okitsu. Sugar supply, hormonal responses and fruitlet abscission were manipulated through full, partial or selective leaf removals at anthesis and thereafter. In developing fruitlets, defoliations reduced soluble sugars (up to 98%), but did not induce nitrogen and water deficiencies. Defoliation-induced abscission was preceded by rises (up to 20-fold) in the levels of abscisic acid (ABA) and 1-aminocyclopropane-1-carboxylic acid (ACC) in fruitlets. Applications to defoliated plants showed that ABA increased ACC levels (2-fold) and accelerated fruitlet abscission, whereas norflurazon and 2-aminoethoxyvinyl glycine reduced ACC (up to 65%) and fruitlet abscission (up to 40%). Only the full defoliation treatment reduced endogenous gibberellin A1 (4-fold), whereas exogenous gibberellins had no effect on abscission. The data indicate that fruitlet abscission induced by carbon shortage in citrus is regulated by ABA and ACC originating in the fruits, while gibberellins are apparently implicated in the maintenance of growth. In this system, ABA may act as a sensor of the intensity of the nutrient shortage that modulates the levels of ACC and ethylene, the activator of abscission. This proposal identifies ABA and ACC as components of the self-regulatory mechanism that adjusts fruit load to carbon supply, and offers a physiological basis for the photoassimilate competition-induced abscission occurring under natural conditions.

Expression of cell cycle regulatory genes and morphological alterations in response to salt stress in Arabidopsis thaliana

Abstract: Hyperosmotic stress severely affects plant growth and development. To examine the effect of salt stress on cell cycle activity in Arabidopsis thaliana (L.) Heynh., the transcriptional regulation of a cyclin-dependent kinase, CDC2aAt, and two mitotic cyclins, Arath;CycB1;1 and Arath;CycA2;1, was studied by using the β-glucuronidase (gus) reporter gene. Moreover, the mRNA abundance of these cell cycle genes as well as CDC2bAt were monitored during salt stress. Upon NaCl treatment, the promoter activities and transcript levels of all cell cycle genes diminished initially in the shoot apex and were subsequently induced during salt-stress adaptation. Additionally, the promoter activities of CDC2aAt and CycA2;1 decreased in the vascular cylinder of the root in correlation with reduced lateral root formation. In the root tips, a regression of CDC2aAt, CycA2;1, and CycB1;1:gus expression was observed, concomitant with a shrinkage of the root meristem and inhibition of root growth. Our data indicate that salt stress interferes with cell cycle regulation at the transcriptional level, resulting in an adaptive growth response.

Fumaric acid: an overlooked form of fixed carbon in Arabidopsis and other plant species

Abstract: Photoassimilates are used by plants for production of energy, as carbon skeletons and in transport of fixed carbon between different plant organs. Many studies have been devoted to characterizing the factors that regulate photoassimilate concentrations in different plant species. Most studies examining photoassimilate concentrations in C3 plants have focused on analyzing starch and soluble sugars. However, work presented here demonstrates that a number of C3 plants, including the popular model organism Arabidopsis thaliana (L.) Heynh., and agriculturally important plants, such as soybean, Glycine max (L.) Merr., contain significant quantities of fumaric acid. In fact, fumaric acid can accumulate to levels of several milligrams per gram fresh weight in Arabidopsis leaves, often exceeding those of starch and soluble sugars. Fumaric acid is a component of the tricarboxylic acid cycle and, like starch and soluble sugars, can be metabolized to yield energy and carbon skeletons for production of other compounds. Fumaric acid concentrations increase with plant age and light intensity in Arabidopsis leaves. Moreover, Arabidopsis phloem exudates contain significant quantities of fumaric acid, raising the possibility that fumaric acid may function in carbon transport.

Effects of ultraviolet radiation on photosynthesis and related enzyme reactions of marine macroalgae

Abstract: Changes in physiological parameters related to photosynthesis were studied in five macroalgal species from Spitsbergen (Monostroma arcticum, Laminaria solidungula, Alaria esculenta, Palmaria palmata, Phycodrys rubens) during a 72-h exposure to UV radiation. Maximal quantum yield of photochemistry (Fv/Fm) and maximal electron transport rate (ETRmax) were measured with a pulse-amplitude-modulated fluorometer; the activity of the Calvin cycle enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were estimated using a photometric test. Proteins of crude extracts were separated by SDS gel electrophoresis and changes in cellular concentrations of Rubisco were determined. Moreover, the concentration of chlorophyll a (Chl a), and protein content, were measured photometrically. In all species, Chl a content, maximal quantum yield as well as ETRmax decreased during the UV treatment. Changes in ETRmax were related to the changes in the overall activity of Rubisco. Analysis of SDS gels showed that in P. rubens, L. solidungula, M. arcticum and A. esculenta decreasing Rubisco activity partly resulted from a degradation of the enzyme. However, in A. esculenta, the formation of a high-molecular-weight polypeptide was observed. In all species, the activity of Rubisco was more strongly impaired than that of G3PDH. Exposure to UV resulted in loss of total protein only in the deepwater species L. solidungula and P. rubens. The different sensitivities to UV exposure of the species tested reflect their zonation pattern in the field.

Evidence for covalent linkage between xyloglucan and acidic pectins in suspension-cultured rose cells.

Abstract: Neutral xyloglucan was purified from the cell walls of suspension-cultured rose (Rosa sp. `Paul's Scarlet') cells by alkali extraction, ethanol precipitation and anion-exchange chromatography on `Q-Sepharose FastFlow'. The procedure recovered 70% of the total xyloglucan at about 95% purity in the neutral fraction. The remaining 30% of the xyloglucan was anionic, as demonstrated both by anion-exchange chromatography at pH 4.7 and by high-voltage electrophoresis at pH 6.5. Alkali did not cause neutral xyloglucan to become anionic, indicating that the anionic nature of the rose xyloglucan was not an artefact of the extraction procedure. Pre-incubation of neutral [3H]xyloglucan with any of ten non-radioactive acidic polysaccharides did not cause the radioactive material to become anionic as judged by electrophoresis, indicating that stable complexes between neutral xyloglucan and acidic polysaccharides were not readily formed in vitro. The anionic xyloglucan did not lose its charge in the presence of 8 M urea or after a second treatment with NaOH, indicating that its anionic nature was not due to hydrogen-bonding of xyloglucan to an acidic polymer. Proteinase did not affect the anionic xyloglucan, indicating that it was not associated with an acidic protein. Cellulase converted the anionic xyloglucan to the expected neutral nonasaccharide and heptasaccharide, indicating that the repeat-units of the xyloglucan did not contain acidic residues. Endo-polygalacturonase converted about 40% of the anionic xyloglucan to neutral material. Arabinanase and galactanase also converted appreciable proportions of the anionic xyloglucan to neutral material. These results show that about 30% of the xyloglucan in the cell walls of suspension-cultured rose cells exists in covalently-linked complexes with acidic pectins.

Intraprotoplasmic and wall-localised formation of arabinoxylan-bound diferulates and larger ferulate coupling-products in maize cell-suspension cultures.

Abstract: Maize (Zea mays L.) cell cultures incorporated radioactivity from [14C]cinnamate into hydroxycinnamoyl-CoA derivatives and then into polysaccharide-bound feruloyl residues. Within 5–20 min, the CoA pool had lost its 14C by turnover and little or no further incorporation into polysaccharides then occurred. The system was thus effectively a pulse–chase experiment. Kinetics of radiolabelling of diferulates (also known as dehydrodiferulates) varied with culture age. In young (1–3 d) cultures, polysaccharide-bound [14C]feruloyl- and [14C]diferuloyl residues were both detectable within 1 min of [14C]cinnamate feeding. Thus, feruloyl residues were dimerised 10 min before secretion, the data show that extensive feruloyl coupling occurred intra-protoplasmically. Exogenous H2O2 (1 mM) caused little additional feruloyl coupling; therefore, wall-localised coupling may have been peroxidase-limited. In older (e.g. 4 d) cultures, less intraprotoplasmic coupling occurred: during the first 2.5 h, polysaccharide-bound [14C]diferuloyl residues were a steady 1.4% of the total polysaccharide-bound [14C]ferulate derivatives. In contrast to the situation in younger cultures, exogenous H2O2 induced a rapid 4- to 6-fold increase in all coupling products, indicating that coupling in the walls was H2O2-limited. In both 2- and 4-d-old cultures, polysaccharide-bound 14C-trimers and larger coupling products exceeded [14C]diferulates 3- to 4-fold, but followed similar kinetics. Thus, although all known dimers of ferulate can now be individually quantified, it appears to be trimers and larger products that make the major contribution to cross-linking of wall polysaccharides in cultured maize cells. We argue that feruloyl arabinoxylans that are cross-linked before and after secretion are likely to loosen and tighten the cell wall, respectively. The consequences for the control of cell expansion and for the response of cell walls to an oxidative burst are discussed.

Chemical composition of apoplastic transport barriers in relation to radial hydraulic conductivity of corn roots (Zea mays L.)

Abstract: The hydraulic conductivity of roots (Lp(r)) of 6- to 8-d-old maize seedlings has been related to the chemical composition of apoplastic transport barriers in the endodermis and hypodermis (exodermis), and to the hydraulic conductivity of root cortical cells. Roots were cultivated in two different ways. When grown in aeroponic culture, they developed an exodermis (Casparian band in the hypodermal layer), which was missing in roots from hydroponics. The development of Casparian bands and suberin lamellae was observed by staining with berberin-aniline-blue and Sudan-III. The compositions of suberin and lignin were analyzed quantitatively and qualitatively after depolymerization (BF(3)/methanol-transesterification, thioacidolysis) using gas chromatography/mass spectrometry. Root Lp(r) was measured using the root pressure probe, and the hydraulic conductivity of cortical cells (Lp) using the cell pressure probe. Roots from the two cultivation methods differed significantly in (i) the Lp(r) evaluated from hydrostatic relaxations (factor of 1.5), and (ii) the amounts of lignin and aliphatic suberin in the hypodermal layer of the apical root zone. Aliphatic suberin is thought to be the major reason for the hydrophobic properties of apoplastic barriers and for their relatively low permeability to water. No differences were found in the amounts of suberin in the hypodermal layers of basal root zones and in the endodermal layer. In order to verify that changes in root Lp(r) were not caused by changes in hydraulic conductivity at the membrane level, cell Lp was measured as well. No differences were found in the Lp values of cells from roots cultivated by the two different methods. It was concluded that changes in the hydraulic conductivity of the apoplastic rather than of the cell-to-cell path were causing the observed changes in root Lp(r).

Effect of heat stress on the inhibition and recovery of the ribulose-1,5-bisphosphate carboxylase/oxygenase activation state.

Abstract: Experiments were conducted to determine the relative contributions of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) activation state vis-a-vis Rubisco activase and metabolite levels to the inhibition of cotton (Gossypium hirsutum L.) photosynthesis by heat stress. Exposure of leaf tissue in the light to temperatures of 40 or 45 °C decreased the activation state of Rubisco to levels that were 65 or 10%, respectively, of the 28 °C control. Ribulose-1,5-bisphosphate (RuBP) levels increased in heat-stressed leaves, whereas the 3-phosphoglyceric acid pool was depleted. Heat stress did not affect Rubisco per se, as full activity could be restored by incubation with CO2 and Mg2+. Inhibition and recovery of Rubisco activation state and carbon dioxide exchange rate (CER) were closely related under moderate heat stress (up to 42.5 °C). Moderate heat stress had negligible effect on Fv/Fm, the maximal quantum yield of photosystem II. In contrast, severe heat stress (45 °C) caused significant and irreversible damage to Rubisco activation, CER, and Fv/Fm. The rate of Rubisco activation after alleviating moderate heat stress was comparable to that of controls, indicating rapid reversibility of the process. However, moderate heat stress decreased both the rate and final extent of CER activation during dark-to-light transition. Treatment of cotton leaves with methyl viologen or an oxygen-enriched atmosphere reduced the effect of heat stress on Rubisco inactivation. Both treatments also reduced tissue RuBP levels, indicating that the amount of RuBP present during heat stress may influence the degree of Rubisco inactivation. Under both photorespiratory and non-photorespiratory conditions, the inhibition of the CER during heat stress could be completely reversed by increasing the internal partial pressure of CO2 (Ci). However, the inhibition of the CER by nigericin, a K+ ionophore, was not reversible when the Ci was increased at ambient or high temperature. Our results indicate that inhibition of photosynthesis by moderate heat stress is not caused by inhibition of the capacity for RuBP regeneration. We conclude that heat stress inhibits Rubisco activation via a rapid and direct effect on Rubisco activase, possibly by perturbing Rubisco activase subunit interactions with each other or with Rubisco.

Isolation and characterization of a jacalin-related mannose-binding lectin from salt-stressed rice (Oryza sativa) plants.

Abstract: A novel plant lectin was isolated from salt-stressed rice (Oryzasativa L.) plants and partially characterized. The lectin occurs as a natural mixture of two closely related isoforms consisting of two identical non-covalently linked subunits of 15 kDa. Both isoforms are best inhibited by mannose and exhibit potent mitogenic activity towards T-lymphocytes. Biochemical analyses and sequence comparisons further revealed that the rice lectins belong to the subgroup of mannose-binding jacalin-related lectins. In addition, it could be demonstrated that the lectins described here correspond to the protein products of previously described salt-stress-induced genes. Our results not only identify the rice lectin as a stress protein but also highlight the possible importance of protein-carbohydrate interactions in stress responses in plants.

Differential activation of H+-ATPase genes by an arbuscular mycorrhizal fungus in root cells of transgenic tobacco.

Abstract: In arbuscular mycorrhizas, H+-ATPase is active in the plant membrane around arbuscules but absent from plant mutants defective in arbuscule development (Gianinazzi-Pearson et al. 1995, Can J Bot 73: S526–S532). The proton-pumping H+-ATPase is encoded by a family of genes in plants. Immunocytochemical studies and promoter-gusA fusion assays were performed in transgenic tobacco (Nicotiana tabacum L.) to determine whether the periarbuscular enzyme activity results from de-novo activation of plant genes by an arbuscular mycorrhizal fungus. The H+-ATPase protein was localized in the plant membrane around arbuscule hyphae. The enzyme was absent from non-colonized cortical cells. Regulation of seven H+-ATPase genes (pma) was compared in non-mycorrhizal and mycorrhizal roots by histochemical detection of β-glucuronidase (GUS) activity. Two genes (pma2, pma4) were induced in arbuscule-containing cells of mycorrhizal roots but not in non-mycorrhizal cortical tissues or senescent mycorrhiza. It is concluded that de-novo H+-ATPase activity in the periarbuscular membrane results from selective induction of two H+-ATPase genes, which can have diverse roles in plant-fungal interactions at the symbiotic interface.

Inoculation and nitrate alter phytohormone levels in soybean roots: differences between a supernodulating mutant and the wild type.

Abstract: The levels of different cytokinins. indole-3-acetic acid (IAA) and abscisic acid (ABA) in roots of Glycine max [L.] Merr. cv. Bragg and its supermodulating mutant nts382 were compared for the first time. Forty-eight hours after inoculation with Bradyrhizobium, quantitative and qualitative differences were found in the root's endogenous hormone status between cultivar Bragg and the mutant nts382. The six quantified cytokinins, ranking similarly in each genotype, were present at higher concentrations (30-196% on average for isopentenyl adenosine and dihydrozeatin riboside, respectively) in mutant roots. By contrast, the ABA content was 2-fold higher in Bragg, while the basal levels of IAA [0.53 mu mol (g DW)(-1), on average] were similar in both genotypes. In 1 mM NO3--fed Bragg roots 48 h post-inoculation, IAA, ABA and the cytokinins isopentenyl adenine, and isopentenyl adenosine quantitatively increased with respect to uninoculated controls. However, only the two cytokinins increased in the mutant. High NO3- (8 mM) markedly reduced root auxin concentration, and neither genotypic differences nor the inoculation-induced increase in auxin concentration in Bragg was observed under these conditions. Cytokinins and ABA, on the other hand, Were little affected by 8 mM NO3-. Root IAA/cytokinin and ABA/cytokinin ratios were always higher in Bragg relative to the mutant, and responded to inoculation (mainly in Bragg) and nitrate (both genotypes). The overall results are consistent with the auxin-burst-control hypothesis for the explanation of autoregulation and supernodulation in soybean. However, they are still inconclusive with respect to the inhibitory effect of NO3-.

Clonal analysis of the Arabidopsis root confirms that position, not lineage, determines cell fate.

Abstract: The cellular organization of the Arabidopsis thaliana (L.) Heynh. root meristem suggests that a regular pattern of cell divisions occurs in the root tip. Deviations from this pattern of division might be expected to disrupt the organization of cells and tissues in the root. A clonal analysis of the 3-d-old primary root meristem was carried out to determine if there is variability in division patterns, and if so to discover their effect on cellular organization in the root. Clones induced in the seedling meristem largely confirmed the predicted pattern of cell divisions. However, the cellular initials that normally give rise to the different cell files in the root were shown to exhibit some instability. For example, it was calculated that a lateral root cap/epidermal initial is displaced every 13 d. Furthermore, the existence of large marked clones that included more than two adjacent cell layers suggests that intrusive growth followed by cell division may occur at low frequency, perhaps in response to local cell deaths in the meristem. These findings support the view that even in plant organs with stereotypical cell division patterns, positional information is still the key determinant of cell fate.

Light quantity controls leaf-cell and chloroplast development in Arabidopsis thaliana wild type and blue-light-perception mutants.

Abstract: Plants acclimate to changes in light quantity by altering leaf-cell development and the accumulation of chloroplast components, such that light absorption is favoured under limiting illumination, and light utilisation under non-limiting conditions. Previous evidence suggests an involvement of a high-light photosynthetic redox signal in the down-regulation of the accumulation of the light-harvesting complexes of photosystem II (Lhcb) and the expression of the Lhcb genes, and of a blue-light signal in the control of leaf development and in the increase in photosynthetic capacity, as affected by the accumulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). We examined the internal anatomy of leaves, the ultrastructure of chloroplasts and accumulation of light-harvesting complexes and Rubisco in wild-type Arabidopsis thaliana (L.) Heynh. and in mutants in each of the three known blue-light photoreceptors, cryptochrome 1, cryptochrome 2 and phototropin, as well as a mutant in both cryptochromes. Our results indicate an extensive capacity of the Arabidopsis mesophyll cells to adapt to high light fluence rate with an increase in palisade elongation. Under high light, chloroplasts showed increased starch accumulation and reductions in the amount of granal thylakoids per chloroplast, in the proportion of chlorophyll b relative to chlorophyll a, and in the accumulation of the major Lhcb polypeptides. The responses were similar for all four mutants, with respect to their wild types. The results are consistent with either a complete redundancy in function between cryptochromes and phototropin, or their absence of involvement in the light-quantity responses tested. We observed minimal effects of light quantity on Rubisco accumulation over the range of fluence rates used, and conclude that elongation of palisade mesophyll cells and accumulation of Rubisco are controlled separately. This indicates that light acclimation must be the result of a number of individual elementary responses. Quantitative differences in the acclimatory responses were observed between the Landsberg erecta and Columbia wild-type ecotypes used.

Chemical-induced apoptotic cell death in tomato cells: involvement of caspase-like proteases.

Abstract: A new system to study programmed cell death in plants is described. Tomato (Lycopersicon esculentum Mill.) suspension cells were induced to undergo programmed cell death by treatment with known inducers of apoptosis in mammalian cells. This chemical-induced cell death was accompanied by the characteristic features of apoptosis in animal cells, such as typical changes in nuclear morphology, the fragmentation of the nucleus and DNA fragmentation. In search of processes involved in plant apoptotic cell death, specific enzyme inhibitors were tested for cell-death-inhibiting activity. Our results showed that proteolysis plays a crucial role in apoptosis in plants. Furthermore, caspase-specific peptide inhibitors were found to be potent inhibitors of the chemical-induced cell death in tomato cells, indicating that, as in animal systems, caspase-like proteases are involved in the apoptotic cell death pathway in plants.

Calcium chloride penetrates plant cuticles via aqueous pores.

Abstract: Penetration of calcium chloride across astomatous cuticular membranes (CMs) isolated from leaves of Pyrus communis L. has been studied. Penetration was a first-order process when calcium chloride concentrations ranged from 2 gl(-1) to 10 gl(-1). Rate constants were increased 10-fold by adding wetting agents but they did not depend on temperature. The accelerators tributyl phosphate and diethyl sebacate had no effect on rates of penetration. Increasing humidity over the salt residue on the CMs from 50 to 90% increased rate constants by about 2-fold. Extracting cuticular waxes from pear leaf CMs increased rate constants by factors of 2 to 3, depending on humidity. Leaf CMs from Malus domestica Borkh., Populus aelha L., Stephanotis floribunda Brongn. and Schefflera actinophylla (Endl.) Harms were also permeable to CaCl2. Highest rate constants were observed with poplar CMs while Schefflera CMs exhibited the lowest permeability. By comparing these results with the well established transport properties of the lipophilic pathway it is concluded that calcium chloride hexahydrate penetrated cuticular membranes via aqueous pores.