Chlorophyll a/b-binding proteins: an extended family.

A large proportion of the chlorophyll in a plant is engaged in harvesting light energy and transferring it to the photochemical reaction centres. These 'antenna' chlorophylls are non-covalently bound to specific proteins to form chlorophyll-protein complexes. The chlorophyll a/b-binding (CAB) polypeptides are encoded by an extended family of nuclear genes. It has recently been discovered that other proteins not known to bind chlorophyll, the early light-inducible proteins (ELIPs), are also related and could be considered part of this family. We suggest that the latter proteins may be involved in pigment biosynthesis or in assembly of the thylakoid membrane.

The Circadian Oscillator Coordinates the Synthesis of Apoproteins and Their Pigments during Chloroplast Development.

Greening has been studied at circadian times of maximal and minimal levels of mRNA for the light-harvesting chlorophyll a/b binding protein in photosystem II (Cab mRNA) after circadian synchronization of etiolated barley plantlets (Hordeum vulgare cv Apex) by heat-shock treatments. It was found that greening occurs faster and without a lag period when illumination was started at the time of maximal Cab mRNA accumulation. This holds true for the rate of accumulation of Cab and early light-inducible protein mRNAs, the levels of their correspondent proteins, and the levels of chlorophyll a and b. When illumination was started at the time of Cab mRNA minimum, a lag in the appearance of all components mentioned above was observed. Under these conditions, the lag in chlorophyll b accumulation was by far more pronounced than that found for chlorophyll a. The circadian oscillation in the capacity of chlorophyll synthesis appears to be controlled via [delta]-aminolevulinic acid ([delta]-ALA) synthesis. [delta]-ALA accumulation after levulinic acid treatment is itself under circadian control; the maxima in stationary concentrations coincide with those of Cab mRNA levels. The amounts of protochlorophyllide and photoconvertible protochlorophyllide showed only minor differences between circadian minima and maxima, the levels being slightly lower during the time of minimum.

Expression of Early Light-Inducible Proteins in Flag Leaves of Field-Grown Barley.

Early light-inducible protein (ELIP) mRNA and protein levels were analyzed during maturation and senescence of barley (Hordeum vulgare L.) flag leaves under field conditions. The data clearly demonstrate that ELIP mRNA levels are related to the sunlight intensity before sample collection. Levels of mRNAs encoding both low and high molecular mass ELIPs fluctuate in parallel. Changes in mRNA levels are accompanied by corresponding changes in protein levels except for days when average temperatures are high. Comparison of flag leaves at different stages of development in spring and winter barley varieties suggests that light-stress-regulated ELIP gene expression is independent of the developmental stage of the leaves. Although chlorophyll content, photosystem II (PSII) efficiency, and 32-kD herbicide-binding protein of PSII levels decrease drastically after the onset of senescence, ELIP mRNA and protein still accumulate to high levels on bright days.

Differential control of xanthophylls and light-induced stress proteins, as opposed to light-harvesting chlorophyll a/b proteins, during photosynthetic acclimation of barley leaves to light irradiance

Barley (Hordeum vulgare L.) plants were grown at different photon flux densities ranging from 100 to 1800 &mgr;mol m-2 s-1 in air and/or in atmospheres with reduced levels of O2 and CO2. Low O2 and CO2 partial pressures allowed plants to grow under high photosystem II (PSII) excitation pressure, estimated in vivo by chlorophyll fluorescence measurements, at moderate photon flux densities. The xanthophyll-cycle pigments, the early light-inducible proteins, and their mRNA accumulated with increasing PSII excitation pressure irrespective of the way high excitation pressure was obtained (high-light irradiance or decreased CO2 and O2 availability). These findings indicate that the reduction state of electron transport chain components could be involved in light sensing for the regulation of nuclear-encoded chloroplast gene expression. In contrast, no correlation was found between the reduction state of PSII and various indicators of the PSII light-harvesting system, such as the chlorophyll a-to-b ratio, the abundance of the major pigment-protein complex of PSII (LHCII), the mRNA level of LHCII, the light-saturation curve of O2 evolution, and the induced chlorophyll-fluorescence rise. We conclude that the chlorophyll antenna size of PSII is not governed by the redox state of PSII in higher plants and, consequently, regulation of early light-inducible protein synthesis is different from that of LHCII.

Greening under high light or cold temperature affects the level of xanthophyll-cycle pigments, early light-inducible proteins, and light-harvesting polypeptides in wild-type barley and the chlorina f2 mutant

Etiolated seedlings of wild type and the chlorina f2 mutant of barley (Hordeum vulgare) were exposed to greening at either 5 degrees C or 20 degrees C and continuous illumination varying from 50 to 800 &mgr;mol m-2 s-1. Exposure to either moderate temperature and high light or low temperature and moderate light inhibited chlorophyll a and b accumulation in the wild type and in the f2 mutant. Continuous illumination under these greening conditions resulted in transient accumulations of zeaxanthin, concomitant transient decreases in violaxanthin, and fluctuations in the epoxidation state of the xanthophyll pool. Photoinhibition-induced xanthophyll-cycle activity was detectable after only 3 h of greening at 20 degrees C and 250 &mgr;mol m-2 s-1. Immunoblot analyses of the accumulation of the 14-kD early light-inducible protein but not the major (Lhcb2) or minor (Lhcb5) light-harvesting polypeptides demonstrated transient kinetics similar to those observed for zeaxanthin accumulation during greening at either 5 degrees C or 20 degrees C for both the wild type and the f2 mutant. Furthermore, greening of the f2 mutant at either 5 degrees C or 20 degrees C indicated that Lhcb2 is not essential for the regulation of the xanthophyll cycle in barley. These results are consistent with the thesis that early light-inducible proteins may bind zeaxanthin as well as other xanthophylls and dissipate excess light energy to protect the developing photosynthetic apparatus from excess excitation. We discuss the role of energy balance and photosystem II excitation pressure in the regulation of the xanthophyll cycle during chloroplast biogenesis in wild-type barley and the f2 mutant.

Dancing the tight rope on the nanoscale--Calibrating a heat flux sensor of a scanning thermal microscope.

We report on a precise in situ procedure to calibrate the heat flux sensor of a near-field scanning thermal microscope. This sensitive thermal measurement is based on 1ω modulation technique and utilizes a hot wire method to build an accessible and controllable heat reservoir. This reservoir is coupled thermally by near-field interactions to our probe. Thus, the sensor's conversion relation V(th)(Q(GS)*) can be precisely determined. V(th) is the thermopower generated in the sensor's coaxial thermocouple and Q(GS)* is the thermal flux from reservoir through the sensor. We analyze our method with Gaussian error calculus with an error estimate on all involved quantities. The overall relative uncertainty of the calibration procedure is evaluated to be about 8% for the measured conversion constant, i.e., (2.40 ± 0.19) µV/µW. Furthermore, we determine the sensor's thermal resistance to be about 0.21 K/µW and find the thermal resistance of the near-field mediated coupling at a distance between calibration standard and sensor of about 250 pm to be 53 K/µW.

Low molecular mass heat-shock proteins of a light-resistant photoautotrophic cell culture.

Two low molecular mass heat-shock proteins (HSPs) of photoautotrophic cell culture (Chenopodium rubrum) have been cloned, sequenced and compared to published sequences. One of these HSPs (23.3 kDa) is posttranslationally transported into chloroplasts and shares homology with the other heat-shock proteins in the last third C-terminal region of the protein, but has a relatively unique sequence in the other two thirds. The correspondent small cytosolic protein of 18.3 kDa is related to all known small HSPs but has a unique DNA-binding domain that has not been described so far in the group of small cytosolic HSPs, it might represent a HSP which is translocated into the nucleus.

The family of light-harvesting-related proteins (LHCs, ELIPs, HLIPs): was the harvesting of light their primary function?

Light-harvesting complex proteins (LHCs) and early light-induced proteins (ELIPs) are essential pigment-binding components of the thylakoid membrane and are encoded by one of the largest and most complex higher plant gene families. The functional diversification of these proteins corresponded to the transition from extrinsic (phycobilisome-based) to intrinsic (LHC-based) light-harvesting antenna systems during the evolution of chloroplasts from cyanobacteria, yet the functional basis of this diversification has been elusive. Here, we propose that the original function of LHCs and ELIPs was not to collect light and to transfer its energy content to the reaction centers but to disperse the absorbed energy of light in the form of heat or fluorescence. These energy-dispersing proteins are believed to have originated in cyanobacteria as one-helix, highly light-inducible proteins (HLIPs) that later acquired four helices through two successive gene duplication steps. We suggest that the ELIPs arose first in this succession, with a primary function in energy dispersion for protection of photosynthetic pigments from photo-oxidation. We consider the LHC I and II families as more recent and very successful evolutionary additions to this family that ultimately attained a new function, thereby replacing the ancestral extrinsic light-harvesting system. Our model accounts for the non-photochemical quenching role recently shown for higher plant psbS proteins.

Posttranscriptional control in the expression of the genes coding for high-light-regulated HL#2 proteins

An antibody was raised against the protein HL#2 which is a nuclear-encoded light-stress-induced protein of barley (Hordeum vulgare L.). The expression of the mRNA and the protein of HL#2 was determined under the influence of high light and methyl jasmonate. The mRNA of HL#2 was induced by high light (1800 &mgr;mol m(-2) s(-1) and 25 degrees C) and the steady-state levels remained elevated for up to 48 h of exposure to high-light stress. In contrast, using an antibody against HL#2 there was no observable change in the level of HL#2 proteins of 18 kDa and 15.5 kDa during the same treatment. These data indicate a pronounced stress-induced control of HL#2 expression at a post-transcriptional level. In the presence of methyl jasmonate (45 &mgr;M), the induction of HL#2 occurred together with that of the two most closely related jasmonate-inducible proteins (JIPs) of 32.6 and 32.7 kDa, as judged by their cross-reactivity with the antibody against HL#2. In contrast to the mRNA and protein levels of early light-inducible proteins (ELIPs) in green barley, those of HL#2 appeared not to be influenced by low temperatures. Therefore, the control of ELIPs and HL#2 by high light fluxes may be measured via the same photoreceptor but must, at least partially, be under the control of two divergent signal transduction chains.

Significance of circadian gene expression in higher plants.

The significance of the circadian clock for living organisms is not fully understood. Recent findings demonstrate circadian control of transcription of quite a number of genes with individual maxima throughout the entire day. Evidence in favor of circadian-clock-controlled translation has also been documented. In this article, we want to promote the idea that in plants the clock functions as a regulator which coordinates critical cellular processes, such as cell division, nitrate reduction, or synthesis of chlorophyll-protein complexes, in such a way that the generation of dangerous, oxidative radicals or exposure to harmful light is minimized. This has been achieved by plant organisms either by confining gene expression to the dark phase or by a tight coordination of different tiers of gene expression during the light phase. This leads to the consequence for the researcher that the time of experimentation needs to be carefully considered and documented. It also follows that one might lose important findings if only a particular portion of the day is investigated.

Circadian synthesis of light-harvesting-chlorophyll-proteins in Euglena gracilis is under translational control.

Two proteins with apparent molecular masses of 17 and 24 kD that are synthesized in a circadian manner in the phytoflagellate Euglena gracilis, were recognized as proteins belonging to the family of light-harvesting-chlorophyll-proteins (LHCPs) of class I (17 kD) and of class II (24 kD). Identification was achieved by N-terminal sequencing of the proteins isolated from two-dimensional polyacrylamide gels and by detection with an anti-LHCP II serum. While it was found that the total amount of LHCPs remains almost constant, when Euglena is grown under diurnal conditions (12 h light and 12 h dark), we could show that the amount of newly synthesized 17 and 24 kD proteins varies about 20-fold with a maximum of synthesis in the light phase. In contrast, the analysis of the mRNA levels at different times revealed only minor differences in the stationary concentration of the LHCP specific mRNA, indicating that the control of LHCP synthesis is at the translational level. Principally, the same finding was obtained using inhibitors of transcription. Thus, it is concluded that the expression of LHCPs in Euglena gracilis in contrast to that of higher plants is primarily regulated at the translational level.

Diurnal and circadian rhythmicity in the expression of light-induced plant nuclear messenger RNAs.

The levels of nuclear mRNAs for three light-inducible proteins (light-harvesting chlorophyll a/b protein, small subunit of ribulose-1,5-bisphosphate carboxylase and early light-induced protein) have been analyzed under light-dark and constant light conditions. The levels of all three mRNAs have been found to vary considerably during the day, both under ligh-dark and under constant light conditions, demonstrating the existence of diurnal and circadian rhythmicity in the expressionoof these nuclear-coded plant proteins. The levels of two of these mRNAs have been found to be enhanced 2 h before the beginning of illumination when active phytochrome levels are still low.

Heat-shock protein synthesis in Chlamydomonas reinhardi. Translational control at the level of initiation of a poly(A)-rich-RNA coded 22-KDa protein in a cell-free system.

The effect of temperature on the in vitro translation of control and heat-shock poly(A)-rich RNA, obtained from Chlamydomonas reinhardi cells, incubated for 2 h at 25 degrees C respectively, was studied using the wheat-germ translation system. Incubation of the cells at 42 degrees C induces the synthesis of RNAs coding for several heat-shock proteins, including a 22-kDa major polypeptide as well as several proteins of 45-94 kDa, as demonstrated by run-off translation of polyribosomes isolated from intact cells. However, the high-molecular-mass heat-shock proteins are poorly translated in the wheat-germ system. The poly(A)-rich RNA coding for the 22-kDa heat-induced polypeptide has an apparent sedimentation coefficient higher than that expected from the molecular mass of its translation product, and was preferentially translated in vitro at temperatures above 31 degrees C as compared with pre-existing RNAs. Raising the temperature of translation, slightly inhibited (10%) the runoff translation of polyribosomes isolated from intact cells. However, when initiation was carried out in vitro for a short time at increasing temperatures and translation continued at 25 degrees C in the presence of aurintricarboxylic acid, the 22-kDa heat-shock polypeptides was preferentially translated. Aurintricarboxylic acid did not significantly inhibit incorporation of [35S]methionine when added to polyribosomes isolated from control or heat-shocked cells. From the above data we conclude that the translation of the 22-kDa heat-shock protein is controlled in vitro at the initiation level.

Gene expression in the developing barley leaf under varying light conditions : The light-harvesting chlorophyll a/b protein and the small subunit of ribulose-1.5-bisphosphate carboxylase.

The expression of proteins within the developmental gradient in wild-type and mutant barley leaves has been studied under different light programs. The analyses have been performed both at the mRNA level by in vitro translation and by separation of proteins. It has been found that in mutant leaves under a normal light cycle, as well as in wild-type leaves under intermittent light conditions, mRNA for light-harvesting chlorophyll a/b protein (LHCP) and the small subunit of ribulose-1.5-bisphosphate carboxylase (RuBPCase) is distributed in the same manner as has been previously described for wild-type barley under a normal light-dark cycle. This observation is surprising considering that, in both cases, LHCP does not accumulate in the membranes. In dark-grown plants as well as in plants grown under intermittent light, synthesis of both LHCP mRNA and protein can be restored in the apical cells of the leaf upon continuous illumination. In such case, integration of LHCP into the membranes occurs without any delay in plants pretreated with intermittent light, in contrast to dark grown plants. For the small subunit of the major soluble protein RuBPCase, no differences in the shape of the gradients both at the level of mRNA or protein have been found under the various light programs, indicating that light exerts only modulating effects on RuBPCase in barley.

A rapidly light-induced chloroplast protein with a high turnover coded for by pea nuclear DNA.

Among the translation products obtained in vitro with mRNAs isolated from etiolated grown pea, or after different times of illumination following the etiolation, a 24000-Mr protein has been observed in the very early phase of greening; its occurrence culminates at 2-4 h after the start of illumination. From these data it is concluded that the corresponding mRNA appears in and disappears from the poly(A)-containing RNA population within hours. The protein product has been characterized as the precursor for a 17000-Mr chloroplast protein; by means of post-translational transport in vitro, the processed product becomes bound to chloroplast membranes. A product of the same size can also be labeled in vivo with a maximum of incorporation of label at 6-8 h after illumination. This product decays with a half-life of about 5 h. These findings imply a regulatory function of the 17000-Mr protein during the process of greening, possibly by synchronization of nuclear and chloroplast genomes. Other possibilities are considered.

Temperature-dependent binding to the thylakoid membranes of nuclear-coded chloroplast heat-shock proteins.

Two nuclear-coded heat-shock proteins (HSP) of pea (Pisum sativum) are synthesized as larger precursors of 26 kDa and 30 kDa in vitro. They are transported post-translationally into isolated, homologous chloroplasts where they are processed to mature proteins of 22 kDa and 25 kDa, respectively. When the chloroplasts used for the transport are isolated from control plants grown at 25 degrees C the 22-kDa and 25-kDa HSPs are located in the stroma of the chloroplasts. However, when chloroplasts are prepared from heat-shocked plants both proteins are found bound to the thylakoid membranes. The transition of the non-binding to the binding status is comparatively sharp and occurs between 36 degrees C and 40 degrees C in the variety 'Rosa Krone'. The transition temperature has been determined at 38 degrees C for 'Rosa Krone' and at 40 degrees C for the variety 'Golf'. At 42 degrees C, 15-min treatment of the plants is sufficient to induce membrane binding, which persists for at least 4-6 h (but not for 24 h) after return to the ambient temperature. Once lost, membrane binding can be reinduced by a second heat-shock treatment in vivo. High light intensities during the heat shock interfere with the binding capacity for heat-shock proteins.

Evidence for the localization of the nuclear-coded 22-kDa heat-shock protein in a subfraction of thylakoid membranes.

The precursor to the nuclear-coded 22-kDa heat-shock protein of chloroplasts (HSP 22) has been transported into isolated intact chloroplasts from heat-shocked plants. The localization of the mature protein in the chloroplast membrane was investigated. We have shown that the processed HSP 22 of pea was not bound to envelopes and found predominantly in thylakoid membranes. The binding of HSP 22 was stable in the presence of high salt concentrations. Solubilization of thylakoid membranes with Triton X-100 and phase partitioning with Triton X-114 indicate an intrinsic localization of HSP 22 or, alternatively, a non-covalent association with integral membrane protein(s). After fractionation into grana and stroma lamellae, HSP 22 was found mostly in the grana-membrane subfraction.

Integration of early light-inducible proteins into isolated thylakoid membranes.

An in-vitro system has been established to study the integration of early light-inducible proteins (ELIP) into isolated thylakoid membranes. The in-vitro-expressed ELIP precursor proteins exist in two forms, a high-molecular-mass aggregate which is accessible to trypsin but no longer to the stromal processing protease and a soluble form which is readily cleaved to the mature form by the stromal protease. The mature form of ELIP is integrated into thylakoid membranes; its correct integration can be deduced from the observation that the posttranslationally transported products and the in-vitro integrated ELIP species are cleaved by trypsin to products of the same apparent molecular mass. Trypsin-resistant fragments of high-molecular-mass and low-molecular-mass ELIP appear to have the same size. The processed ELIP species, as well as an engineered mature form of ELIP, are integrated into isolated thylakoid membranes. Integration of the mature protein occurs in the absence of stroma, into sodium-chloride-washed, and trypsin-treated thylakoid membranes. The process of integration is almost temperature independent over 0-30 degrees C. Analysis of the time course of integration leads to the conclusion that, under in-vitro conditions, processing but not integration into membranes is the rate-limiting step. In the absence of stroma, the ELIP precursor is bound to the thylakoid membranes, however, it is no longer accessible to the stromal maturating protease when added after binding has occurred. In conclusion, integration of ELIP differs in many essential details from that of its relatives, the light-harvesting chlorophyll a/b protein family.