Light metabolism and chloroplast structure in chlorophyll-deficient tobacco mutants

In tobacco mutants which contain 1/8 to 1/30 of the normal chlorophyll content per leaf area the content of yellow pigments (carotenoids) is also diminished but less in proportion to the chlorophyll content. The pale yellow-green mutant grows and matures provided that light intensity and temperature make up for the chlorophyll deficiency. In most green plants and algae light saturation of photosynthesis is reached between 5000 and 12,000 ergs/sec.cm2. The mutants continue to give higher photosynthetic rates until the incident intensity reaches 50,000 ergs/sec.cm2. While often unable to compensate their respiration at intensities at which the normal green plant approaches saturation, the pale yellow-green leaves are able to provide the mutant plant with two to three times the absolute amount of carbon dioxide assimilation per hour and leaf area at 50,000 ergs/sec.cm2 and 20 degrees to 25 degrees C. These observations are valid for red light lambda > 600 m mu. In blue light lambda < 575 m mu (below saturation levels) the mutants separate into two classes, one in which absorption by some carotenoid enhances the photosynthetic rate and the other in which the absorbing pigments are inactive and therefore depress the rate strongly. The unusual kinetics of photosynthesis in these chlorophyll-deficient tobacco mutants is reflected in the structure of their chloroplasts which we found to be of a kind thus far not described for healthy, normally growing, higher plants.     

Characterization of a virescent chloroplast mutant of tobacco

Virescent mutations produce plants in which young leaves have reduced chlorophyll levels but accumulate nearly normal amounts of chlorophyll as they age; they are predominantly nuclear mutations. We describe here a virescent mutation (designated Vir-c) found in a somatic hybrid line derived from Nicotiana tabacum L. and Nicotiana suaveolens Lehm. This mutation is inherited maternally. Young, half-expanded Vir-c leaves contained three to six times less chlorophyll than did control leaves, and reached maximum chlorophyll levels much later in development. Chlorophyll synthesis rates and chloroplast numbers per cell in Vir-c were similar to the control, and carotenoid content in Vir-c was sufficient to protect chlorophyll from photo-oxidation. Photosynthetic rates of Vir-c at low light intensities suggested a reduced ability to collect light. Electron micrographs of Vir-c chloroplasts from half-expanded leaves showed a significant reduction in thylakoids per granum. The decrease in granal thylakoids was strongly associated with low chlorophyll levels; mature Vir-c leaves with nearly normal chlorophyll content showed normal granal profiles. These results are discussed in relation to virescent mutants previously described.     

Molecular cloning of tobacco chloroplast ribosomal RNA genes

Summary Tobacco chloroplast ribosomal RNAs were shown to be hybridized with two EcoRI fragments of tobacco chloroplast DNA. These DNA fragments having molecular weights of 1.9x10⁶ and 2.8x10⁶ daltons were cloned using the bacterial plasmid pMB9 as a vector and E. coli HB101 as host bacteria. The recombinant plasmids containing either or both of these fragments were constructed and characterized.Abbreviations rRNA ribosomal RNA - EDTA ethylenediamine tetraacetic acid - SSC 0.15 M NaCl-0.015 M sodium citrate - EcoRI and HindIII restriction endonucleases isolated from E. coli RY13 and Haemophilus influenzae Rd, respectively     

Lamellar dispersion and phase separation of chloroplast membrane lipids by negative staining electron microscopy

Aqueous dispersions of lipids isolated from spinach chloroplast membranes were studied by electron microscopy after negative staining with phosphotungstic acid. Influence of low temperature (5°C for 24 h) was also investigated. It was observed that when contacted with water, these lipids, as such, formed multilamellar structures. Upon sonication, these multilamellar structures gave rise to a clear suspension of unilamellar vesicles varying in size (diameter) between 250 and 750 Å. When samples of sonicated unilamellar vesicles were stored at 5°C for 24 h or more, they revealed a variety of lipid aggregates including liposomes, cylindrical rods (about 100 Å wide and up to 3600 Å long), and spherical micellar structures (100–200 Å in diameter)—thus indicating phase separation of lipids.     

Direct sequencing of tobacco chloroplast genome by the polymerase chain reaction

We have developed a polymerase chain reaction (PCR) method for sequencing of tobacco chloroplast genome. In a mixture containing chloroplast DNA, 5-end-labeled oligonucleotide primer, Taq DNA polymerase and reaction buffer, we were able to sequence a segment of chloroplast 16S rRNA gene. The results showed that the 750 bp of DNA sequenced were identical to the sequence reported, indicating that direct sequencing method that we have developed is useful for the sequencing of chloroplast genome. To analyze the chloroplast genome more rapidly in those in vitro grown plantlets, we also developed a simple method which is applicable for the amplifications and sequencing of chloroplast 16S rRNA fragment from either 0.15 g of tobacco leaf or stem tissue. The readable sequences obtained from the presented methods were consistent with the published sequence.     

Mapping of transfer RNA genes on tobacco chloroplast DNA

Tobacco chloroplast tRNAs have been purified by two-dimensional polyacrylamide gel electrophoresis, identified by aminoacylation, labelled at their 3-end and hybridized to tobacco chloroplast DNA restriction fragments, in order to establish a tRNA gene map. These hybridization studies have revealed the localization of at least seven genes in each inverted repeat region, a minimum of 22 tRNA genes in the large single copy region and one tRNA gene in the small single copy region. Comparison of the tobacco chloroplast tRNA gene map to that of maize shows many similarities, but also some differences suggesting that DNA sequence rearrangements have occurred in the chloroplast genome during evolution.     

A novel dual-specificity protein kinase targeted to the chloroplast in tobacco

The NtDSK1 cDNA encoding a novel chloroplast-targeted protein kinase was identified in Nicotiana tabacum. It contains the kinase domain at the C-terminus and a putative regulatory domain at the N-terminus. The recombinant NtDSK1 underwent autophosphorylation of serine, threonine, and tyrosine residues, indicating that NtDSK1 encodes a functional dual-specificity protein kinase. The NtDSK1-green fluorescent protein fusion protein was targeted to chloroplasts. Furthermore, the NtDSK1 protein was immunodetected in chloroplast fractions isolated from tobacco seedlings. The NtDSK1 mRNA expression was developmentally regulated in different tissues, including anthers and germinating seeds, and strongly stimulated by gibberellin. The mRNA was rapidly light responsive during seedling growth. NtDSK1 may play a role in a light-regulated signaling process in tobacco.     

Effects of freezing on the structure of chloroplast membranes

Electron spin resonance (ESR) spectra of stearic acid spin labels incorporated into spinach thylakoids can be used to monitor membrane changes during freezing. Changes in the ESR parameters can be directly correlated to the extent of functional freeze damage. Freeze-induced changes in the ESR parameters strongly depend on the osmotic conditions of the incubation medium. Similar changes as on freezing can be observed by transferring thylakoids from an isotonic to a hypotonic medium, i.e., by swelling osmotically flattened thylakoids. This and computer simulations of spin label ESR spectra, which allow for variation of vesicle shape, lead to the conclusion that freeze-induced ESR spectral changes are due to swelling of the thylakoids. Indeed, van't Hoff plots of thylakoid packed volume indicate a freeze-induced increase in the apparent number of osmotically active molecules within the intrathylakoid lumen. During freezing, salt and/or sugar leak into the lumen. Simultaneously, proton channels are irreversibly opened. As the structural alterations obtained upon freezing are not accompanied by a change in bulk fluidity, these data are interpreted in terms of a local action of cryotoxic agents on critical microstructures, possibly at the rims of the thylakoid membranes.     

'Senescence-associated vacuoles' are involved in the degradation of chloroplast proteins in tobacco leaves

Massive degradation of photosynthetic proteins is the hallmark of leaf senescence; however the mechanism involved in chloroplast protein breakdown is not completely understood. As small 'senescence-associated vacuoles' (SAVs) with intense proteolytic activity accumulate in senescing leaves of soybean and Arabidopsis, the main goal of this work was to determine whether SAVs are involved in the degradation of chloroplastic components. SAVs with protease activity were readily detected through confocal microscopy of naturally senescing leaves of tobacco (Nicotiana tabacum L.). In detached leaves incubated in darkness, acceleration of the chloroplast degradation rate by ethylene treatment correlated with a twofold increase in the number of SAVs per cell, compared to untreated leaves. In a tobacco line expressing GFP targeted to plastids, GFP was re-located to SAVs in senescing leaves. SAVs were isolated by sucrose density gradient centrifugation. Isolated SAVs contained chloroplast-targeted GFP and the chloroplast stromal proteins Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) and glutamine synthetase, but lacked the thylakoid proteins D1 and light-harvesting complex II of the photosystem II reaction center and photosystem II antenna, respectively. In SAVs incubated at 30 degrees C, there was a steady decrease in Rubisco levels, which was completely abolished by addition of protease inhibitors. These results indicate that SAVs are involved in degradation of the soluble photosynthetic proteins of the chloroplast stroma during senescence of leaves.