Hubert Greppin

Regulation of antenna structure and electron transport in Photosystem II of Pisum sativum under elevated temperature probed by the fast polyphasic chlorophyll a fluorescence transient: OKJIP

Abstract Chlorophyll a (Chl a) florescence induction kinetics from the minimum yield F0 to the maximum yield Fm provide information on the filling up of the plastoquinone pool with reducing equivalents. In this paper, we have examined the effect of high temperature (above 40°C) on Chl a fluorescence rise kinetics starting from 40 μs (to 1 s) in pea leaves (Pisum sativum). The variable Chl a fluorescence is strongly quenched after heat treatment. With increasing temperature or the duration of heat treatment a typical O-J-I-P transient (Strasser et al. (1995) Photochem. Photobiol., 61, 32–42) is transformed into an O-K-J-I-P transient, with an additional rapid step called K detected in the 200–300 μs range. After prolonged heat treatment, the K-step becomes a dominant peak in the Chl a fluorescence transient followed by a large dip. We have investigated the origin and the possible interpretation of these changes by using NH2OH which acts as an electron donor to PS II, and DCMU which is known to block the PS II electron transport chain by displacing QB. From the present data we propose that the appearance of this K-step is due to two effects: (1) inhibition of the water splitting system that leads to a much slowed turn over of the reduction of QA; (2) changes in the architecture of the antenna of PS II which affect the energy migration properties within the photosynthetic unit. The K-step can thus be used as an indicator of the heterogeneity of photosynthetic units and as an indicator for the physiological state of the photosynthetic sample.

Plant hormones and plant growth regulators in plant tissue culture

SummaryThis is a short review of the classical and new, natural and synthetic plant hormones and growth regulators (phytohormones) and highlights some of their uses in plant tissue culture. Plant hormones rarely act alone, and for most processes— at least those that are observed at the organ level—many of these regulators have interacted in order to produce the final effect. The following substances are discussed: (a) Classical plant hormones (auxins, cytokinins, gibberellins, abscisic acid, ethylene and growth regulatory substances with similar biological effects. New, naturally occurring substances in these categories are still being discovered. At the same time, novel structurally related compounds are constantly being synthesized. There are also many new but chemically unrelated compounds with similar hormone-like activity being produced. A better knowledge of the uptake, transport, metabolism, and mode of action of phytohormones and the appearance of chemicals that inhibit synthesis, transport, and action of the native plant hormones has increased our knowledge of the role of these hormones in growth and development. (b) More recently discovered natural growth substances that have phytohormonal-like regulatory roles (polyamines, oligosaccharins, salicylates, jasmonates, sterols, brassinosteroids, dehydrodiconiferyl alcohol glucosides, turgorins, systemin, unrelated natural stimulators and inhibitors), as well as myoinositol. Many of these growth active substances have not yet been examined in relation to growth and organized developmentin vitro.

Analysis and expression of the class III peroxidase large gene family in Arabidopsis thaliana

Higher plants possess a large set of the classical guaiacol peroxidases (class III peroxidases, E.C. 1.11.1.7). These enzymes have been implicated in a wide array of physiological processes such as H(2)O(2) detoxification, auxin catabolism and lignin biosynthesis and stress response (wounding, pathogen attack, etc.). During the last 10 years, molecular cloning has allowed the isolation and characterization of several genes encoding peroxidases in plants. The achievement of the large scale Arabidopsis genome sequencing, combined with the DNA complementary to RNA (cDNA) expressed sequence tags projects, provided the opportunity to draw up the first comprehensive list of peroxidases in a plant. By screening the available databases, we have identified 73 peroxidase genes throughout the Arabidopsis genome. The evolution of the peroxidase multigene family has been investigated by analyzing the gene structure (intron/exon) in correlation with the phylogenetic relationships between the isoperoxidases. An evolutionary pattern of extensive gene duplications can be inferred and is discussed. Using a cDNA array procedure, the expression pattern of 23 peroxidases was established in the different organs of the plant. All the tested peroxidases were expressed at various levels in roots, while several were also detected in stems, leaves and flowers. The specific functions of these genes remain to be determined.

A theoretical and experimental analysis of the qP and qN coefficients of chlorophyll fluorescence quenching and their relation to photochemical and nonphotochemical events

The initial (F0), maximal (FM) and steady-state (FS) levels of chlorophyll fluorescence emitted by intact pea leaves exposed to various light intensities and environmental conditions, were measured with a modulated fluorescence technique and were analysed in the context of a theory for the energy fluxes within the photochemical apparatus of photosynthesis. The theoretically derived expressions of the fluorescence signals contain only three terms, X=J2p2F/(1−G), Y=T/(1−G) and V, where V is the relative variable fluorescence, J2 is the light absorption flux in PS II, p2F is the probability of fluorescence from PS II, G and T are, respectively, the probabilities for energy transfer between PS II units and for energy cycling between the reaction center and the chlorophyll pool: F0=X, FM=X/(1−Y) and FS=X(1+(YV/(1−Y))). It is demonstrated that the amplitudes of the previously defined coefficients of chlorophyll fluorescence quenching, qP and qN, reflect, not just photochemical (qP) or nonphotochemical (qN) events as implied in the definitions, but both photochemical and nonphotochemical processes of PS II deactivation. The coefficient qP is a measure of the ratio between the actual macroscopic quantum yield of photochemistry in PS II (41-1) in a given light state and its maximal value measured when all PS II traps are open (41-2) in that state, with 41-3 and 41-4. When the partial connection between PS II units is taken into consideration, 1-qP is nonlinearily related to the fraction of closed reaction centers and is dependent on the rate constants of all (photochemical as well as nonphotochemical) exciton-consuming processes in PS II. On the other hand, 1-qN equals the (normalized) ratio of the rate constant of photochemistry (k2b) to the combined rate constant (kN) of all the nonphotochemical deactivation processes excluding the rate constant k22 of energy transfer between PS II units. It is demonstrated that additional (qualitative) information on the individual rate constants, kN-k22 and k2b, is provided by the fluorescence ratios 1/FM and (1/F0)−(1/FM), respectively. Although, in theory, 41-5 is determined by the value of both k2b and kN-k22, experimental results presented in this paper show that, under various environmental conditions, 41-6 is modulated largely through changes in kN, confirming the idea that PS II quantum efficiency is dynamically regulated in vivo by nonphotochemical energy dissipation.

Extracellular ascorbic acid and enzyme activities related to ascorbic acid metabolism in Sedum album L. leaves after ozone exposure

Ascorbic acid (AA) and glutathione (GSH) contents and enzymatic activities of dehydroascorbate (DHA) reductase and glutathione reductase were measured in the apoplast and whole leaves of Sedum album L. plants after a 2-hr exposure to different ozone (O3) concentrations (0.2, 0.4 and 0.6 μl/l O3). Although the reduced AA level decreased in the apoplast of exposed plants, the total amount of ascorbic acid (AA+DHA) increased in that compartment following O3 exposure. The increase depended on the O3 concentration and could reach twice the control values. These results suggested the existence of a continuous supply of AA from cells in response to oxidant exposure. AA and GSH levels were depleted in the whole leaves during the exposure to O3 but rapidly recovered after exposure. Neither dehydroascorbate reductase nor glutathione reductase activities could be detected in the apoplast compartment. In cell extracts, dehydroascorbate reductase was activated and glutathione reductase was not affected by O3 exposure. These results are discussed in the context of a cycle of reactions involving ascorbate peroxidase, dehydroascorbate reductase, glutathione reductase and glucose 6-P dehydrogenase, which would operate in the apoplast and in the interior of the cell. The rapid recovery of AA and GSH levels after O3 exposure suggests a prominent role for these enzymes in cell protection against oxidative damage.

Functioning of photosystems I and II in pea leaves exposed to heat stress in the presence or absence of light Analysis using in-vivo fluorescence, absorbance, oxygen and photoacoustic measurements

Fluorimetric, photoacoustic, polarographic and absorbance techniques were used to measure in situ various functional aspects of the photochemical apparatus of photosynthesis in intact pea leaves (Pisum sativum L.) after short exposures to a high temperature of 40 ° C. The results indicated (i) that the in-vivo responses of the two photosystems to high-temperature pretreatments were markedly different and in some respects opposite, with photosystem (PS) II activity being inhibited (or down-regulated) and PSI function being stimulated; and (ii) that light strongly interacts with the response of the photosystems, acting as an efficient protector of the photochemical activity against its inactivation by heat. When imposed in the dark, heat provoked a drastic inhibition of photosynthetic oxygen evolution and photochemical energy storage, correlated with a marked loss of variable PSII-chlorophyll fluorescence emission. None of the above changes were observed in leaves which were illuminated during heating. This photoprotection was saturated at rather low light fluence rates (around 10 W · m−2). Heat stress in darkness appeared to increase the capacity for cyclic electron flow around PSI, as indicated by the enhanced photochemical energy storage in far-red light and the faster decay of P700+(oxidized reaction center of PSI) monitored upon sudded interruption of the far-red light. The presence of light during heat stress reduced somewhat this PSI-driven cyclic electron transport. It was also observed that heat stress in darkness resulted in the progressive closure of the PSI reaction centers in leaves under steady illumination whereas PSII traps remained largely open, possibly reflecting the adjustment of the photochemical efficiency of undamaged PSI to the reduced rate of photochemistry in PSII.

Identification of a Ca(2+)-pectate binding site on an apoplastic peroxidase

An apoplastic isoperoxidase from zucchini (APRX) was shown to bind strongly to polygalacturonic acid in their Ca2+-induced conformation. By homology modeling, we were able to identify a motif of four clustered arginines (positions 117, 262, 268, and 271) that could be responsible for this binding. To verify the role of these arginine residues in the binding process, we prepared three mutants of APRX (M1, R117S; M2, R262Q/R268S; and M3, R262Q/R268S/R271Q). APRX and the three mutants were expressed as recombinant glycoproteins by the baculovirus–insect cell system. This procedure yielded four active enzymes with similar molecular masses that were tested for their ability to bind Ca2+-pectate. Recombinant wild-type APRX exhibited an affinity for the pectic structure comparable to that of the native plant isoperoxidase. The mutations impaired binding depending on the number of arginine residues that were replaced. M1 and M2 showed intermediate affinities, whereas M3 did not bind at all. This was demonstrated using an in vitro binding test and on cell walls of hypocotyl cross-sections. It can be concluded that APRX bears a Ca2+-pectate binding site formed by four clustered arginines. This site could ensure that APRX is properly positioned in cell walls, using unesterified domains of pectins as a scaffold.

Involvement of indole-3-acetic acid in the circadian growth of the first internode of Arabidopsis

Abstract. The extension rate of the first inflorescence node of Arabidopsis was measured during light/dark or continuous light exposure and was found to exhibit oscillations which showed a circadian rhythmicity. Decapitation induced a strong inhibition of stem extension. Subsequent application of IAA restored growth and the associated extension–rate oscillations. In addition, IAA treatments, after decapitation, re-established the circadian rhythmicity visible in the intact plants during free run. This indicates that the upper zone of the inflorescence has a major influence on the extension rate of floral stems and implies a role for auxin. Application of N-(1-naphthyl)phthalamic acid, an IAA transport inhibitor, to an intact floral stem inhibited growth and the rhythmicity in the extension rate oscillations, indicating that IAA polar transport may play a role in the dynamics of stem elongation. Furthermore, IAA-aspartate application, after decapitation, did not restore growth and rhythmicity. Nevertheless, biochemical analysis of IAA and IAA-aspartate demonstrated circadian fluctuations of the endogenous levels of both compounds. These observations suggest that IAA metabolism is an essential factor in the regulation of the circadian growth rhythm of Arabidopsis floral stems.

‘Waldsterben’, part IV (continuing series)

Peroxidase and superoxide dismutase activities, and ascorbic acid content, were measured in both intercellular fluid and cell material of current and 1-year-old needles of Norway spruce saplings treated with ozone, ambient air and activated carbon-filtered air in outdoor fumigation chambers. Ethylene evolution was also compared. Plants from carbon-filtered air treatments had significantly lower enzyme activities and higher ascorbic acid content. These changes were more marked in intercellular fluid than in cell material. Significant changes were noted at ozone levels typical of ambient air quality in a typical urban area. These results suggest the need for simultaneous screening of several biochemical markers as a way of overcoming the lack of specificity of any single marker for the identification of a perturbation by a particular stress, such as ozone.

Confirmation of the role of auxin and calcium in the late phases of adventitious root formation

Poplar shoots raised in vitro were induced to root by incubation on an auxin (NAA) containing medium for 7 h. After 13 days on an auxin-free medium, 97% of the treated shoots had rooted. The introduction of known antiauxins (PCIB, PBA, POAA) into the rooting expression auxin-free medium, after the 7-h induction by NAA, completely (PCIB and PBA) or severely (POAA) inhibited rooting. The exclusion of calcium from the expression auxin free medium reduced the percentage of rooting by about 42%. The inhibition was still higher in the presence of EGTA, a calcium chelator. Lanthanum chloride, a calcium channel blocker, also completely inhibited rooting, when incorporated into the auxin free medium, with or without calcium. These results support previous hypotheses about the implication of both endogenous auxin and calcium in the late phases of the adventitious rooting process.