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Fruit tree production is challenged by climate change, which is characterized by heat waves, warmer winters, increased storms, and recurrent droughts. The technology of top netting may provide a partial solution, as it alleviates climatic effects by microclimate manipulation. The tree physiological performance is improved under the nets, with an increased productivity and quality. The application of photoselective nets, which also alter the light spectrum, may result in additional horticultural improvements. We present the results of a 5-year experimental study on Valencia oranges, examining three nets: red, pearl, and transparent. Each net was tested at three fertigation conditions: a field standard (100%, I100) and two reduced fertigation regimes, which were 80% (I80) and 60% (I60) of the standard. The average multi-annual yield under the red and pearl nets with I100 and I80 and transparent net with I100 was significantly higher than that of the control trees. While the multi-annual yield increase under the red net I80 was due to the increase in the fruit number, in other treatments, the effect was mostly due to induction in the individual fruit weight. The data presented here show that an increased productivity of orange trees grown under photoselective nets, particularly the red net, with its specific spectral properties, was achieved with a considerable water-saving effect.

In the past decade, light-emitting diodes (LEDs) have been replacing most other types of light sources. One emerging use for LEDs in horticulture is ‘intra-canopy illumination’ or ‘LED-interlighting’, feasible owing to their low heat output and small physical size. The interlighting technique, typically implemented in environmentally-controlled greenhouses also supplemented with overhead lighting, is mostly relevant for high-wire vegetable cultivation, in which self-shading results in light limitations for a large fraction of the canopy. Interlighting has been shown to increase the yield and/or improve fruit quality in all-year round greenhouse crops such as tomato, cucumber and sweet pepper. In this work, we utilized daytime supplemental intra-canopy LED illumination for sweet pepper grown in high-density ‘Spanish’ trellis systems within passive high tunnels in the Jordan Valley, Israel (latitude ˜32 °N). While canopy top at these conditions is not light-limited, extensive deep shading of the inner canopy is a disadvantage. In two experiments carried out in two separate seasons, the supplemental lighting, which enhanced the photosynthetic rates of the inner canopy foliage by 3.5- to 5.7-fold, resulted in significant increase (˜30%) of the fruit yield during the spring season. The added yield was attained by higher fruit numbers, with no notable effects on fruit size or weight. Our results raise the prospect that LED-interlighting may be a useful practical tool for maximizing fruit production, even in geographical regions of ample sunlight.

Annual and perennial plants represent two different evolutionary strategies based on differential synchronization of their reproductive development. The mobile signal protein FLOWERING LOCUS T (FT) plays a central role in mediating the onset of reproduction in both plant types. Two novel FT-like genes from pear (Pyrus communis)-PcFT1 and PcFT2-were isolated, and their expression profiles were determined for one annual cycle. The effects of PcFT2 on flowering were investigated in annual (tobacco) and perennial (apple) plants by means of grafting and generating transgenic plants. Long-distance graft transmission of PcFT2 in both annual and perennial plants was confirmed using a 35S::PcFT2-YFP construct. Ectopic overexpression of PcFT2 caused early flowering in tobacco but not in apple. Transgenic apples were less sensitive to short-day-induced dormancy, and this phenotype was also observed in wild-type apples grafted onto the transgenic plants. Comparison of PcFT2 protein structure to the paralogous FT proteins from apple and pear showed alterations that could influence protein structure and thus the florigen-activation complex. PcFT2 protein seems to function by promoting flowering as all other FT proteins in the annual plant tobacco while in the perennial plant apple PcFT2 does not promote flowering but delays senescence. This observation may hint to a modified function of FT2 in perennial plants.

Sunscald of apple (Malus × domestica Borkh.) fruit is a major cause for economical losses. It is widely accepted that exposure to high temperature and light increases the incidence and severity of this fruit injury, but the separate contribution of each factor and the mechanisms involved in the injury development in apples are not well understood. The present study aimed at the quantification of both temperature and light required for photo-oxidative sunscald development and for tolerance acquisition, under controlled conditions. In mid-summer, immature green ‘Smoothie’ apples from the inner (shaded) part of the canopy were picked biweekly and placed in the dark for 48 h at 25 °C for decondidting. Fruit were then exposed to a variety of temeprature and light regimes. Sunscald injury developed only when peel temperature exceeded 40 °C and was light-dependent. Maximum tolerance to sunscald was acquired in fruit pre-exposed to 38 °C for 24 h. This temporary tolerance gradually diminished with storage time at 25 °C. Development of brown stains within the bleached peel areas increased with temperature. Fv/Fm was negatively correlated with PFD and the duration of exposure to temperatures above 40 °C. Threshold values of Fv/Fm required for bleaching and browning of the peel were 0.11 and 0.072, respectively. A ‘photo-destructive quantum’ (PDQ) was characterized as the minimum absorbed energy required for peel bleaching. The calculated PDQ diminished with the rise in temperature. Pre-treatment with 5 and 10 mg L−1 methyl viologen increased fruit susceptibility to sunscald compared to controls, whereas 1 mg L−1 increased fruit tolerance to the combined injurious effect of light and heat. We conclude that sunscald is an expression of photo-oxidative stress in the fruit peel which is facilitated by peel temperatures above 40 °C. Short-term field tolerance is acquired by insolation at sub-injurious temperatures, but this tolerance is rather weak and cannot accommodate the harsh atmospheric conditions which prevail in the Israeli summer.

Most banana screenhouses are currently equipped with transparent screens, with a nominal shading of 8–15%. Two types of screens are widely used, woven or knitted, which differ in their texture and hence in their radiative and aerodynamic properties. These differences may induce different micro-climatic conditions and hence different atmospheric water demand, plant response and yield. We have investigated four types of screens based on different shading levels (8%, 10% or 13%) and different screen textures (woven or knitted). A large commercial banana plantation in Northern Israel was covered with patches of the four different screen types, each in 4–5 replicates, randomly located. Air velocity, temperature, humidity and net radiation were measured simultaneously below two out of the four screen types: the 10% woven and 10% knitted. The results showed that both the net radiation and air temperature were similar under these two screens. Nevertheless, under the knitted screen the horizontal mean air velocity was 18% higher and the specific humidity 8% lower than under the woven screen. Leaf lamina tearing (typical wind damage) and estimated reference evapotranspiration were higher under the knitted screen; the latter mainly during the fall–winter season. However, the horticultural measures of flowering and fruit yield characteristics were the same under all four screen types, with results typical of screenhouse banana plantations in this region. Hence, the results suggest a potential increase in water use efficiency under the woven as compared to the knitted screen.

Red sweet pepper (Capsicum annuum L. cv. Vergasa) was grown under Pearl or black (control) nettings with 35% shading to compare their effects on postharvest fruit quality. Fully colored fruits were harvested four times from October to December, and after each harvest they were stored at 7 °C for 16 days, followed by 20 °C for 3 days. Compared with black netting, the Pearl netting significantly reduced water loss, decay incidence and titratable acidity, and increased fruit firmness, elasticity, ascorbic acid level and antioxidant activity, but did not change the external quality, except for a decrease in chroma. The effects of the Pearl netting were most pronounced on late-season-harvested fruits. Significant interactions were obtained between the shade netting type and harvest date. Analysis of correlations among the quality traits indicated that increased antioxidant activity and ascorbic acid content were tightly associated with the postharvest fruit quality attributes affected by the Pearl netting. Comparison between climatic parameters under the two netting treatments revealed no significant difference in air temperature and humidity, but the Pearl netting increased not only transmittance of long-waveband light but also light intensity within plant canopy. The findings suggest that the Pearl netting is more effective in maintaining postharvest sweet pepper fruit quality than the traditional black netting, especially at late-season harvests. The effects of the Pearl netting can be related to alteration of antioxidant levels induced by the pre-harvest light environment manipulation.

Photoselective nets provide a new tool for light-quality manipulation in protected horticulture. We have studied the photosynthetic activity and leaf characteristics of sweet pepper (Capsicum annuum L. 'Tirza') grown under Pearl, Yellow and Red ChromatiNet® of the same shading factor. Their transmitted light is enriched by diffused light of 390, 520 and 590 nm and up, under the Pearl, Yellow and Red net, respectively, compared to an equivalent black shade net. Leaves of the same age and position were found to respond to this light quality manipulation in the following way: The highest net assimilation (Anet) and stomatal conductance (g(sw)) at midday were obtained under the Pearl net, followed by the Red and Yellow nets; the effective quantum yield (ΔF/F'm) was lowest under the Red net; despite of no significant difference in stomatal density, stomatal width was largest under the Pearl net, and smallest under the Yellow; both leaf thickness and palisade/sponge ratio were lowest under the Yellow net; the specific leaf weight (SLW) was largest under the Pearl net; chlorophyll content was lowest, while chlorophyll/carotenoid ratio was highest under the Red net. Therefore, photoselective shading can differentially affect leaf parameters and activities in sweet pepper. From the aspect of photosynthesis, the Pearl net appears to be more suitable for sweet pepper summer production in Israel, or equivalent areas.

SummaryThe effect of coloured shade-nets on the growth characteristics of several ornamental crops for the cut flower industry was tested. Overall, seven cultivars of four different species (lisianthus, sunflower, Trachelium, and Ornithogalum) were grown under four different coloured nets. There was a marked impact of net colour on flowering stem length and cut flower weight in three of the four species. Compared to the black, neutral net, growth under red and yellow coloured shade-nets caused a significant increase in flowering stem-length in three lisianthus and two sunflower cultivars. This increase in stem-length was accompanied by an increase in cut flower weight in the lisianthus cultivars grown under the yellow net. Plants grown under the blue net had a shorter stem-length in two sunflower cultivars and in Trachelium. This effect was accompanied by a lower weight of Trachelium cut flowers grown under the blue net. The diameters of Trachelium and sunflower inflorescences were smaller, and those of l...

To determine whether the color of illumination under which plants are grown, affects the structure of photosynthetic antennae, pea plants were grown under either blue-enriched, red-enriched, or white light. Carotenoid content of isolated chloroplasts was found to be insensitive to the color of illumination during growth, while chlorophyll a/b ratio in chloroplasts isolated from young illuminated leaves showed susceptibility to color. Color of illumination affects the LHCII chiral macroaggregates in intact leaves and isolated chloroplasts, providing light-induced alteration of the handedness of the LHCII chiral macroaggregate, as measured with circular dichroism and circularly polarized luminescence. The susceptibility of handedness to current illumination (red light excitation of chlorophyll fluorescence) is dependent on the color under which the plants were grown, and was maximal for the red-enriched illumination. We propose the existence of a long-term (growth period) color memory, which influences the susceptibility of the handedness of LHCII chiral macroaggregates to current light.

SummaryMany green decorative branches (“greens”), which form an important part of ornamental bouquets, originatefrom shade plants, and are commercially grown in Israel under black shade nets. We are studying the possibility to manipulate the nature of the vegetative growth of greens to improve the yield and quality desirable for various ornamental crops.The present paper summarizes the results obtained for Pittosporum variegatum grown under shade nets of various optical properties: green, red, blue, grey, black, and reflective. The knitting density and design of the nets were adjusted to give the same transmittance of sunlight in the PAR (photosynthetically active radiation, 400–700.nm) region. The experiments were carried out in a commercial plot. A single layer of 50% shade net covered the plot during thewinter season, and two layers in the summer, according to common practice. Data were collected mostly during the second growth year. The main results obtained included: (i) pronounced stimulation of bra...

Circular polarization of luminescence (CPL; Steinberg IZ (1978) Annu Rev Biophys Bioeng 7: 113–137) was applied to study pea chloroplasts in different structural states. The structural changes of chloroplasts were induced by variation of osmotic pressure, concentration of magnesium-ions or photoinhibition. Both large CPL and psi-type circular dichroism (psi, polymerization and salt induced) signals appeared in the presence of granal macrostructure and were sensitive to structural changes of the grana. The relation was studied between the amount of CPL expressed as an emission anisotropy factor gem and amplitudes of the red psi-type CD bands. The positive psi-type CD band was not directly correlated with gem possibly due to a large contribution of circular intensity differential scattering to the measured CD spectra. However, a linear correlation between the amplitude of the negative psi-type CD band and gem was found. The CPL signal of pea chloroplasts was attributed to a psi-type origin, which is observed in macroaggregates with densely packed chromophores with a long-range chiral order, and directly depends on the level of macroorganization. With the use of CPL-based microscopy, the long-range packing of LHC II particles can be studied in individual chloroplasts in future. In addition, the CPL method in general allows the study of the macro-organization of grana in green leaves, where conventional light-transmission methods fail.

A sulfide-quinone oxidoreductase (SQR, EC 1.8.5.'.) has been purified to homogeneity from chromatophores of the non-sulfur purple bacterium Rhodobacter capsulatus DSM 155. It is composed of a single polypeptide with an apparent molecular mass of about 55 kDa, exhibiting absorption and fluorescence spectra typical for a flavoprotein and similar to the SQR from the cyanobacterium Oscillatoria limnetica. From N-terminal and tryptic peptide sequences of the pure protein a genomic DNA clone was obtained by polymerase chain reaction amplification. Its sequence contains an open reading frame of 1275 base pairs (EMBL nucleotide sequence data base, accession no. X97478X97478) encoding the SQR of R. capsulatus. The deduced polypeptide consists of 425 amino acid residues with a molecular mass of 47 kDa and a net charge of +9. The high similarity (72%)/identity (48%) between the N termini of the cyanobacterial and the bacterial enzyme was confirmed and extended. Both enzymes exhibit the FAD/NAD(P) binding betaalphabeta-fold (Wierenga, R. K., Terpstra, P., and Hol, W. G. S. (1986) J. Mol. Biol. 187, 101-107). The complete sequence of the SQR from R. capsulatus shows further similarity to flavoproteins, in particular glutathione reductase and lipoamide dehydrogenase. The cloned sqr was expressed in Escherichia coli in a functional form.

We investigated the effect of photoinhibitory illumination on the chiral macroorganization of the chromophores in spinach thylakoid membranes. By measuring circular dichroism (CD), we found that prolonged (15 min) illumination of membranes with intense white light led to irreversible diminishment of the main CD bands originating from the chiral macroorganization of the chromophores. The irreversible decrease of the main CD bands showed a nearly linear correlation with the extent of photoinhibition which was determined by chlorophyll fluorescence induction. CD measurements also revealed that the excitonic CD bands, which are given rise by short-range interactions between the chromophores inside the complexes or particles, were largely insensitive to the photoinhibitory illumination of the membranes. These data show that, whereas photoinhibitory treatment has no perceptible effect on the molecular architecture of the bulk of the pigment–protein complexes, it leads to a disorganization of their macroarray, and an irreversible disassembly of the chirally organized macrodomains.

Insects use optical cues for host finding, flight orientation and navigation. Therefore, manipulation of these optical cues can interfere with host finding and dispersal of insect pests. Sucking insect pests, such as aphids, whiteflies and thrips, cause great economic losses for growers of agricultural crops worldwide. These pests cause direct feeding damages and often transmit viruses to crop plants. These insects have receptors for UV light (peak sensitivity at 360 nm) and for green-yellow light (peak sensitivity at 520–540 nm). The absence of UV deters these pests and decreases their dispersal rate. Green-yellow color induces landing and favors settling (arresting) of these insects. High level of reflected sunlight (above 25% of sun radiation) also deters landing of these insects. Thus, optical cues can be used to divert pests away from crop plants. This can be achieved by incorporating optical additives to mulches (below plants), to cladding materials (plastic sheets, nets and screens above plants) or to other objects elsewhere in the growing environment. The optical properties such as size, shape, and contrast of the color cue greatly affect the response of the insect. Non-persistent viruses can be transmitted only within minutes to a few hours after aphids acquired them. Thus, a delay of the viruliferous aphids with an arresting color can reduce the efficacy of viral transmission. Results of many studies indicate that optical manipulation can reduce infestation levels of sucking pests and the incidence of viral diseases they transmit. Future development of this technology must be compatible with the requirements for plant production and biocontrol. Optical manipulations can be a part of integrated pest management (IPM) programs for both open field and protected crops. This chapter includes a review of the published literature, results of our studies, and suggestions for future research and development of this technology.

The reduction by sulfide of exogenous ubiquinone is compared to the reduction of cytochromes in chromatophores of Rhodobacter capsulatus. From titrations with sulfide values for Vmax of 300 and 10 μmoles reduced/mg bacteriochlorophyll a·h, and for Km of 5 and 3 μM were estimated, for decyl-ubiquinone-and cytochrome c-reduction, respectively. Both reactions are sensitive to KCN, as has been found for sulfide-quinone reductase (SQR) in Oscillatoria limnetica, which is a flavoprotein. Effects of inhibitors interfering with quinone binding sites suggest that at least part of the electron transport from sulfide in R. capsulatus employs the cytochrome bc 1-complex via the ubiquinone pool.

Olive leaves are known to mature slowly, reaching their maximum photosynthetic activity only after full leaf expansion. Poor assimilation rates, typical to young olive leaves, were previously associated with low stomata conductance. Yet, very little is known about chloroplast biogenesis throughout olive leaf development. Here, the photosynthetic activity and plastids development throughout leaf maturation is characterized by biochemical and ultrastructural analyses. Although demonstrated only low photosynthetic activity, the plastids found in young leaves accumulated both photosynthetic pigments and proteins required for photophosphorylation and carbon fixation. However, Rubisco (ribulose-1,5-bisphosphate carboxylase-oxygenase), which catalyzes the first major step of carbon fixation and one of the most abundant proteins in plants, could not be detected in the young leaves and only slowly accumulated throughout development. In fact, Rubisco levels seemed tightly correlated with the observed photosynthetic activities. Unlike Rubisco, numerous proteins accumulated in the young olive leaves. These included the early light induced proteins, which may be required to reduce the risk of photodamage, because of light absorption by photosynthetic pigments. Also, high levels of ribosomal L11 subunit, transcription factor elF-5A, Histones H2B and H4 were observed in the apical leaves, and in particular a plastidic-like aldolase, which accounted for approximately 30% of the total proteins. These proteins may upregulate in their levels to accommodate the high demand for metabolic energy in the young developing plant tissue, further demonstrating the complex sink-to-source relationship between young and photosynthetically active mature leaves.

This paper describes measurements of electrical potentials generated by renal Na/K-ATPase reconstituted into proteoliposomes, utilizing the anionic dye, oxonol VI. Calibration of absorption changes with imposed diffusion potentials allows estimation of absolute values of electrogenic potentials. ATP-dependent Nacyt/Kexc exchange in K-loaded vesicles generates large potentials, up to 250 mV. By comparing initial rates or steady-state potentials with ATP-dependent 22Na fluxes in different conditions, it is possible to infer whether coupling ratios are constant or variable. For concentrations of Nacyt (2-50 mM) and ATP (1-1000 microM) and pH's (6.5-8.5), the classical 3Nacyt/2Kexc coupling ratio is maintained. However, at low Nacyt concentrations (less than 0.8 mM), the coupling ratio is apparently less than 3Nacyt/2Kexc. ATP-dependent Nacyt/congenerexc exchange in vesicles loaded with Rb, Cs, Li and Na is electrogenic. In this mode congeners, including Naexc, act as Kexc surrogates in an electrogenic 3Nacyt/2congenerexc exchange. (ATP + Pi)-dependent Kcyt/Kexc exchange in K-loaded vesicles is electroneutral. ATP-dependent "uncoupled" Na flux into Na- and K-free vesicles is electroneutral at pH 6.5-7.0 but becomes progressively electrogenic as the pH is raised to 8.5. The 22Na flux shows no anion specificity. We propose that "uncoupled" Na flux is an electroneutral 3Nacyt/3Hexc exchange at pH 6.5-7.0 but at higher pH's the coupling ratio changes progressively, reaching 3Na/no ions at pH 8.5. Slow passive pump-mediated net K uptake into Na- and K-free vesicles is electroneutral, and may also involve Kcyt/Hexc exchange. We propose the general hypothesis that coupling ratios are fixed when cation transport sites are saturated, but at low concentrations of transported cations, e.g., Nacyt in Na/K exchange and Hexc in "uncoupled" Na flux, coupling ratios may change.

Circularly polarized chlorophyll luminescence (CPL) may serve as a measure of chiral macroaggregates of the light-harvesting chlorophyll-protein complexes (LHC II) in both isolated chloroplasts and intact leaves (Gussakovsky et al (2000) Photosynth Res 65: 83–92). In the present work, we applied the CPL approach to study the effect of fast (1–2 min) thermal impacts on LHC II macroaggregates. The results revealed unexpected temperature-response kinetics, composed of initial bell-shaped changes in the CPL signal, followed by degradation down to a steady state (equilibrium). The bell-shape effect was dependent upon illumination, and vanished in the dark. A mathematical analysis of the temperature-response kinetics uniquely indicated that LHC II chiral macroaggregates may persist in both left- and right-handed forms. These forms differ in their response to high temperatures. Both forms are more thermostable in leaves than in isolated chloroplasts. The cooperative degradation of LHC II macroaggregates, which is induced by the thermal impact, is irreversible. It is therefore suggested that the native LHC II macroaggregates are stable, stationary, non-equilibrium, spatially heterogeneous (dissipative) structures. The dissipative properties probably allow the interconversion between left- and right-handed forms under perturbation by certain factors. Illumination probably serves as one such perturbation factor, initiating the interconversion of dark-adapted, left-handed to light-dependent, right-handed LHC II macroaggregates. The chiral heterogeneity of the LHC II macroaggregates is a newly revealed aspect which needs to be taken into consideration in future circular dichroism or CPL studies.

Circularly polarized luminescence (CPL) is a powerful technique to study the macroorganization of photosynthetic light-harvesting apparatus in vivo and in vitro. It is particularly useful for monitoring environmental stress induced molecular re-organization of thylakoid membranes in green leaves. The current study focuses on two questions which are important to perform and interpret such experiments: how does CPL depend on the excitation wavelength and how on the orientation of the granal thylakoids. CPL and circular dichroism (CD) of pea chloroplasts were complementarily applied when chloroplasts were either in suspension or trapped in a polyacrylamide gel (PAAG) after alignment in a magnetic field. In contrast to the CD spectrum, the CPL signal was found to be independent of the excitation wavelength in both the Soret and the Qy absorption region for chloroplasts in both suspension and PAAG. The improved resolution of luminescence measurements revealed a relatively small negative CPL band in addition to the previously described large positive band. No effect of photoselection upon excitation on the CPL spectra was detected. The CPL intensity at 690 nm at the edge of the granal thylakoids was found to be higher than at the face of the grana suggesting the CPL anisotropy.

Fructokinases catalyze the key step of fructose phosphorylation in plants. LeFRK2, the major fructokinase-encoding gene in tomato plants, is abundantly expressed in roots, stems, and fruits. To analyze the role of LeFRK2 in plant development, we analyzed transgenic tomato plants with sense and antisense expression of StFRK, the potato homolog of LeFRK2. Increased fructokinase activity had no effect. However, plants in which LeFRK2 was specifically suppressed, either via antisense suppression or via co-suppression, exhibited growth inhibition and wilting of young leaves at daytime. Grafting experiments indicated that a stem interstock of antisense plants was sufficient to inhibit growth and cause leaf wilting. Stem secondary xylem exhibited particular suppression of LeFRK2 and the area of active xylem, estimated by eosin uptake, was significantly smaller in antisense stem compared to that of wild-type plants. These results suggest that LeFRK2 might be required for proper development of xylem that affected growth and wilting.

Most of the symplastic water transport in plants occurs via aquaporins, but the extent to which aquaporins contribute to plant water status under favorable growth conditions and abiotic stress is not clear. To address this issue, we constitutively overexpressed the Arabidopsis plasma membrane aquaporin, PIP1b, in transgenic tobacco plants. Under favorable growth conditions, PIP1b overexpression significantly increased plant growth rate, transpiration rate, stomatal density, and photosynthetic efficiency. By contrast, PIP1b overexpression had no beneficial effect under salt stress, whereas during drought stress it had a negative effect, causing faster wilting. Our results suggest that symplastic water transport via plasma membrane aquaporins represents a limiting factor for plant growth and vigor under favorable conditions and that even fully irrigated plants face limited water transportation. By contrast, enhanced symplastic water transport via plasma membrane aquaporins may not have any beneficial effect under salt stress, and it has a deleterious effect during drought stress.

The role of increased oxidation induced by successive stresses of chilling and high light in the induction of leaf abscission was studied in Ixora coccinea plants in relation to auxin metabolism and oxidative processes. Exposure of plants following dark chilling (7 degrees C for 3 days) to high light (500-700mgr;mol m-2 s-1 photosynthetically active radiation) for 5 h at 20-25 degrees C enhanced chilling-induced leaf abscission. This abscission was inhibited by pretreatment with the antioxidant butylated hydroxyanisole, alpha-naphthaleneacetic acid or the ethylene action inhibitor, 1-methylcyclopropene. The oxidative processes initiated during the low light period following the dark chilling period, such as indoleacetic acid (IAA) decarboxylation and lipid peroxidation, were further enhanced by subsequent exposure to high light. Photoinhibition, expressed by the reduction of the chlorophyll fluorescence parameter Fv/Fm, was evident following exposure to high light, irrespective of the temperature of the pretreatment, but this reduction persisted only in chilled plants. This suggests that oxidative processes generated during and after the chilling period might have inhibited the recovery from photoinhibition. The chilling stress under darkness induced a 60% reduction in superoxide dismutase (SOD) activity and significant increases (130-600%) in the activities of several other antioxidative enzymes. These data suggest that the chilling-induced reduction in SOD activity may well be responsible for the increase in the oxidative stress induced by the subsequent light treatment, as expressed by the increased enzymatic activities. Taken together, this study provides further support for the involvement of oxidative processes in the events occurring in tissues exposed to sequential chilling and light stresses, leading to reduction in free IAA content in the abscission zone and to leaf abscission.

Chilling-sensitivity is an important agricultural problem in both the U.S. and Israel. Most research attention has focused so far on herbaceous crop plants, even though the problem is also acute in the fruit tree industry. Under BARD funding we made substantial progress in identifying the mechanisms involved in the disruption of photosynthesis following a chill in mango. Our investigation with fruit trees has been substantially accelerated by drawing on our knowledge and experience with herbaceous crops. The four original research objectives, focused or discovering the underlying mechanisms of chill-induced inhibition of photosynthesis in fruit trees, and the main achievements are listed below. [1] Separating stomatal from non-stomatal components of chilling on photosynthesis in fruit trees. We found evidence that the dark chill-induced inhibition of photosynthesis in mango was E combination of both stomatal and mesophyll components. [2] Differentiating photo damage from light-induced photo protection of photosystem II (PSII). Dark chilling exacerbate high light photoinhibition, as a result of primary inhibition in the carbor reduction cycle. Nevertheless, in Israeli orchards we observed chronic photoinhibition of PSII photochemistry in the winter. This photo damage was reversible over a few days if sunlight was attenuated with filters or night temperature rose. Practical implications of this finding deserve further investment. Additional achievement was the development of a new biophysical tool to study macro-structural changes of LHCII particles in intact, attached leaves. [3] Determine the role of oxidative stress in the dark-chilling-induced inhibition, with emphasis on oxygen radical scavenging, lipid peroxidation and redox-controlled carbon-cycle enzymes. We found an increase in lipid peroxidation following a dark chill, and partial protective effects or an antioxidant. However, the photoinhibition observed in mango orchards in Israel during the winter did not appear to be a general oxidative stress. [4] Investigate whether chilling interferes with the diurnal and circadian rhythm of gene expression of key photosynthetic proteins as has been shown for chilling-sensitive crop plants. The results indicated that most of the circadian rhythm in photosynthesis was due to reduced lea: internal CO2 concentrations during the subjective night, as a result of rhythmic stomatal closure Chilling-induced interference with circadian timing in mango, does not play the central role in chilling inhibition of photosynthesis that has previously been demonstrated in certain chilling sensitive herbaceous plants. Practical implications of the research achievements are feasible, but require few more years of research.

The effect of a cold night on photosynthesis in herbaceous chilling-sensitive crops, like tomato, has been extensively studied and is well characterized. This investigation examined the behaviour of the sub-tropical fruit tree, mango, to enable comparison with these well-studied systems. Unlike tomato, chilling between 5 degrees C and 7 degrees C overnight produced no significant inhibition of light-saturated CO(2) assimilation (A:) during the first hours following rewarming, measured either under controlled environment conditions or in the field. By midday, however, there was a substantial decline in A:, which could not be attributed to photoinhibition of PSII, but rather was associated with an increase in stomatal limitation of A: and lower Rubisco activity. Overnight chilling of tomato can cause severe disruption in the circadian regulation of key photosynthetic enzymes and is considered to be a major factor underlying the dysfunction of photosynthesis in chilling-sensitive herbaceous plants. Examination of the gas exchange of mango leaves maintained under constant conditions for 2 d, demonstrated that large depressions in A: during the subjective night were primarily the result of stomatal closure. Chilling did not disrupt the ability of mango leaves to produce a circadian rhythm in stomatal conductance. Rather, the midday increase in stomatal limitation of A: appeared to be the result of altered guard cell sensitivity to CO(2) following the dark chill.

The gene encoding sulfide-quinone reductase (SQR; E.C.1.8.5.′), the enzyme catalyzing the first step of anoxygenic photosynthesis in the filamentous cyanobacterium Oscillatoria limnetica , was cloned by use of amino acid sequences of tryptic peptides as well as sequences conserved in the Rhodobacter capsulatus SQR and in an open reading frame found in the genome of Aquifex aeolicus . SQR activity was also detected in the unicellular cyanobacterium Aphanothece halophytica following sulfide induction, with a V max of 180 μmol of plastoquinone-1 (PQ-1) reduced/mg of chlorophyll/h and apparent K m values of 20 and 40 μM for sulfide and quinone, respectively. Based on the conserved sequences, the gene encoding A. halophytica SQR was also cloned. The SQR polypeptides deduced from the two cyanobacterial genes consist of 436 amino acids for O. limnetica SQR and 437 amino acids for A. halophytica SQR and show 58% identity and 74% similarity. The calculated molecular mass is about 48 kDa for both proteins; the theoretical isoelectric points are 7.7 and 5.6 and the net charges at a neutral pH are 0 and −14 for O. limnetica SQR and A. halophytica SQR, respectively. A search of databases showed SQR homologs in the genomes of the cyanobacterium Anabaena PCC7120 as well as the chemolithotrophic bacteria Shewanella putrefaciens and Thiobacillus ferrooxidans . All SQR enzymes contain characteristic flavin adenine dinucleotide binding fingerprints. The cyanobacterial proteins were expressed in Escherichia coli under the control of the T7 promoter. Membranes isolated from E. coli cells expressing A. halophytica SQR performed sulfide-dependent PQ-1 reduction that was sensitive to the quinone analog inhibitor 2 n -nonyl-4-hydroxyquinoline- N -oxide. The wide distribution of SQR genes emphasizes the important role of SQR in the sulfur cycle in nature.

Anoxygenic photosynthesis with sulfide serving as the electron donor is a property unique to prokaryotes. Most photosynthetic bacteria can grow photoautotrophically using sulfide (as well as few other inorganic sulfur compounds) as electron donors for CO2 fixation (see ref. 1 for recent review). Cyanobacteria are exceptional in the world of phototrophic prokaryotes. With the closely related prochlorophytes, they are the only eubacteria that can perform plant-type oxygenic photosynthesis, using two photosystems in series and water as the electron donor. However, some species of cyanobacteria can facultatively shift to anoxygenic, sulfide-dependent photosynthesis in which only PS I is involved2-5. This unique capacity to shift between anoxygenic and oxygenic photosynthesis was discovered in various strains of cyanobacteria, evolutionarily distant from each other3. It was considered to represent a primitive relic of the evolution of photosynthesis6. Of these strains, the filamentous cyanobacterium Oscillatoria limnetica has been studied most extensively. O. limnetica shifts to anoxygenic photosynthesis 2—3 hours after incubation in the presence of sulfide and light, in an inducible process specific to sulfide7, 8. Depending on the growth conditions, the induced cells perform several sulfide-dependent reactions: CO2 fixation, 3, 7-9 H2 evolution10 or N2 fixation. 11

Inorganic reduced sulfur compounds serve as electron donors in many phototrophic and chemotrophic bacteria1’2. Sulfide-quinone reductase (SQR; E.C.1.8.5. “.) is one of the main enzymes involved in the oxidation of sulfide to sulfur. This sulfide induced membrane bound flavoprotein with an apparent MW of approximately 56-kDa was shown to be widely distributed among bacteria3. SQR was first purified from thylakoids of the cyanobacterium Oscillatoria limnetica4 and, more recently, from membranes of the ”non-sulfur“ purple bacterium Rb. capsulatus5. The sqr-gene of Rb. capsulatus had been sequenced and functionally expressed in E. coli5. Rb. capsulatus oxidizes sulfide only to the redox level of sulfur. However, it is not known, whether SQR is the only sulfide oxidizing enzymatic system in this bacterium. Here we demonstrate that Rb. capsulatus looses its capability of sulfide dependent growth after inactivation of SQR. In former investigations it was shown that expression of SQR in Rb. capsulatus depends on sulfide and is possibly modulated by oxygen6. This results were achieved using a mutant strain with the genes of the luciferase of Vibrio fischeri (luxAB) inserted behind the putative sqrpromotor. However, the activity of the luciferase itself depends on oxygen. For this reason a second reporter gene, the alkaline phosphatase of E. coli (PhoA), was used in this investigation.

A chiral molecule characterized by different absorption of left- and right-handed polarized light (CD), is expected to emit circularly polarized light at non-polarized excitation (circular polarization of luminescence, CPL) [1,2]. The CPL is expressed by the emission anisotropy factor gem which is the weighted difference between the left- and right-handed circularly polarized components of light emitted: (1) where p and m are the electric and magnetic dipole moment operators respectively, A’ and B’ are the quantum states involved in the emission processes, Ple and Pre are probabilities of left- and right-handed circularly polarized emission, respectively, f and Δf are the total luminescence intensity and its circularly polarized part, respectively. When the left-handed part is larger, the gem-factor is positive. More detailed information on the CPL method was reviewed [1–3].

Reduction of cytochromes in chlorosome-free membranes of Chlorobia was studied anaerobically, with an LED array spectrophotometer. For Chlorobium tepidum these membranes contained 0.2 moles cytochrome per mole of bacteriochlorophyll a. The observed change upon complete reduction of oxidized membranes with dithionite could be satisfactorily fitted with three cytochrome components having absorption peaks at 553 (cyt c), 558 and 563 nm (cyt b), in relative amounts of 5:1:2. About 20% of total cytochrome 553 were reducible by ascorbate. Menaquinol reduced all of the 553-component, and this reduction was sensitive to stigmatellin, NQNO and antimycin A. The reduction was insensitive to KCN. However, it was transient at low concentrations of menaquinol in the absence of KCN, but permanent in its presence, demonstrating that electron transport into an oxidation pool was blocked. The 563-component was only slightly reduced by menaquinol unless NQNO or antimycin were present. The stimulation of cytochrome 563-reduction by these inhibitors was more pronounced in the presence of ferricyanide. This phenomenon reflects ‘oxidant-induced reduction’ of cytochrome b and demonstrates that a Q-cycle is operative in Chlorobia. Also, sulfide fully reduced cytochrome 553, but more slowly than menaquinol. KCN inhibited in this case, as did stigmatellin, NQNO and antimycin A. NQNO was a better inhibitor than antimycin A. Cytochrome 563 again was hardly reduced unless antimycin A was added. The effect was more difficult to observe with NQNO. This supports the conclusion that sulfide oxidation proceeds via the quinone pool and the cytochrome bc-complex in green sulfur bacteria.

Circularly polarized chlorophyll luminescence (CPL) was recently shown to be an effective tool for the study of chiral macroaggregate formation of the light-harvesting chlorophyll a/b pigment–protein complexes (LHCIIs) in isolated chloroplasts. The CPL measuring system was modified to study green leaves. Spectral and intensity features of pea leaf CPL signals suggested that CPL indeed detected chiral macroaggregates. The signals were found to depend on the excitation vs emission optical alignment, as well as on the side (adaxial or abaxial) of the leaf. Illumination of attached leaves with either low intensity or strong photoinhibitory light did not affect the chiral macroaggregation status. In contrast, the induction of drought stress in detached leaves led to full disruption of the chiral macroaggregates. The disruption developed gradually during slow dehydration, whereas under fast, heat-stimulated dehydration it was manifested in cooperative diminution of both CPL and photochemical capacity. Simultaneous exposure of the leaves to photoinhibitory illumination and fast dehydration resulted in negative CPL signals. The results demonstrate the potency of CPL as a non-destructive tool for structural studies of LHCII in intact leaves under varying environmental conditions.

Many green-decorative branches (“Greens”) and cut flowers are commercially grown under shade nets, for both the reduction of the natural intercepted sunlight as well as physical protection. The most commonly used are black nets, which do not affect the visible light spectrum. In the work presented here we have studied the effects of shade nets of varying optical properties on the vegetative and flowering responses of ornamental plants, searching for nets that will specifically induce a desired behavior, thus gaining benefits in addition to the mere shading. Nets of different transmittance spectra, light scattering, reflectance and thermal properties are being studied for their effect on the vegetative growth of several Greens: Pittosporum variegatum, Ruscus hypoglossum, and Leather-leaf fern. The knitting density of all nets has been adjusted to have the same percent shading in the PAR (photosynthetically active radiation) range of the spectrum for all nets investigated. Experiments were carried out in commercial plots. Data were collected for microclimate, physiological and horticultural parameters. The main results obtained so far: i) pronounced stimulation of the vegetative growth under the Red net; ii) dwarfing by the Blue net; iii) the Grey net markedly enhanced branching, yielding “bushy” plants with short side branches; iv) the reflective, thermal net (Aluminet®) enhanced side, long branching (in Pittosporum). Recently we have applied a similar approach to cut flowers such as Lupinus luteus, Lisianthus eustoma, and Dubium ornitugalum, and obtained dramatic effects of some of the nets on both the vegetative development and flowering behavior. The results to be presented, suggest that sophistication of the use of shade nets can lead to better agricultural performance.

Sugars are key regulatory molecules that affect diverse processes in higher plants. Hexokinase is the first enzyme in hexose metabolism and may be a sugar sensor that mediates sugar regulation. We present evidence that hexokinase is involved in sensing endogenous levels of sugars in photosynthetic tissues and that it participates in the regulation of senescence, photosynthesis, and growth in seedlings as well as in mature plants. Transgenic tomato plants overexpressing the Arabidopsis hexokinase-encoding gene AtHXK1 were produced. Independent transgenic plants carrying single copies of AtHXK1 were characterized by growth inhibition, the degree of which was found to correlate directly to the expression and activity of AtHXK1. Reciprocal grafting experiments suggested that the inhibitory effect occurred when AtHXK1 was expressed in photosynthetic tissues. Accordingly, plants with increased AtHXK1 activity had reduced chlorophyll content in their leaves, reduced photosynthesis rates, and reduced photochemical quantum efficiency of photosystem II reaction centers compared with plants without increased AtHXK1 activity. In addition, the transgenic plants underwent rapid senescence, suggesting that hexokinase is also involved in senescence regulation. Fruit weight, starch content in young fruits, and total soluble solids in mature fruits were also reduced in the transgenic plants. The results indicate that endogenous hexokinase activity is not rate limiting for growth; rather, they support the role of hexokinase as a regulatory enzyme in photosynthetic tissues, in which it regulates photosynthesis, growth, and senescence.

Photoinhibition (PI; light stress) was studied in leaves of 3-year-old, potted, mango plants, following changes in chlorophyll fluorescence characteristics. Iron deficiency was induced by calcareous soil mix. It was found that during summer days, the photochemical quantum yield (measured as Fv: Fm) was reduced by 15% to 30% in sun-exposed leaves by noon (under 2000 μmol·m–2·s–1). This apparent PI was mostly recovered by the next day. In Fe-deficient plants, the reversible PI was enhanced further. In addition, Fe-deficiency increased the “I” phase (i.e., the QB-nonreducing centers) and the non-photochemical quenching (qN) and reduced the photochemical quenching (qP). Photosynthesis rates in the deficient plants were half the control rates. Overnight chilling at 7C, which by itself did not affect the Fv: Fm ratio much, dramatically increased the susceptibility of the plants to photodamage on the following day (indicated by fluorescence and gas exchange assays). This postchilling PI was mostly irreversible but could slowly (within about 1 week) be recovered in a thermostated greenhouse. These results agree with our observations of field-grown Fe-deficient mango trees that develop severe necrosis and leaf-drop each autumn in the Jordan Valley when the days are still sunny and warm and the nights turn cold.

Energy dependent reverse electron flow reactions in isolated thylakoids provide a unique tool to study, in the dark, the coupling between the ATP synthase, proton transport and the electron transfer system. Appropriate experimental conditions have been established to follow experimentally the following reactions: 1. ATP driven proton uptake into the inner-thylakoid space, which requires preactivation of the ATP synthase. 2. ATP driven reverse electron transport, which involves proton transport as an intermediate, and results in the reduction of QA by an externally added electron donor. 3. ATP driven luminescence, which requires the presence of an oxidized partner on the water side of photosystem II, and involves electron transport from QB to QA. 4. ΔpH driven reverse electron flow, which does not require the participation of the ATP synthase, and uses reduced intermediates between the two photosystems as electron donors for the reduction of QA. 5. ΔpH driven luminescence which again uses reduced intermdiates between the two photosystems as electron donors for QA reduction, and requires the presence of an oxidized partner on the water side of photosystem II. Several of these reactions have been shown to occur in intact chloroplasts and may provide an important regulatory mechanism in vivo.

1. Quercetin, a flavonoid which acts as an energy transfer inhibitor in photophosphorylation is shown to inhibit the P-ATP exchange activity of membrane-bound CF1 and the ATPase activity of isolated CF1. Quercetin, affects also the proton uptake in chloroplasts in a manner similar to that of dicyclohexyl-carbodiimide. 2. The light-dependent proton uptake in EDTA-treated chloroplasts is stimulated by quercetin. In untreated chloroplasts quercetin has a dual effect: it enhances at pH above 7.5 while at lower pH values it decreases the extent of H+ uptake. Similar effects were obtained with dicyclohexylcarbodiimide. 3. Like quercetin, dicyclohexylcarbodiimide was also found to inhibit the ATPase activity of isolated CF1. 4. Quercetin inhibits uncoupled electron transport induced by either EDTA-treatment of chloroplasts or by addition of uncouplers. Quercetin restores H+ uptake in both types of uncoupled chloroplasts. 5. The mode of action of quercetin and dicyclohexylcarbodiimide in photo-phosphorylation is discussed, and interaction with both CF1 and F0 is suggested.

BAL (2,3-dithiopropan-1-ol) treatment of chloroplasts has previously been reported to induce a block in electron transport from water to NADP+ at a site preceding plastocyanin [Belkin et al. (1980) Biochim. Biophys. Acta 766, 563-569]. In the present work the block was further characterized. The following properties of BAL treatment are described. Inhibition of electron transport from water to lipophilic acceptors but not to silicomolybdate. Inhibition of the slow, sigmoidal phase of chlorophyll a fluorescence induction. Inability of N,N,N',N',-tetramethyl-p-phenylenediamine to bypass the inhibition of NADP+ photoreduction with water as the electron donor. Inhibition of electron transport from externally added quinols to NADP+. Inhibition of cytochrome f reduction by photosystem II, but not its oxidation by photosystem I. Inhibition of cytochrome b6 turnover and cytochrome f rereduction after single-turnover flash illumination under cyclic electron-flow conditions. The BAL-induced block is therefore located between the secondary quinone acceptor (QB) and the cytochrome b6f complex. It was further found that (a) the isolated cytochrome complex is not inhibited after BAL treatment; (b) BAL-reacted plastoquinone-1 inhibits electron transport in chloroplasts; (c) BAL does not inhibit electron transport in chromatophores of Rhodospirilum rubrum or Rhodopseudomonas capsulata. It is suggested that the inhibition of electron transport in chloroplasts results from specific reaction of BAL with the endogenous plastoquinone.

Utilizing oxonol VI as a transmembrane electric potential indicating dye, chloroplasts are shown to develop rapid transient light-induced and ATP-induced potentials. Following the large transient signal smaller steady-state potentials are maintained with either driving system. The ATP-induced potential in the dark depends upon preactivation of the light-triggered ATPase of the chloroplasts, and is inhibited by uncouplers, ionophores such as valinomycin, and energy-transfer inhibitors such as tentoxin, Dio-9 or DCCD. Nigericin increased the signal of both the light- and the ATP-induced reactions. The fact that relatively large transient membrane potentials are induced by either a dark-to-light transition or ATP in the dark provides an explanation for previously observed phenomena such as early kinetics of photophosphorylation and the ATP-induced luminescence.

The effect of 2,3-dimercaptopropan-1-ol (BAL) on photosynthetic electron transport was studied in Oscillatoria limnetica cells as well as in spinach chloroplasts. Preincubation of the cyanobacterial cells with BAL under aerobic conditions caused inhibition of the sulfide-dependent H 2 evolution. The inhibited site was bypassed by N , N , N ′, N ′-tetramethyl- p -phenylene diamine. Similarly, NADP photoreduction by spinach chloroplasts was inhibited by BAL pretreatment and restored by the addition of reduced 2,6-dichlorophenol-indophenol. These results accord previous data demonstrating damage of mitochondrial Rieske iron-sulfur protein after BAL treatment (Slater, E.C. and De Vries, S. (1980) Nature 288, 717–718). However, in chloroplasts there was no change in the Rieske center ESR spectrum after BAL treatment. When added anaerobically, BAL served as an electron donor for light-dependent, H 2 evolution in cyanobacteria or for NADP reduction in spinach chloroplasts. These reactions were insensitive to 3-(3′,4′-dichlorophenyl)-1,1dimethylurea (DCMU), but sensitive to 2,5-dibromothymoquinone or to aerobic pretreatment with BAL. The Rieske protein ESR signal of chloroplasts ( g = 1.89) which disappeared upon illumination in the presence of DCMU and methyl viologen, was restored by BAL. It is suggested that as opposed to its aerobic inhibitory effect, BAL donates electrons to the Rieske protein under anaerobic conditions.

Formation of a transmembrane electric potential coupled to ATP hydrolysis is demonstrated in chloroplast ATPase complex containing proteoliposomes. The ATP-induced signals were detected through absorbance changes of the membrane potential-responding dye oxonol VI. They were inhibited by the specific energy-transfer inhibitor, tentoxin and the ionophore valinomycin while stimulated by nigericin. Calibration of the transmembrane potential signal was possible by the application of a proton diffusion potential. The ATP-induced transmembrane potential was estimated to be 40–50 mV.

When the cytochrome b6/f complex from chloroplasts is incorporated into liposomes, reduction of external plastocyanin by plastoquinol is stimulated under uncoupling conditions. An extra H+/e− is ejected from the vesicles during the reaction, in addition to the scalar proton liberated from plastoquinol. This is stimulated by valinomycin/K+ and abolished under uncoupling conditions. Furthermore, the formation of a membrane potential during the reaction, negative inside the vesicles, is observed with the help of carbocyanine dyes. We conclude that the cytochrome b6/f complex, like the cytochrome bc1 from mitochondria, functions as an electrogenic proton translocator.

Cyclic electron flow around photosystem I plays an important role in providing sufficient ATP for the photosynthetic assimilation of COz [ 1,2]. Cyclic activity may be observed in isolated intact chloroplasts [2,3] but is lost, along with NADP+ reduction, during the isolation of naked thylakoids; however, addition of ferredoxin has been reported [4,5] to restore NADP+ reduction and cyclic photophosphorylation (i.e. phosphorylation resulting from cyclic electron flow around photosystem I). This reconstituted cyclic phosphorylation was found to resemble cyclic phosphorylation in intact chloroplasts in being inhibited by antimycin (a specific inhibitor of cyclic electron flow [1,6,7]), by the quinone-analogue DBMIB [6,8] or by poor redox poising [ 1,5,6,9,10]. The reconstituted cyclic system has special value in studies of the cyclic pathway because there is no permeability barrier isolating substrates, inhibitors and membrane probes from the outer thylakoid surface. It has, however, the disadvantage of requiring large amounts of exogenous ferredoxin while still not giving restoration of cyclic electron flow at the rates seen in intact chloroplasts. An improved procedure is reported here that gives envelope-free thylakoids retaining almost full activity of cyclic electron flow. A spectroscopic characteristic of intact chloroplasts is the slow (milliseconds rise time) electrochromic