The Effects of Benzyladenine on the Accumulation of Messenger RNAs That Encode the Large and Small Subunits of Ribulose-l,5-bisphosphate Carboxylase/oxygenase and Light-Harvesting Chlorophyll a/b Protein in Excised Cucumber Cotyledons

Benzyladenine promoted rapid accumulation of mRNAs that encodedthe small subunit of ribulose-l,5-bisphosphate carboxylase/oxygenase(RuBisCO) and light-harvesting chlorophyll a/b protein in etiolatedcucumber cotyledons, but only after prior incubation of thecotyledons in water. However, benzyladenine hardly affectedthe level of mRNA for the large subunit of RuBisCO. ¹ Present address: Shonan Junior College, Yokosuka, Kanagawa238, Japan (Received September 3, 1990; Accepted March 4, 1991)     

Expression of photosynthesis-related genes and their regulation by light during somatic embryogenesis in<Emphasis Type="Italic"> Daucus carota</Emphasis>

To clarify the spatial and temporal pattern of gene expression for photosynthesis-associated proteins during somatic embryogenesis in Daucus carota L., the localization of mRNAs for three genes, rbcL, Lhcb and por, was examined in dark-grown and light-irradiated somatic embryos by in situ hybridization. The three mRNAs were expressed in common in the mesophyll precursor cells of light-irradiated embryos at the late torpedo and plantlet stages, but characteristic expression patterns of each photosynthesis-related gene were also observed. Expression of rbcL mRNA first occurred throughout the embryo but gradually became localized in the mesophyll precursor cells and cortex during early embryogenesis. Localization of Lhcb mRNA in the mesophyll precursor cells and shoot apical meristem became clear in the early torpedo stage. Expression of Lhcb mRNA was not affected by light during early embryogenesis, but could be induced by light in the torpedo stage, suggesting that light-inducible expression of Lhcb mRNA arises within the torpedo stage. At the late torpedo stage, clear localization of por mRNA started in mesophyll precursor cells of the cotyledon in light-irradiated embryos. Greening potency of the embryo also appeared first at this stage. Therefore, greening and initial differentiation of photosynthetic tissues during somatic embryogenesis seem to be associated with coordinated expression of mRNA for rbcL, Lhcb and por in late torpedo-shaped embryos.Abbreviations DIG Digoxigenin - Lhcb3 Gene encoding a type-III light-harvesting chlorophyll a/b-binding protein of photosystem II - LHCII Light-harvesting chlorophyll a/b-binding protein of photosystem II - POR Protochlorophyllide oxidoreductase - rbcL Gene encoding the large subunit of Rubisco - Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase     

Photosynthetic gene expression and cellular differentiation in developing maize leaves

We have exploited the positional gradient of cellular differentiation in Zea mays leaves to study the accumulation of mRNAs encoding subunits of the two CO₂-fixing enzymes and the major chlorophyll-binding protein. These three proteins are differentially compartmentalized in the two photosynthetically active cell types of the leaf. Previous studies have shown that accumulation of the two carboxylases commences 2 to 4 cm from the base of the leaf (Mayfield SP, WC Taylor Planta 161: 481-486) at a position where bundle sheath and mesophyll cells show morphological evidence of maturation. The light-harvesting chlorophyll a/b protein accumulates progressively from the leaf base, as does its mRNA, in spite of its localization in mesophyll cells after cellular differentiation occurs. While small quantities of phosphoenolpyruvate carboxylase mRNA are detectable in the basal region of the leaf, significant mRNA accumulation is coincident with that of the polypeptide at 4 to 6 cm from the leaf base, the region where bundle sheath and mesophyll cells exhibit fully differentiated morphologies. mRNAs encoding the small and large subunits of ribulose 1,5-bisphosphate carboxylase accumulate to significant levels before bundle sheath cells are fully differentiated and before their polypeptides are detectable. Cytological examination indicates that this is the position at which the maturation of intermediate vascular bundles is first evident. Cytosolically localized small subunit mRNA and chloroplast-localized large subunit mRNA are complexed with polyribosomes at all positions of the leaf.     

The effect of heat shock on morphogenesis in barley : coordinated circadian regulation of mRNA levels for light-regulated genes and of the capacity for accumulation of chlorophyll protein complexes

The effect of daily heat-shock treatments on gene expression and morphogenesis of etiolated barley (Hordeum vulgare) was investigated. Heat-shock treatments in the dark induced shortening of the primary leaves and the coleoptiles to the length of those in light-grown plantlets. In addition, the mRNA levels of the light-induced genes that were investigated were raised under these conditions and showed distinct oscillations over a period of at least 3 d. While the mRNA levels for chlorophyll a/b binding protein (LHC II), plastocyanin, and the small subunit of ribulose-1,5-bisphosphate carboxylase had maxima between 8 and 12 pm (12-16h after the last heat-shock treatment), the mRNA levels for thionin oscillated with a phase opposed to that of LHC II. Etiolated barley, the circadian oscillator of which was synchronized by cyclic heatshock treatments, was illuminated for a constant interval at different times of the day; this led to the finding that greening was fastest at the time when the maximal levels of mRNA for LHC II were also observed. Whereas accumulation of chlorophyll a during a 4-h period of illumination oscillated by a factor of 3, chlorophyll b accumulation changed 10- to 15-fold. Similarly, accumulation of LHC II was highest when pigments accumulated maximally. Hence, greening or, in other words, thylakoid membrane assembly is under control of the circadian oscillator.     

Evolutionary Changes in Chlorophyllide a Oxygenase (CAO) Structure Contribute to the Acquisition of a New Light-harvesting Complex in Micromonas

Chlorophyll b is found in photosynthetic prokaryotes and primary and secondary endosymbionts, although their light-harvesting systems are quite different. Chlorophyll b is synthesized from chlorophyll a by chlorophyllide a oxygenase (CAO), which is a Rieske-mononuclear iron oxygenase. Comparison of the amino acid sequences of CAO among photosynthetic organisms elucidated changes in the domain structures of CAO during evolution. However, the evolutionary relationship between the light-harvesting system and the domain structure of CAO remains unclear. To elucidate this relationship, we investigated the CAO structure and the pigment composition of chlorophyll-protein complexes in the prasinophyte Micromonas. The Micromonas CAO is composed of two genes, MpCAO1 and MpCAO2, that possess Rieske and mononuclear iron-binding motifs, respectively. Only when both genes were introduced into the chlorophyll b-less Arabidopsis mutant (ch1-1) was chlorophyll b accumulated, indicating that cooperation between the two subunits is required to synthesize chlorophyll b. Although Micromonas has a characteristic light-harvesting system in which chlorophyll b is incorporated into the core antennas of reaction centers, chlorophyll b was also incorporated into the core antennas of reaction centers of the Arabidopsis transformants that contained the two Micromonas CAO proteins. Based on these results, we discuss the evolutionary relationship between the structures of CAO and light-harvesting systems.     

Antenna ring around trimeric Photosystem I in chlorophyll b containing cyanobacterium Prochlorothrix hollandica

Prochlorothrix hollandica is one of the three known species of an unusual clade of cyanobacteria (formerly called “prochlorophytes”) that contain chlorophyll a and b molecules bound to intrinsic light-harvesting antenna proteins. Here, we report the structural characterization of supramolecular complex consisting of Photosystem I (PSI) associated with the chlorophyll a/b-binding Pcb proteins. Electron microscopy and single particle image analysis of negatively stained preparations revealed that the Pcb-PSI supercomplex consists of a central trimeric PSI surrounded by a ring of 18 Pcb subunits. We conclude that the formation of the Pcb ring around trimeric PSI represents a mechanism for increasing the light-harvesting efficiency in chlorophyll b-containing cyanobacteria.     

Accumulation of Chlorophyll, Chloroplastic Proteins, and Thylakoid Membranes during Reversion of Chromoplasts to Chloroplasts in Citrus sinensis Epicarp

In vitro culture of pericarp segments from fruit of Citrus sinensis (L.) Osbeck cv Valencia was used to determine the temporal sequence in development of chloroplasts from chromoplasts during regreening of the epicarp. Regreening of chromoplasts closely resembled greening of etioplasts, except that regreening proceeded much more slowly. Chlorophyll, the light-harvesting chlorophyll a/b binding protein of photosystem II, the chlorophyll a binding protein of reaction center P-700 of photosystem I, thylakoid membranes, and adenosine triphosphate synthetase were all detected at very low levels in degreened epicarp. All of these increased in parallel during regreening of the epicarp. Ribulose 1,5-bisphosphate carboxylase (RuBPCase) levels were high in degreened epicarp and declined for the first 10 days of culture before reaccumulating in the regreening segments. Light was necessary for the accumulation of all of the chloroplastic components. A lack of exogenous nitrogen did not prevent the accumulation of any chloroplastic component except Ru-BPCase, although accumulation of the other components was reduced. Sucrose at 150 millimolar in media lacking nitrogen markedly inhibited the accumulation of chlorophyll and light-harvesting chlorophyll a/b-protein.     

Phytochrome Regulation of Greening in Pisum: Chlorophyll Accumulation and Abundance of mRNA for the Light-Harvesting Chlorophyll a/b Binding Proteins

A brief pulse of red light eliminates or reduces the lag in chlorophyll accumulation that occurs when dark-grown pea seedlings are transferred to continuous white light. The red light pulse also induces the accumulation of specific mRNAs. We compared time courses, escape from reversal by far-red light, and fluence-response behavior for induction of mRNA for the light-harvesting chlorophyll a/b binding proteins (Cab mRNA) with those for induction of rapid chlorophyll accumulation in seedlings of Pisum sativum cv Alaska. In both cases the time courses of low fluence and very low fluence responses diverged from each other in a similar fashion: the low fluence responses continued to increase for at least 24 hours, while the very low fluence responses reached saturation by 8 to 16 hours. Both responses escaped from reversibility by far-red slowly, approaching the red control level after 16 hours. The fluence-response curve for the Cab mRNA increase, on the other hand, showed threshold and saturation at fluences 10-fold lower than threshold and saturation values for the greening response. Therefore, the level of Cab mRNA, as measured by the presence of sequences hybridizing to a cDNA probe, does not limit the rate of chlorophyll accumulation after transfer of pea seedlings to white light. The Cab mRNA level in the buds of seedlings grown under continuous red light remained high even when the red fluence rate was too low to allow significant greening. In this case also, abundance of Cab mRNA cannot be what limits chlorophyll accumulation.     

The effects of water stress on the development of the photosynthetic apparatus in greening leaves

The effects of low and high relative humidity and of polyethylene glycol-induced root water stress on chlorophyll accumulation, on formation of the lamellar chlorophyll-protein complexes, and on the development of photosynthetic activity during chloroplast differentiation were examined. Low relative humidity or polyethylene glycol-induced root water stress (stress conditions) resulted in a 3 to 4 hour lag in chlorophyll accumulation, retarded the rate of chlorophyll b accumulation, and reduced the rate of formation of the light-harvesting chlorophyll a/b protein. All of these effects could be overcome by high relative humidity (nonstress) conditions. Concomitant measurement of leaf water potential showed that under stress conditions greening leaves were subjected to initial water deficits of −8 bars which decreased to −5 bars after 3 to 4 hours of illumination corresponding to the end of the lag phase. Leaves greening under nonstress conditions did not experience leaf water deficits greater than about −5 bars. It seems that the attainment of a minimum leaf water potential of −5 bars may be critical in the control of early chloroplast development. These results demonstrate that the lag phase is not indicative of a programmed event in chloroplast development, but rather is attributable to environmental conditions prevailing during leaf development and greening.     

Fe deficiency induces phosphorylation and translocation of Lhcb1 in barley thylakoid membranes

HvLhcb1 a major light-harvesting chlorophyll a/b-binding protein in barley, is a critical player in sustainable growth under Fe deficiency. Here, we demonstrate that Fe deficiency induces phosphorylation of HvLhcb1 proteins leading to their migration from grana stacks to stroma thylakoid membranes. HvLhcb1 remained phosphorylated even in the dark and apparently independently of state transition, which represents a mechanism for short-term acclimation. Our data suggest that the constitutive phosphorylation-triggered translocation of HvLhcb1 under Fe deficiency contributes to optimization of the excitation balance between photosystem II and photosystem I, the latter of which is a main target of Fe deficiency.     

Development of Photosynthetic Activity following Anaerobic Germination in Rice-Mimic Grass (Echinochloa crus-galli var oryzicola)

Shoots of anaerobically germinated Echinochloa crus-galli var oryzicola are nonpigmented whether germinated in light or dark, and chlorophyll synthesis is minimal for the first 12 to 18 hours of greening after exposure to ambient conditions. When chlorophyll development is compared between greening anoxic and etiolated shoots, there is a 100-fold difference in chlorophyll levels at 8 hours, an 8-fold difference at 24 hours, but roughly equal amounts at 60 hours. The chlorophyll a/b ratio approaches 3 earlier in greening anoxic shoots than in greening etiolated shoots, relative to total chlorophyll. The long lag in chlorophyll synthesis can be shortened by giving dark-grown anoxic shoots a 24-hour midtreatment of air before light.

Development of photosynthetic activity in etiolated shoots, determined by CO₂ gas exchange, ¹⁴CO₂ uptake, and activity of carboxylating enzymes closely parallels development of chlorophylls. However, development of photosynthetic capability in greening anoxic shoots does not parallel chlorophyll development; ability to fix carbon lags behind chlorophyll synthesis. A reason for this lag is the very low activity of RuBP carboxylase during the first 36 hours of greening in anoxic shoots. The activity of phosphoenolpyruvate carboxylase is also delayed, but its kinetics more closely match those of chlorophyll development.

     

Biogenesis of thylakoid membranes with emphasis on the process in Chlamydomonas

Recent results obtained by electron microscopic and biochemical analyses of greening Chlamydomonas reinhardtii y1 suggest that localized expansion of the plastid envelope is involved in thylakoid biogenesis. Kinetic analyses of the assembly of light-harvesting complexes and development of photosynthetic function when degreened cells of the alga are exposed to light suggest that proteins integrate into membrane at the level of the envelope. Current information, therefore, supports the earlier conclussion that the chloroplast envelope is a major biogenic structure, from which thylakoid membranes emerge. Chloroplast development in Chlamydomonas provides unique opportunities to examine in detail the biogenesis of thylakoids.Abbreviations Rubisco ribulose bisphosphate carboxylase/oxygenase - CAB Chl a/b-binding (proteins) - Chlide chlorophyllide - LHC I light-harvesting complex of PS I - LHC II light-harvesting complex of PS II - Pchlide protochlorophyllide     

Photosynthetic Genes of Petunia (Mitchell) Are Differentially Expressed during the Diurnal Cycle

The petunia (Petunia [Mitchell]) chloroplast proteins, the chlorophyll a/b-binding (Cab) proteins, and the small subunit of ribulose bisphosphate carboxylase (RbcS) are encoded by nuclear genes that are expressed in a light-dependent manner. The steady-state concentrations of five cab mRNAs vary with a dramatic circadian rhythm in plants grown under a constant diurnal cycle (10 hours light, 14 hours dark). cab mRNA levels reach their maximum during the light period, but begin to drop prior to the dark period. These RNAs fall to their minimum concentration during the dark period and then begin to increase again in anticipation of the light. Within this general pattern, there are variations in expression among specific classes of cab genes. The light harvesting complex of photosystem II LHCII-type 1 cab mRNAs rise to a well-defined maximum at 2 hours prior to the dark period. All but one of these genes are expressed in anticipation of the light period. The LHCII type 2 cab mRNA and the LHC of photosystem I cab mRNA are expressed at more constant levels throughout the light period. The expression of these genes anticipates the light more than does the expression of the LHCII type 1 genes. The steady state mRNA levels for the petunia rbcS genes show no significant diurnal fluctuation.     

Photocontrol of the Accumulation of Plastid Polypeptides during Greening of Tomato Cotyledons : Potentiation by a Pulse of Red Light

A pulse of red light acting through phytochrome accelerates the formation of chlorophyll upon subsequent transfer of dark-grown seedlings to continuous white light. Specific antibodies were used to follow the accumulation of representative subunits of the major photosynthetic complexes during greening of seedlings of tomato (Lycopersicon esculentum). The time course for accumulation of the various subunits was compared in seedlings that received a red light pulse 4 h prior to transfer to continuous white light and parallel controls that did not receive a red light pulse. The light-harvesting chlorophyll-binding proteins of photosystem II (LHC II), the 33-kD extrinsic polypeptide of the oxygen-evolving complex (OEC1), and subunit II of photosystem I (psaD gene product) all increased in the light, and did so much faster in seedlings that received the inductive red light pulse. The red light pulse had no significant effect on the abundance of the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), nor on several plastid-encoded polypeptides: the large subunit of Rubisco, the β subunit of the CF₁ complex of plastid ATPase, and the 43- and 47-kD subunits of photosystem II (CP43, CP47). Subunits I (cytochrome b₆f) and III (Rieske Fe-S protein) of the cytochrome b₆f complex showed a small or no increase as a result of the red pulse. The potentiation of greening by a pulse of red light, therefore, is not expressed uniformly in the abundance of all the photosynthetic complexes and their subunits.     

The Expression of Light-Regulated Genes in the High-Pigment-1 Mutant of Tomato

Three light-regulated genes, chlorophyll a/b-binding protein (CAB), ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit, and chalcone synthase (CHS), are demonstrated to be up-regulated in the high-pigment-1 (hp-1) mutant of tomato (Lycopersicon esculentum Mill.) compared with wild type (WT). However, the pattern of up-regulation of the three genes depends on the light conditions, stage of development, and tissue studied. Compared with WT, the hp-1 mutant showed higher CAB gene expression in the dark after a single red-light pulse and in the pericarp of immature fruits. However, in vegetative tissues of light-grown seedlings and adult plants, CAB mRNA accumulation did not differ between WT and the hp-1 mutant. The ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit mRNA accumulated to a higher level in the hp-1 mutant than WT under all light conditions and tissues studied, whereas CHS gene expression was up-regulated in de-etiolated vegetative hp-1-mutant tissues only. The CAB and CHS genes were shown to be phytochrome regulated and both phytochrome A and B1 play a role in CAB gene expression. These observations support the hypothesis that the HP-1 protein plays a general repressive role in phytochrome signal transduction.