Absorption and circular dichroism spectra of chloroplast membrane fragments from spinach,barley and a barley mutant at room temperature and liquid nitrogen temperature

The absorption and CD spectra of chloroplast fragments from spinach, barley and a barley mutant (chlorophyll b-minus) were studied at temperatures of 23°C and ?196°C. The CD spectrum of wild type barley and spinach at ?196°C showed troughs at 640, 653, 676 and 695 nm and a maximum at 667 nm. The CD spectrum of the barley mutant at ?196°C consisted of a large trough at 684 nm, a small trough at 695 nm and a positive peak at 670 nm. A new feature observed at ?196°C but not at 23°C is the trough at 640 nm. This 640 nm CD signal is missing in the CD spectrum of the barley mutant. It is attributable to the light-harvesting chlorophyll $\text{a}\text{b}$ protein which appears to be missing in the mutant. Another new feature, the trough at 695 nm, was observed in the CD spectra of spinach, barley and the barley mutant at ?196°C. The 695 nm trough appears to be sensitive to detergents and it may be due to a labile chlorophyll a·protein complex. Possible interpretations of these data are discussed.     

Effect of aVirido-Xantha mutation in barley on the content of individual plastid pigments

Summary A virido-xantha (vx) barley mutant with reduced chlorophyll content when grown at 15°C was investigated to determine quantitative and qualitative changes in the plastid pigments. Both chlorophyll and the capacity to produce protochlorophyllide from exogeneously supplied δ-aminolevulinic acid are reduced in the mutant. The total carotenoid content is also reduced in the mutant, but individual carotenoids are not reduced coordinately. Antheraxanthin actually accumulates in the mutant, and zeaxanthin was found in significant amounts in mutant seedlings but could not be detected in normal barley seedlings. Included in the methods is a procedure for the preliminary identification of zeaxanthin employing spectrophotometric analysis of fractions eluted from a sucrose column. The presence of recessive suppressors of thevx mutant results in increased contents of the chlorophyll pigments accompanied by a partial reversal of the changes in carotenoid indicated above. The changes in chlorophyll and carotenoid contents are also partially reversed when mutant seedlings are grown at 21°C. Research supported under contract AT(11-1)-332 with the United States Atomic Energy Commission.     

Chlorophyll accumulation and breakdown in light-grown barley transferred to darkness: effect of seedling age

Diurnally grown barley (Hordeum vulgare L. cv. Clipper) seedlings of various ages (3–4, 5–6 and 10–11-days-old) were transferred to darkness for 17 h and changes in leaf fresh weight, chlorophyll a, chlorophyll b and protochlorophyllide measured. The results were consistent with previous evidence of a light-independent chlorophyll biosynthetic pathway in light-grown barley. There was a net gain in chlorophyll (μg leaf^-1) in 5–6- and 10–11-day-old plants after 17 h dark treatment. The amounts of chlorophyll that accumulated were similar (5.9 and 4.3 μg Chl leaf^-1), despite a twofold difference in leaf size at T₀. The rate of leaf expansion in 5–6-day-old plants greatly exceeded the rate of chlorophyll accumulation and leaves were noticeably paler after dark treatment i.e. there was a reduction in chlorophyll concentration (μg g fresh weight^-1) in spite of an increase in chlorophyll content (μg leaf^-1). The ability of light-grown barley to accumulate chlorophyll in darkness was a function of seedling age. Very young seedlings (3–4-day-old) generally lost chlorophyll in darkness. The decrease in chlorophyll per leaf resulted mainly from loss of chlorophyll b. Preferential loss of chlorophyll b resulted in dramatic increases in the chlorophyll a:b ratio. Since 3–4-day-old seedlings (1) accumulated 5-aminolevulinic acid in the presence of levulinic acid at a rate comparable to older seedlings, and (2) converted exogenous 5-aminolevulinic acid to chlorophyll in the absence of light, it is unlikely that failure of the youngest plants to accumulate chlorophyll in darkness was due to blocks at these steps in the pathway. Net loss of chlorophyll (μg leaf^-1) in 3–4-day-old seedlings in darkness was eliminated by the addition of chloramphenicol, which occasionally produced a small, but significant, gain in total chlorophyll. These results imply that chlorophyll degradation in young barley leaves is strongly influenced by the chloroplast genome, and is a major factor influencing changes in chlorophyll levels in darkness. The present findings are consistent with the suggestion that the failure of 3–4-day-old barley seedlings to accumulate chlorophyll in darkness may be due to chlorophyll turnover in which the rate of degradation exceeds the rate of synthesis.     

A second infA plastid gene point mutation shows a compensatory effect on the expression of the cytoplasmic line 2 (CL2) syndrome in barley

The IF1 protein is one of the factors controlling translation initiation in bacteria. This protein is encoded by the infA gene, which, in several higher plants, is located in the plastome. Cytoplasmic Line 2 (CL2), an alboviridis barley mutant, was the first to be proposed as an infA gene mutation (T 157 C) in higher plants. This mutant was isolated from a chloroplast mutator genotype (cpm/cpm) and was made genetically stable by backcrosses with a wild-type nuclear genotype. In the present work, genetically stable CL2 plants were backcrossed as females by cpm/cpm plants in order to regain the mutator activity. Interestingly, a seedling carrying a first leaf blade with a darker green stripe on a typical CL2-mutant background was observed in the F(4) generation. The T 157 C transition was confirmed in tissues from the CL2 background, whereas a second transition (A 178 G) was also found in the darker stripe. Two clearly different levels of CL2 syndrome were observed in the seedlings of the F(5) and F(6) progenies. Those of the greener group carried both transitions. These results suggest a compensatory effect of the second mutation and support the involvement of the infA plastid gene in CL2 syndrome, confirming CL2 as the first mutant of this gene reported in higher plants.     

Mutation mechanism of chlorophyll-less barley mutant <Emphasis Type="Italic">NYB</Emphasis>

NYB is chlorophyll-less barley mutant, which is controlled by a recessive nuclear gene. The mutation mechanism is revealed. The activities of enzymes transforming 5-aminolevulinic acid into protochlorophyllide were the same in both NYB and the wild type (WT), but the activity of the protochlorophyllide oxidoreductase (POR) in WT was much higher than that of NYB. Most of the photosystem 2 apoproteins were present in both WT and NYB, suggesting that the capability of protein synthesis was probably fully preserved in the mutant. Thus chlorophyll (Chl) biosynthesis in NYB was hampered at conversion form protochlorophyllide (Pchlide) into chlorophyllide. The open reading frame of porB gene in NYB was inserted with a 95 bp fragment, which included a stop codon. The NYB mutant is a very useful material for studies of Chl biosynthesis, chloroplast signalling, and structure of light-harvesting POR-Pchlide complex (LHPP).     

The Chloroplast Function Database: a large‐scale collection of Arabidopsis Ds/Spm‐ or T‐DNA‐tagged homozygous lines for nuclear‐encoded chloroplast proteins,and their systematic phenotype analysis

A majority of the proteins of the chloroplast are encoded by the nuclear genome, and are post‐translationally targeted to the chloroplast. From databases of tagged insertion lines at international seed stock centers and our own stock, we selected 3246 Ds/Spm (dissociator/suppressor–mutator) transposon‐ or T‐DNA‐tagged Arabidopsis lines for genes encoding 1369 chloroplast proteins (about 66% of the 2090 predicted chloroplast proteins) in which insertions disrupt the protein‐coding regions. We systematically observed 3‐week‐old seedlings grown on agar plates, identified mutants with abnormal phenotypes and collected homozygous lines with wild‐type phenotypes. We also identified insertion lines for which no homozygous plants were obtained. To date, we have identified 111 lines with reproducible seedling phenotypes, 122 lines for which we could not obtain homozygotes and 1290 homozygous lines without a visible phenotype. The Chloroplast Function Database presents the molecular and phenotypic information obtained from this resource. The database provides tools for searching for mutant lines using Arabidopsis Genome Initiative (AGI) locus numbers, tagged line numbers and phenotypes, and provides rapid access to detailed information on the tagged line resources. Moreover, our collection of insertion homozygotes provides a powerful tool to accelerate the functional analysis of nuclear‐encoded chloroplast proteins in Arabidopsis. The Chloroplast Function Database is freely available at http://rarge.psc.riken.jp/chloroplast/ . The homozygous lines generated in this project are also available from the various Arabidopsis stock centers. We have donated the insertion homozygotes to their originating seed stock centers.     

Rice Chlorina-1 and Chlorina-9 encode ChlD and ChlI subunits of Mg-chelatase, a key enzyme for chlorophyll synthesis and chloroplast development

Photosynthetic organisms exhibit a green color due to the accumulation of chlorophyll pigments in chloroplasts. Mg-protoporphyrin IX chelatase (Mg-chelatase) comprises three subunits (ChlH, ChlD and ChlI) and catalyzes the insertion of Mg²⁺ into protoporphyrin IX, the last common intermediate precursor in both chlorophyll and heme biosyntheses, to produce Mg-protoporphyrin IX (MgProto). Chlorophyll deficiency in higher plants results in chlorina (yellowish-green) phenotype. To date, 10 chlorina (chl) mutants have been isolated in rice, but the corresponding genes have not yet been identified. Rice Chl1 and Chl9 genes were mapped to chromosome 3 and isolated by map-based cloning. A missense mutation occurred in a highly conserved amino acid of ChlD in the chl1 mutant and ChlI in the chl9 mutant. Ultrastructural analyses have revealed that the grana are poorly stacked, resulting in the underdevelopment of chloroplasts. In the seedlings fed with aminolevulinate-dipyridyl in darkness, MgProto levels in the chl1 and chl9 mutants decreased up to 25% and 31% of that in wild-type, respectively, indicating that the Mg-chelatase activity is significantly reduced, causing the eventual decrease in chlorophyll synthesis. Furthermore, Northern blot analysis indicated that the nuclear genes encoding the three subunits of Mg-chelatase and LhcpII in chl1 mutant are expressed about 2-fold higher than those in WT, but are not altered in the chl9 mutant. This result indicates that the ChlD subunit participates in negative feedback regulation of plastid-to-nucleus in the expression of nuclear genes encoding chloroplast proteins, but not the ChlI subunit.Haitao Zhang and Jinjie Li contributed equally to this work     

Development of synthetic Brassica amphidiploids by reciprocal hybridization and comparison to natural amphidiploids

In a previous study we proposed that cytoplasmic genomes have played an important role in the evolution of Brassica amphidiploid species. Based on this and other studies, we hypothesized that interactions between the maternal cytoplasmic genomes and the paternal nuclear genome may cause alterations in genome structure and/or gene expression of a newly synthesized amphidiploid, which may play an important role in the evolution of natural amphidiploid species. To test this hypothesis, a series of synthetic amphidiploids, including all three analogs of the natural amphidiploids B. napus, B. juncea, and B. Carinata and their reciprocal forms, were developed. These synthetic amphidiploids were characterized for morphological traits, chromosome number, and RFLPs revealed by chloroplast, mitochondrial, and nuclear DNA clones. The maternal transmission of chloroplast and mitochondrial genomes was observed in all of the F₁ hybrids examined except one hybrid plant derived from the B. rapa x B. oleracea combination, which showed a biparental transmission of organelles. However, the paternal chloroplast and mitochondrial genomes were not observed in the F₂ progeny. Nuclear genomes of synthetic amphidiploids had combined RFLP patterns of their parental species for all of the nuclear DNA clones examined. A variation in fertility was observed among self-pollinated progenies of single amphidiploids that had completely homozygous genome constitutions. Comparisons between natural and synthetic amphidiploids based on restriction fragment length polymorphism (RFLP) patterns indicated that natural amphidiploids are considerably more distant from the progenitor diploid species than the synthetic amphidiploids. The utility of these synthetic amphidiploids for investigating the evolution of amphidiploidy is discussed.     

Regulation of synthesis of the photosystem I reaction center

The in vivo biosynthesis of the P700 chlorophyll a-apoprotein was examined to determine whether this process is light regulated and to determine its relationship to chlorophyll accumulation during light- induced chloroplast development in barley (Hordeum vulgare L.). Rabbit antibodies to the 58,000-62,000-mol-wt apoprotein were used to measure relative synthesis rates by immunoprecipitation of in vivo labeled leaf proteins and to detect apoprotein accumulation on nitrocellulose protein blots. 5-d-old, dark-grown barley seedlings did not contain, or show net synthesis of, the 58,000-62,000-mol-wt polypeptide. When dark- grown barley seedlings were illuminated, net synthesis of the apoprotein was observed within the first 15 min of illumination and accumulated apoprotein was measurable after 1 h. After 4 h, P700 chlorophyll a-apoprotein biosynthesis accounted for up to 10% of the total cellular membrane protein synthesis. Changes in the rate of synthesis during chloroplast development suggest coordination between production of the 58,000-62,000-mol-wt polypeptide and the accumulation of chlorophyll. However, when plants were returned to darkness after a period of illumination (4 h) P700 chlorophyll a-apoprotein synthesis continued for a period of hours though at a reduced rate. Thus we found that neither illumination nor the rate of chlorophyll synthesis directly control the rate of apoprotein synthesis. The rapidity of the light-induced change in net synthesis of the apoprotein indicates that this response is tightly coupled to the primary events of light-induced chloroplast development. The data also demonstrate that de novo synthesis of the apoprotein is required for the onset of photosystem I activity in greening seedlings.     

Somatic hybridization in Nicotiana: Restoration of photoautotrophy to an albino mutant with defective plastids

Protoplasts of a cytoplasmic albino mutant of Nicotiana tabacum L. characterized by a deficient chloroplast genome were fused with protoplasts of a nitrate-reductase deficient mutant (NR^-) of N. tabacum. Somatic hybrids were obtained where the genome of the NR^- mutant was complemented by the cytoplasmic albino mutant which could synthesize an active nitrate reductase, and the chlorophyll deficiency in the albino mutant was restored by the chloroplasts from the NR^- mutant. Cybrids were also obtained in which the deficient plastids of the cytoplasmic albino mutant were replaced by normal chloroplasts from the NR^- mutant. The system used permitted a simple selection of the hybrids and the cybrids. The NR^- mutant was excluded at the cellular level by transfer of the cells to medium deficient in reduced nitrogen. The cytoplasmic albino mutant grew well on the selective nitrate medium. However, during callus formation, clear differences in the morphology and pigmentation of the calli were found which permitted selection for photoautotrophy at the callus level. The hybrid or cybrid nature of the plants was confirmed by examination of their morphology and chromosome number. Although the fusion partners come from the same species, only one plant showed the white-green variegated pattern typical of that of the cytoplasmic albino parent, indicating that segregation of plastids occurred during development of the calli and regeneration of the plants.     

Light-independent accumulation of chlorophyll a and b and protochlorophyllide in green barley (Hordeum vulgare)

Barley ( Hordeum vulgare L. cvs Clipper, Procter, Astrix) seedlings were transferred from daylight to darkness and changes in chlorophyll a , chlorophyll b , protochlorophyllide and chlorophyllide (μ leaf⁻¹) in either the first or second leaf determined spectrophotometrically after separating the esterified from unesterified pigments by partitioning between ammoniacal acetone and light petroleum ether. Chlorophyll a and b as well as protochlorophyllide accumulated in the dark. The ratio of chlorophyll to protochlorophyllide formed in the absence of light was 18:1. 5-aminolevulinic acid (10 m M ) promoted the synthesis of chlorophyll a and b and protochlorophyllide. Pigment synthesis and response to 5-aminolevulinic acid addition was related to tissue age. Mature tissue in the apical third of the leaf accumulated most chlorophyll, but per μg chlorophyll present at the time of transfer to darkness, was less efficient than immature tissue towards the base of the leaf. Immature tissue was also most responsive to added 5-aminolevulinic acid. Chlorophyll synthesis in the dark was accompanied by chloroplast development. Chloroplasts in immature leaf tissue increased in size and extent of thylakoid development when transferred from daylight to darkness. The results indicate that chlorophyll synthesis and chloroplast membrane development in light-grown barley continue into the dark phase of the diurnal cycle. A light-independent protochlorophyllide reductase in light-grown barley seedlings is postulated.     

Translation and stability of proteins encoded by the plastid psbA and psbB genes are regulated by a nuclear gene during light-induced chloroplast development in barley

We have characterized a nuclear mutant of barley, viridis-115, lacking photosystem II (PSII) activity and compared it to wild-type seedlings during light-induced chloroplast development. Chloroplasts isolated from wild-type and viridis-115 seedlings illuminated for 1 h synthesized similar polypeptides and had similar protein composition. After 16 h of illumination, however, mutant plastids exhibited reduced ability to radiolabel D1, CP47, and several low Mr membrane polypeptides, and by 72 h, synthesis of these proteins was undetectable. Immunoblot analysis showed that plastids of dark-grown wild-type barley lacked several PSII proteins (D1, D2, CP47, and CP43) and that 16 h of illumination resulted in the accumulation of these polypeptides. In contrast, these polypeptides did not accumulate in illuminated viridis-115 seedlings, although mutant plastids accumulated two PSII proteins that participate in oxygen evolution, oxygen-evolving enhancers 1 and 3. Northern analysis showed that the levels of psbA and psbB mRNA in mutant plastids were equal to or greater than levels in wild-type plastids throughout the developmental period examined here. These results indicate that the nuclear mutation present in viridis-115 affects the translation and stability of the chloroplast-encoded D1 and CP47 polypeptides and that its influence is expressed after the onset of light-induced chloroplast development.