Developmental changes in spatial distribution of in vivo fluorescence and epidermal UV absorbance over Quercus petraea leaves.

BACKGROUND AND AIMS: Epidermal phenolic compounds (mainly flavonoids) constitute a vital screen that protects the leaf from damage by natural ultraviolet (UV) radiation. The effectiveness of epidermal UV-screening depends on leaf anatomy, the content of UV-screening compounds and their spatial uniformity over the leaf area. To investigate in vivo the spatial pattern of the epidermal UV-screen during leaf development, a fluorescence imaging method was developed to map the epidermal UV-absorbance at a microscopic scale. This study was done on oak (Quercus petraea) leaves that were used as a model of woody dicotyledonous leaves. METHODS: The leaf development of 2-year-old trees, grown outdoors, was monitored, at a macroscopic scale, by in vivo measurements of chlorophyll content per unit area and epidermal UV-absorbance using two optical leaf-clip meters. The distribution of pigments within leaves was assessed in vivo spectroscopically. The microscopic images of UV-induced fluorescence and UV-absorbance acquired in vivo during leaf development were interpreted from spectral characteristics of leaves. KEY RESULTS: At a macroscopic scale, epidermal UV-absorbance was high on the upper leaf side during leaf development, while it increased on the lower leaf side during leaf expansion and reached the adaxial value at maturity. At a microscopic scale, in immature leaves, for both leaf sides, the spatial distribution of epidermal UV-absorbance was heterogeneous, with a pattern depending on the flavonoid content of vacuoles in developing epidermal cells. At maturity, epidermal UV-absorbance was uniform. CONCLUSIONS: The spatial pattern of epidermal UV-screen over the area of oak leaves is related to leaf anatomy during development. In vivo spectroscopy and fluorescence imaging of the leaf surface showed the distribution of pigments within the leaf and hence can provide a tool to monitor optically the leaf development in nature.

Responses of epidermal phenolic compounds to light acclimation: in vivo qualitative and quantitative assessment using chlorophyll fluorescence excitation spectra in leaves of three woody species.

Chlorophyll fluorescence (ChlF) excitation spectra were measured to assess the UV-sunscreen compounds accumulated in fully expanded leaves of three woody species belonging to different chemotaxons, (i.e. Morus nigra L., Prunus mahaleb L. and Lagerstroemia indica L.), grown in different light microclimates. The logarithm of the ratio of ChlF excitation spectra (logFER) between two leaves acclimated to different light microclimates was used to assess the difference in epidermal absorbance (EAbs). EAbs increased with increasing solar irradiance intercepted for the three species. This epidermal localisation of UV-absorbers was confirmed by the removal of the epidermis. It was possible to simulate EAbs as a linear combination of major phenolic compounds (Phen) identified in leaf methanol extracts by HPLC-DAD. Under UV-free radiation conditions, shaded leaves of M. nigra accumulated chlorogenic acid. Hydroxybenzoic acid (HBA) derivatives and hydroxycinnamic acid (HCA) derivatives greatly increased with increasing PAR irradiance under the low UV-B conditions found in the greenhouse. These traits were also observed for the HCA of the two other species. Flavonoid (FLAV) accumulation started under low UV-A irradiance, and became maximal in the adaxial epidermis of sun-exposed leaves outdoors. A decrease in the amount of HCA was observed concomitantly to the intense accumulation of FLAV for both leaf sides of the three species. Judging from the logFER, under low UV-B conditions, larger amounts of HCA are present in the epidermis in comparison to FLAV for the three species. Upon transition from the greenhouse to full sunlight outdoors, there was a decrease in leaf-soluble HCA that paralleled FLAV accumulation in reaction to increasing solar UV-B radiation in the three species. In M. nigra, that contains large amounts of HCA, the logFER analysis showed that this decrease occurred in the adaxial epidermis, whereas the abaxial epidermis, which is protected from direct UV-B radiation, continued to accumulate large amounts of HCA.

Light-induced changes of NADPH fluorescence in isolated chloroplasts: a spectral and fluorescence lifetime study.

Isolated chloroplasts show a light-induced reversible increase in blue-green fluorescence (BGF), which is only dependent on NADPH changes. In the present communication, we report a time-resolved and spectral analysis of this BGF in reconstituted chloroplasts and intact isolated chloroplasts, in the dark and under actinic illumination. From these measurements we deduced the contribution of the different forms of NADPH (free and bound to proteins) to the light-induced variation of BGF and conclude that this variation is due only to the redox change of the NADP pool. A simple model estimating the distribution of NADPH between the free and bound form was designed, that explains the differences measured for the BGF of reconstituted chloroplasts and intact chloroplasts. From the decay-associated spectra of the chloroplast BGF, we also deduced the participation of flavins to the green peak of chloroplast fluorescence emission spectrum, and the existence of excitation energy transfer from proteins to bound NADPH in chloroplasts. In addition, we re-examined the use of chloroplast BGF as a quantitative measure of NADPH concentration, and confirmed that chloroplast BGF can be used for non-destructive, continuous and probably quantitative monitoring of light-induced changes in NADP redox state.

Characterization of Blue-Green Fluorescence in the Mesophyll of Sugar Beet (Beta vulgaris L.) Leaves Affected by Iron Deficiency.

The mesophyll of sugar beet (Beta vulgaris L.) leaves emits red (chlorophyll a) fluorescence and blue-green fluorescence when excited with ultraviolet light. The intensity of blue-green fluorescence was increased in mesophylls affected by iron deficiency. This increase was large and progressive. It was concomitant with a decrease of photosynthetic pigments per unit of leaf area. Most of the increase in blue-green fluorescence can be explained by the decrease of the screening of ultraviolet light by chlorophylls and carotenoids. In addition, chlorophylls selectively reabsorb blue fluorescence, which leads to a change in the form of the fluorescence emission spectra. This effect induces an increase of the blue-to-green fluorescence ratio in control mesophylls that was concomitant with the decrease of chlorophyll per unit of leaf area. Iron deficiency induced a decrease of the blue-to-green fluorescence ratio that may be attributed to an accumulation of flavins fluorescing in the green. Time-resolved fluorescence measurements indicate that they are mostly riboflavin and/or flavin mononucleotide phosphate. Our data also indicate that the blue-green fluorescence emitted from the mesophyll contains fluorescence of nicotinamide nucleotides.

Photoinactivation of the photosynthetic electron transport chain by accumulation of over-saturating light pulses given to dark adapted pea leaves.

The effect of cumulative over-saturating pulses (OSP) of white light (1 s, >10 000 mumol photons m(-2) s(-1)), applied every 20 min on pea leaves, was investigated during a complete diurnal cycle of 24 h. In dark-adapted leaves, this treatment leads to a progressive decline of the optimum Photosystem II (PS II) quantum yield. Continuous low background light (except far-red light) had a protective effect against this OSP-induced photoinactivation. The lack of far-red effect could be due to its absorption mainly in PS I and not in PS II, but could be also due to the general low absorption in this wavelength region. The photoinactivation was enhanced in leaves that had been previously infiltrated with chloramphenicol. The quantum yield of CO(2) assimilation, but not its maximal capacity, was inhibited by the OSP treatment. The most spectacular effects observed, in addition to an irreversible quenching of Fm, was a strong inhibition of Q(A) (-) reoxidation revealed by a large increase in the Fs level and consequently by a decrease of DeltaF/Fm'. Under such conditions, we observed that the electron flow deduced from DeltaF/Fm' underestimated the real electron flow to CO(2). Time-resolved Chlorophyll a fluorescence measurements showed that the reduced capacity of Q(A) (-) reoxidation in OSP treated leaves was accompanied by the appearance of a 4.7 ns component attributed to PS II charge recombination. We suggest that a modification at the Q(B) site may influence the redox potential of Q(A)/Q(A) (-), facilitating the reversion of the primary charge separation. In addition, a 1.2 ns fluorescence component accumulated, which appeared to be responsible for the underestimation of PS II electron flow. The observed photoinactivation seemed to be different from the photoinhibition often described in the literature, which occurs under continuous light.

The effect of decreasing temperature up to chilling values on the in vivo F685/F735 chlorophyll fluorescence ratio in Phaseolus vulgaris and Pisum sativum: the role of the photosystem I contribution to the 735 nm fluorescence band.

The effect of leaf temperature (T), between 23 and 4 degrees C, on the chlorophyll (Chl) fluorescence spectral shape was investigated under moderate (200 microE m-2 s-1) and low (30-35 microE m-2 s-1) light intensities in Phaseolus vulgaris and Pisum sativum. With decreasing temperature, an increase in the fluorescence yield at both 685 and 735 nm was observed. A marked change occurred at the longer emission band resulting in a decrease in the Chl fluorescence ratio, F685/F735, with reducing T. Our fluorescence analysis suggests that this effect is due to a temperature-induced state 1-state 2 transition that decreases and increases photosystem II (PSII) and photosystem I (PSI) fluorescence, respectively. Time-resolved fluorescence life-time measurements support this interpretation. At a critical temperature (about 6 degrees C) and low light intensity a sudden decrease in fluorescence intensity was observed, with a larger effect at 685 than at 735 nm. This is probably linked to a modification of the thylakoid membranes, induced by chilling temperatures, which can alter the spill-over from PSII to PSI. The contribution of photosystem I to the long-wavelength Chl fluorescence band (735 nm) at room temperature was estimated by both time-resolved fluorescence lifetime and fluorescence yield measurements at 685 and 735 nm. We found that PSI contributes to the 735 nm fluorescence for about 40, 10 and 35% at the minimal (F0), maximal (Fm) and steady-state (Fs) levels, respectively. Therefore, PSI must be taken into account in the analysis of Chl fluorescence parameters that include the 735 nm band and to interpret the changes in the Chl fluorescence ratio that can be induced by different agents.

Relationships between optically assessed polyphenols and chlorophyll contents, and leaf mass per area ratio in woody plants: a signature of the carbon-nitrogen balance within leaves?

Chlorophyll (Chl) and epidermal polyphenol (EPhen) contents were estimated in vivo using two optical leaf-clips, SPAD-502 and Dualex, respectively. The area-based measurements were transformed into mass-based data by taking into account the leaf dry mass per area (LMA). Measurements were performed on forest trees and on saplings grown under controlled conditions. While LMA increased with irradiance along a vertical transect in a beech canopy or in saplings grown under different and increasing irradiance levels, mass-based EPhen (EPhen(m)) increased, whereas mass-based Chl (Chl(m)) decreased. This was a signature of a gradual switch of investment from protein into polyphenol production. A similar signature was obtained in saplings grown on nitrogen-deficient soil with respect to fertilized controls. However, nitrogen effects remained moderate compared to irradiance-induced effects. EPhen(m) and Chl(m) both declined with plant ageing-induced increases in LMA, under all tested growth conditions. This was a signature of an accumulation of dry matter that diluted Chl and EPhen. The described competition between Chl and EPhen in leaves fits well with the predictions of the Protein Competition Model (PCM), that is, that the total leaf mass-based polyphenols content (Phen(t)) is controlled by the competition between protein and polyphenol biosynthetic pathways and its metabolic regulation.

Induction of oscillations in chlorophyll fluorescence by re-illumination of intact isolated pea chloroplasts.

Oscillations in chlorophyll fluorescence yield were observed upon re-illumination of intact isolated pea (Pisum sativumL.) chloroplasts that had attained their maximal rate of photosynthesis and had spent a short period in darkness. The oscillations depended on the length of the previous dark period, the length of previous illumination, and the reaction temperature. This finding confirms the presence of an "oscillatory center" in the chloroplasts temselves.

An improved procedure for the isolation of intact chloroplasts of high photosynthetic capacity.

An improved procedure for the mechanical isolation of chloroplasts of high degree of intactness (90-95%) and photosynthetic capacity (25-50 mmol of O2/s per mol of chlorophyll) is described. The combination of pea plants (Pisum sativum L.) as starting material and the high reproducibility of the procedure readily and cheaply yields reliable intact chloroplasts for photosynthetic studies.

UV-induced blue-green and far-red fluorescence along wheat leaves: a potential signature of leaf ageing.

Under UV-excitation, leaves emit red (RF) and far-red (FRF) fluorescence from chlorophyll and blue-green fluorescence (BGF) from hydroxycinnamic acids. In this study, the aim was to develop a fluorescence signature of wheat leaf ageing after the emergence of the lamina. FRF and BGF were examined in the first three leaves of 2-week-old wheat plants. It was investigated how FRF and BGF vary as leaf and tissue aged by spectroscopic measurements, time-resolved BGF analysis and microscopic imaging of the leaf surface. It was found that FRF decreased with leaf and tissue ageing because of an accumulation of UV-absorbers in the epidermis. BGF also decreased, but without changes either in the shape of excitation and emission spectra or in the fluorescence lifetime. So, BGF emanated from the leaf surface, without changes in fluorophore composition during leaf ageing. The shape of the BGF spectrum indicates that ferulic acid bound to the cell wall is the main blue-green fluorophore. The effects of pH and solvents on BGF from intact leaves and ferulic acid in solution were similar, confirming the hydroxycinnamic acid origin of BGF. UV-fluorescence microscopic imaging of the surface of intact leaves showed that different epidermis cell types and sclerenchyma bands emitted BGF. The decreasing gradient of BGF from the base to the apex of the lamina could be related to the decrease in the surface of the fluorescent sclerenchyma bands. The significance of FRF and BGF as potential signatures of wheat lamina growth are discussed.

Photosynthetic oxygen evolution and chlorophyll fluorescence in intact isolated chloroplasts on a solid support: the influence of orthophosphate.

We devised recently a method to trap intact isolated chloroplasts on a solid support consisting of membrane filters made of cellulose nitrate (Cerovic et al., 1987, Plant Physiol. 84, 1249-1251). The addition of alkaline phosphatase to the reaction medium enabled continuous photosynthesis by spinach (Spinacia oleracea L.) chloroplasts to be sustained by hydrolysis of newly produced and exported triose phosphates and recycling of orthophosphate. In this system, simultaneous measurements of chlorophyll fluorescence and oxygen evolution were performed and their dependence on orthophosphate concentration was investigated. Optimal photosynthesis was obtained at a much higher initial orthophosphate concentration (2-4 mM) compared to intact chloroplasts in suspension. Secondary kinetics of chlorophyll fluorescence yield were observed and were shown to depend on the initial orthophosphate concentration.

Photosynthesis by intact isolated chloroplasts on solid support.

A new approach to measurements of photosynthesis by isolated chloroplasts has been devised. Intact isolated chloroplasts were trapped in the cavities of membrane filters. The thin layers of chloroplasts so obtained were assayed for O(2) evolution and CO(2) assimilation in leaf-chambers. Photosynthetic gas exchange could be demonstrated to take place either in a closed or a flow-through system. The chloroplasts were morphologically intact as shown by light or scanning electron microscopy and displayed stable rates of photosynthesis in the presence of phosphate and alkaline phosphatase. The methods described open the way to in vitro measurement of photosynthesis, by chloroplasts under conditions more closely resembling those in leaves.

The role of photophosphorylation in SO2 and SO 3 (2-) inhibition of photosynthesis in isolated chloroplasts.

Sulphur dioxide inhibits noncyclic photophosphorylation in isolated envelope-free chloroplasts. This inhibition was shown to be reversible and competitive with phosphate, with an inhibitor constant of Ki=0.8mM. The same inhibition characteristics were observed when phosphoglycerate (PGA)- or ribulose-1,5-bisphosphate (RuBP)-dependent oxygen evolution was examined in a reconstituted chloroplast system in the presence of SO 3 (2-) . Using an ATP-regenerating system (phosphocreatine-creatine kinase), it was demonstrated that the inhibition of PGA-dependent oxygen evolution is solely the result of inhibited photophosphorylation. It is concluded that at low SO2 and SO 3 (2-) concentrations the inhibition of photophosphorylation is responsible for the inhibition of photosynthetic oxygen evolution.

A post-translational modification of the photosystem II subunit CP29 protects maize from cold stress.

The resistance of maize plants to cold stress has been associated with the appearance of a new chlorophyll a/b binding protein in the thylakoid membrane following chilling treatment in the light. The cold-induced protein has been isolated, characterized by amino acid sequencing, and pulse labeled with radioactive precursors, showing that it is the product of post-translational modification by phosphorylation of the minor chlorophyll a/b protein CP29 rather than the product of a cold-regulated gene or an unprocessed CP29 precursor. We show here that the CP29 kinase activity displays unique characteristics differing from previously described thylakoid kinases and is regulated by the redox state of a quinonic site. Finally, we show that maize plants unable to perform phosphorylation have enhanced sensitivity to cold-induced photoinhibition.

Simultaneous measurement of changes in red and blue fluorescence in illuminated isolated chloroplasts and leaf pieces: The contribution of NADPH to the blue fluorescence signal.

A newly developed nitrogen laser fluorimeter insensitive to actinic illumination was used to follow simultaneously the light induced changes in red and blue fluorescence of intact isolated spinach chloroplasts and leaf pieces. The recorded variable blue fluorescence was linked to a water soluble component of intact isolated chloroplasts, depended on Photosystem I, and was related to changes in carbon metabolism. From the comparison of changes in intact and broken chloroplasts and from fluorescence spectra under different conditions, it was concluded that the variation in NADPH was the major cause for the changes in blue fluorescence. This study opens a path towards continuous and non-destructive monitoring of NADPH redox state in chloroplasts and leaves.