Cab Gene Expression Research Articles

Signal transduction mutants of arabidopsis uncouple nuclear CAB and RBCS gene expression from chloroplast development

Chloroplast development requires coordinate nuclear and chloroplast gene expression. A putative signal from the chloroplast couples the transcription of certain nuclear genes encoding photosynthesis-related proteins with chloroplast function. We have identified at least three Arabidopsis nuclear genes ( GUN1, GUN2, and GUN3) necessary for coupling the expression of some nuclear genes to the functional state of the chloroplast. Homozygous recessive gun mutations allow nuclear gene expression in the absence of chloroplast development and furthermore may interfere with the switch from dark-grown to light-grown development. Other reports suggest this intracellular crosstalk also involves mitochondrial interactions. The GUN genes thus define steps in one specific branch of a complex interorganellar regulatory network.

Shedding light on clock controlled cab gene transcription in higher plants

Research from several laboratories has revealed that the circadian clock regulates the transcription of many higher plant genes. Amongst the most well characterized of these genes are those that encode the chlorophyll a/b binding (Cab) proteins of the light harvesting complexes of photosystem II. Analysis of cab gene expression in both etiolated and green plants reveals a complex interaction between phytochrome(s) and the circadian clock, in which phytochrome appears to regulate the level of cab transcription and the circadian clock regulates the phase. The development of a non-invasive assay for cab transcription based on firefly luciferase expression in transgenic plants has not only made it possible to analyze clock regulated transcription in living plants, but also provides a means to screen for clock mutants in Arabidopsis.

Light-independent developmental regulation of cab gene expression in Arabidopsis thaliana seedlings.

We found a transient increase in the amount of mRNA for four nuclear genes encoding chloroplast proteins during early development of Arabidopsis thaliana. This increase began soon after germination as cotyledons emerged from the seed coat; it occurred in total darkness and was not affected by external factors, such as gibberellins or light treatments used to stimulate germination. Three members of the cab gene family and the rbcS-1A gene exhibited this expression pattern. Because timing of the increase coincided with cotyledon emergence and because it occurred independently of external stimuli, we suggest that this increase represents developmental regulation of these genes. Further, 1.34 kilobases of the cab1 promoter was sufficient to confer this expression pattern on a reporter gene in transgenic Arabidopsis seedlings. The ability of the cab genes to respond to phytochrome preceded this developmental increase, showing that these two types of regulation are independent.

A rice cab gene promoter contains separate cis-acting elements that regulate expression in dicot and monocot plants.

The major light-harvesting chlorophyll a/b binding proteins of the photosynthetic apparatus are encoded by families of nuclear cab genes. The expression of most cab genes is tissue specific and photoregulated in angiosperms. In transgenic tobacco plants, expression of the reporter gene beta-glucuronidase (GUS) is photoregulated and tissue specific from 5' upstream sequences of the rice cab1R gene; deletion of sequences upstream from position -170 with respect to the transcription start site eliminates the enhanced and photoregulated expression in the transgenic plants. Using an in situ transient expression assay, we have determined that the sequence OCT-R, an octamer repeat that lies within the -269 to -170 region of cab1R, is essential for photoregulated expression of the chimeric GUS gene in leaf cells of maize and rice but is not required for expression in illuminated tobacco leaves. Conversely, box III*- and G-box-like sequences found near OCT-R in cab1R are necessary for high-level transient expression of the reporter gene in tobacco leaf tissue but are not required for transient expression in maize or rice leaves.

The Greening Process in Cress Seedlings. III. Age-Dependent Changes in the Capacity of the Tetrapyrrole Pathway

Accumulation of protochlorophyllide (Pchlide) was determined after incubation of etiolated seedlings of garden cress (Lcpidium sativum L.) with exogenous 5-aminolevulinate for 24 h. Young seedlings (start of incubation 0-12 h after imbibition) accumulated only moderate amounts (<0.2 nmol per seedling) of Pchlide. Maximum capacity of Pchlide accumulation (ca. 14 nmol per seedling) was reached at 30-36 h after imbibition. At 60-72 h after imbibition, the capacity of Pchlide formation from exogenous ALA was only 0.2-0.3 nmol per seedling. Uptake of ALA via roots was high in young and old seedlings. The carotenoid content and pattern did not change by ALA incubation. Precursors of Pchlide were also accumulated by ALA treatment. Predominant products were protoporphyrin and magnesium protoporphyrin (MgP) in 30 h old seedlings but magnesium protoporphyrin monomethylester (MgPMe) in 72 h old seedlings. The previously described interaction of Pchlide precursors with cab gene expression cannot be correlated with a single porphyrin. A possible correlation with the ratio MgPMe: MgP is discussed.

Antagonistic blue- and red-light regulation of cab-gene expression during photosynthetic adaptation in Scenedesmus obliquus

During adaptation of the photosynthetic apparatus of the green alga Scenedesmus obliquus to various light qualities, the accumulation of chlorophyll and pigment—protein complexes (with specific consideration of chlorophyll a/b-binding ( Cab) proteins) and cab-gene expression were determined. The fluence rate dependences for chlorophyll accumulation and cab-gene expression were very different. Very low fluence rates of violet (404 nm), blue (461 nm) and red (650 nm) light below the photosynthetic threshold, i.e. between 10 −3 and 10 −1 μmol m −2 s −1, inhibited all of these reactions in cells grown under heterotrophic conditions. At elevated fluence rates (above 1 μmol m −2 s −1), red light retained its negative regulation, whereas blue light stimulated pigment accumulation. Under autotrophic conditions the pattern was more complex, because chlorophyll accumulation was unaffected by light below the photosynthetic threshold. However, the expression of cab-genes was inhibited by red light but stimulated by blue light. Cells adapted to fluence rates, which ensured photosynthetic energy supply (above 1 μmol m −2 s −1), showed an increase in chlorophyll accumulation, blue light being more effective than red light. The results confirm and extend our previous discovery of two antagonistically acting photoreceptors in Scenedesmus which mediate and coordinate the complex functional and structural changes associated with photosynthetic adaptation. One of these receptor pigments is a blue-light receptor with positive action; the other is a violet—red-light receptor which can operate far below the photosynthetic threshold and exerts a negative regulation.

Abscisic Acid Negatively Regulates Expression of Chlorophyll a/b Binding Protein Genes during Soybean Embryogeny

The levels of abscisic acid (ABA) during embryogenesis in the soybean (Glycine max) cultivar Dare were quantitated. An increase in the quantity of ABA per cotyledon was correlated with a decrease in the chlorophyll a/b binding (Cab) protein gene mRNA population. Soybean cotyledons were cultured in vitro in the presence or absence of ABA. Quantitation of cotyledonary ABA levels and Cab mRNA levels indicated that the application of 5 x 10(-5) molar and 5 x 10(-6) molar exogenous ABA decreased Cab mRNA prevalences. S1 nuclease protection experiments demonstrated that exogenous ABA modulated the level of Cab3 mRNA. These data strongly suggest that one of the developmental regulators of Cab gene expression during soybean embryogeny is the plant hormone, ABA; ABA negatively regulates Cab mRNA accumulation.

Cop1: a regulatory locus involved in light-controlled development and gene expression in Arabidopsis.

Light signals from the environment are perceived by specific regulatory photoreceptors in plants and are transduced by unknown mechanisms to genes that control growth and development. We have identified a genetic locus in Arabidopsis thaliana, which appears to play a central role in this transduction process. Mutations in this locus, designated cop1 (constitutively photomorphogenic), result in dark-grown seedlings with the morphology of wild-type seedlings grown in the light. In addition, these mutations lead to constitutive expression of an array of normally light-regulated genes in dark-grown seedlings and in light-grown adult plants placed in darkness. Promoter-reporter fusion constructs of some of these genes are constitutively expressed in dark-grown transgenic cop1 seedlings, indicating that the aberrant behavior of these genes results primarily from aberrant modulation of their promoter activities in the mutant. In contrast, light control of seed germination and diurnal control of cab gene expression is normal in the cop1 mutants. Because these mutations are recessive, we conclude that in seedlings and adult plants, the wild-type cop1 gene product normally acts in darkness to repress the expression of genes involved in the dark-adaptive developmental and that regulatory photoreceptors act to reverse this action upon exposure to light. However, photocontrol of seed germination and diurnal rhythms is apparently exerted via one or more separate pathways not involving the cop1 product. one or more separate pathways not involving the cop1 product.

ANALYSIS OF THE DIURNAL EXPRESSION PATTERNS OF THE TOMATO CHLOROPHYLL alb BINDING PROTEIN GENES. INFLUENCE OF LIGHT and CHARACTERIZATION OF THE GENE FAMILY*

Steady-state mRNA levels of the chlorophyll a/b binding (cab) proteins oscillate substantially during a diurnal cycle in tomato leaves. This accumulation pattern is also observed in complete darkness, supporting the hypothesis that the expression of cab genes is at least partially regulated by an endogenous rhythm ("biological clock"). The amplitude of the cab mRNA accumulation is dependent on the duration of illumination and the circadian phase in which light was applied to the tomato plants. These results at the molecular level correlate well with the photoperiodic phenomenon. The characterization of the expression pattern of individual members of the cab gene family was attempted. Distinct primer extension products were detected using specific oligonucleotides homologous to the cab 1, cab 4, cab 5 and cab 8 genes. Based on this analysis the transcription start sites of these genes were determined to be between position -70 and -9 upstream of the ATG codon. During the diurnal cycle the cab 1 and cab 4 genes exhibit the same expression pattern; no transcripts detected at 3 and 6 a.m., maximum mRNA levels were measured at noon and decreasing levels in the afternoon.

PHYTOCHROME‐REGULATED EXPRESSION OF GENES ENCODING LIGHT‐HARVESTING CHLOROPHYLL a/b‐PROTEIN IN TWO LONG HYPOCOTYL MUTANTS and WILD TYPE PLANTS OF Arabidopsis thaliana*

The cab genes which encode the light-harvesting chlorophyll a/b-protein (LHCP) are expressed normally with respect to phytochrome regulation in the hy-3 and hy-5 long hypocotyl mutants of Arabidopsis thaliana. In etiolated seedlings of these mutants as well as of the wild type, 1 min of red light elevates cab mRNA levels substantially within 2 h; this increase is reversed if far-red light is given immediately after the red light treatment. We conclude that the genetic defects in these mutants do not affect steps in the signal transduction pathway leading to the regulated expression of cab genes. Additionally, the mRNA from one of the three known A. thaliana cab genes, AB140, is similar in quantity to the mRNAs from the other two, AB165 and AB180, in dark-grown seedlings of hy-3 and hy-5 as well as the parent A. thaliana (Landsberg) after a brief red light treatment. This aspect of cab gene expression differs from the strain Columbia of A. thaliana in which AB140 mRNA is the predominant message. In mature white light-grown plants of the strain Columbia, AB140 as well as a combination of AB165 and AB180 mRNAs are expressed at high levels, suggesting that AB165 and/or AB180 may be developmentally regulated.

Expression of cab Genes in Douglas-Fir Is Not Strongly Regulated by Light

Dark-grown Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) seedlings had approximately 30% of the major polypeptide of the light-harvesting chlorophyll a/b binding protein, 30% of cab mRNA, 54% of psbA mRNA, and 14% of total chlorophyll, in comparison with amounts in light-grown seedlings. Seedlings entrained under a 24-hour photoperiod of light and dark showed small diurnal fluctuations in cab and psbA mRNA levels and, when transferred to continuous conditions, no circadian rhythms in mRNA levels were apparent. These results suggest that regulation of cab gene expression in Douglas-fir differs from regulation in angiosperms, because in the latter, both light and circadian factors strongly influence the expression of cab genes.

Light to Dark Transition Modulates the Phase of Antenna Chlorophyll Protein Gene Expression

The genes coding for the light-harvesting chlorophyll a/b-binding protein of photosystem II (cab) in wheat are regulated by phytochrome and an endogenous circadian clock. The regulation by these factors has been shown to be at the transcriptional level. Here we show that the phase of cab gene expression is reset by light to dark transition of the previous day. The time of light to dark transition is linearly related to the time of maximal cab gene expression under free running conditions in darkness. We propose that in nature the onset of darkness resets the phase of the circadian clock, which regulates the timing of cab gene expression, so as to anticipate the time of sunrise in the following day.

Phytochrome activation of two nuclear genes requires cytoplasmic protein synthesis.

We have investigated the effects of protein synthesis inhibitors on light-induced expression of two plant nuclear genes, Cab and rbcS, in wheat, pea and transgenic tobacco. Light activation of these two genes is very sensitive to cycloheximide, an inhibitor of cytoplasmic protein synthesis but not to chloramphenicol, an inhibitor of organellar protein synthesis. Studies with chimeric gene constructs in transgenic tobacco seedlings show that cycloheximide exerts its effect at the transcriptional level. As a control, we show that the expression of the cauliflower mosaic virus (CaMV) 35S promoter is enhanced by cycloheximide treatment, irrespective of the coding sequence used. Escape-time analyses with green wheat seedlings show that the cycloheximide block for Cab gene expression is after the primary signal transduction step linked to phytochrome photoconversion. Our results suggest that phytochrome activation of Cab and rbcS is mediated by a labile protein factor(s) synthesized on cytoplasmic ribosomes.

Rice Phytochrome Is Biologically Active in Transgenic Tobacco.

To investigate the mechanisms of phytochrome action in vivo, we have overexpressed rice phytochrome in transgenic tobacco plants. A full-length rice phytochrome cDNA was fused to the cauliflower mosaic virus 35S promoter and transferred to tobacco. The progeny of some of the transgenic plants contain large amounts of rice phytochrome mRNA in green leaves. Extracts prepared from overexpressing plants contain twofold to fivefold more spectrophotometrically detectable phytochrome than extracts from control plants. Species-specific, anti-phytochrome monoclonal antibodies were used in immunoblots to discriminate between rice and tobacco phytochrome apoproteins in fractions eluted from a DEAE-Sepharose column. Red minus far-red difference spectra of the partially purified rice phytochrome from the transgenic plants indicate that the rice phytochrome assembles with chromophore and is photoreversible. Analysis of the circadian pattern of Cab mRNA levels in transgenic plants versus controls demonstrates that the overproduction of rice phytochrome extends the duration of the free-running rhythm of Cab gene expression. The rice phytochrome is, therefore, biologically active in the transgenic tobacco plant, which establishes a system for in vivo functional analysis of phytochrome.

Isolation and characterization of a maize chlorophyll a/b binding protein gene that produces high levels of mRNA in the dark

A cDNA library prepared using mRNA isolated from red-light irradiated maize seedlings was screened by a difference procedure for clones that represent red-light regulated mRNA. Two such clones were found to represent mRNA for a chlorophyll a/b binding protein (CAB), and one of these (pAB1084) was used to screen a maize genomic library. One positive genomic clone (lambda AB1084) was isolated and sequenced. The gene represented by lambda AB1084, which we designate maize cab-1, contains extensive nucleotide homology within its protein coding region to CAB genes from other species. The boundaries of the transcribed region of the cab-1 gene were determined by S1 nuclease mapping. The 5' terminus of cab-1 mRNA is located 52-54 nucleotides (nt) upstream of the translation start site and 34 nt downstream of a TATA box. As in the case of petunia CAB genes, several poly(A) addition sites are present in mRNA from the cab-1 gene. The 5' flanking DNA of cab-1 contains sequences related to elements that have been implicated in the light-regulated expression of CAB and rbcS genes in other plant systems. Quantitative Northern blot hybridization analysis using a gene specific probe for cab-1 indicates that the mRNA for this gene is present at 0.4% of the total mRNA and up to 80% of the total CAB mRNA in the leaves of dark-grown seedlings. In consequence, although the degree of up-regulation by white light is only moderate (3- to 6-fold), cab-1 transcripts account for approximately 2% of the mRNA in the leaves of light-grown seedlings.