Negative staining of chloroplast lamellae inAcanthus leaves below the compensation point

Summary Leaves ofAcanthus kept in an environment with a low concentration of carbon dioxide but connected to plants growing in open air show at electron microscopy level chloroplasts with anomalous stain of the thylakoids. Intra- and interthylakoidal spaces are electron opaque, while the outer protein layers appear formed by electron translucent globular units on which a dark deposit is visible in correspondence of the end-granal membranes and frets. It is suggested that the stain is in some way related to compounds active in light dependent photosynthesis which strongly reduce the osmium tetroxide.Supported by a grant of C.N.R. (Rome).     

A rapid,single leaf,nucleic acid assay for determining the cytoplasmic organelle complement of rapeseed and related Brassica species

Summary An assay is described whereby Eco RI restriction fragment length polymorphisms of mitochondrial and chloroplast DNAs can definitively identify cytoplasms of interest in Brassica crop development. Restrictable mitochondrial and chloroplast DNA is extracted from as little as 2–3 g and 0.5 g leaf tissue, respectively, and the donor plants are able to continue to develop in a normal manner. An unknown cytoplasm can be identified in three days, which is a considerable saving in time and labor compared to the several years required by traditional methods. The assay is very inexpensive and should be established as a routine procedure in laboratories involved in sexual or somatic Brassica hybrid production.     

Chloroplast development and the synthesis of chlorophyll and protochlorophyllide in Zostera transferred to darkness

Intact plants and isolated leaves of Zostera capricornii Martens ex Aschers were transferred from daylight to darkness. Substantial amounts of chloropyll a and b continued to accumulate in immature and mature tissue in the same ratio as in the light and were incorporated into chlorophyll-protein complexes in the thylakoids. A small amount of protochlorophyllide also accumulated in immature tissue in the dark. Proplastids and immature chloroplasts continued to develop into mature chloroplasts in the dark in the normal manner but prolamellar bodies, which are a conspicuous feature of immature chloroplasts, took longer to disperse than in the light. Protochlorophyllide accumulation and prolamellar-body formation were not correlated. The results indicate that Zostera has a genetic capacity for dark chlorophyll synthesis which is expressed in immature and mature leaf tissue and enables this plant to continue synthesising chlorophyll and assembling chloroplasts at night.Abbreviations Chl chlorophyll - T _o time of transfer to darkness     

The chloroplast gene encoding ribosomal protein S4 in Chlamydomonas reinhardtii spans an inverted repeat — unique sequence junction and can be mutated to suppress a streptomycin dependence mutation in ribosomal protein S12

The ribosomal protein gene rps4 was cloned and sequenced from the chloroplast genome of Chlamydomonas reinhardtii. The N-terminal 213 amino acid residues of the S4 protein are encoded in the single-copy region (SCR) of the genome, while the C-terminal 44 amino acid residues are encoded in the inverted repeat (IR). The deduced 257 amino acid sequence of C. reinhardtii S4 is considerably longer (by 51–59 residues) than S4 proteins of other photosynthetic species and Escherichia coli, due to the presence of two internal insertions and a C-terminal extension. A short conserved C-terminal motif found in all other S4 proteins examined is missing from the C. reinhardtii protein. In E. coli, mutations in the S4 protein suppress the streptomycin-dependent (sd) phenotype of mutations in the S12 protein. Because we have been unable to identify similar S4 mutations among suppressors of an sd mutation in C. reinhardtii S12 obtained using UV mutagenesis, we made site-directed mutations [Arg₆₈ (CGT) to Len (CTG and CTT)] in the wild-type rps4 gene equivalent to an E. coli Gln₅₃ to Len ribosomal ambiguity mutation (ram), which suppresses the sd phenotype and decreases translational accuracy. These mutants were tested for their ability to transform the sd S 12 mutation of C. reinhardtii to streptomycin independence. The streptomycin-independent isolates obtained by biolistic transformation all possessed the original sd mutation in rps12, but none had the expected donor Leu₆₈ mutations in rps4. Instead, six of 15 contained a Gln₇₃ (CAA) to Pro (CCA) mutation five amino acids downstream from the predicted mutant codon, irrespective of rps4 donor DNA. Two others contained six- and ten-amino acid, in-frame insertions at S4 positions 90 and 92 that appear to have been induced by the biolistic process itself. Eight streptomycin-independent isolates analyzed had wild-type rps4 genes and may possess mutations identical to previously isolated suppressors of sd that define at least two additional chloroplast loci. Cloned rps4 genes from streptomycin-independent isolates containing the Gln₇₃ to Pro mutation and the 6-amino acid insertion in r-protein S4 transform the sd strain to streptomycin independence.     

Chromosomal and cell size analysis of cold tolerant maize

Summary C-band number, guard cell length, and chloroplast number per guard cell were determined for eight maize populations. These populations consisted of maize selected for cold tolerance at the University of Nebraska as well as the original unselected populations. The genome size of these populations had previously been determined. C-band number fluctuated concertedly with the changes in genome size indicating that deletions and additions of constitutive heterochromatin occurred during selection, resulting in altered genome sizes. Guard cell size of all the cold tolerant populations was greater than the cell size of the respective nonselected populations. Chloroplast number per guard cell was also higher in all the cold tolerant populations than in their parental populations, but the increases were not statistically significant. The results indicate that changes in genome size that occurred during selection for cold tolerance are the result of changes in amounts of C-band heterochromatin and that the selection process results in an increase in cell size in the cold tolerant populations.     

Connection between the rate of cooling and fluorescence properties at 77 K of isolated chloroplasts

Cooling of chloroplasts to ?196°C can under certain circumstances lead to an erroneous analysis of energy distribution. After minimizing influences of sample geometry and effects of plastid concentration it is shown that externally induced membrane change leads to an increase in the ratio $\text{F}{}_{740}\text{F}{}_{687}$ of the fluorescence emission spectrum. Similar alterations can be observed by variation of the rate of cooling the plastids to 77 K, especially if whole chloroplasts are used. The differences in emission ratios are indicative also of changes in initial energy distribution between the photosystems, given here by the value α_N. This is inferred from experiments with either osmotically induced thylakoid disturbances or those effected through a slow cooling process. The circumstances and the significance of these observations are discussed.     

Characterization of the complete chloroplast genome of Zephyranthes phycelloides (Amaryllidaceae,tribe Hippeastreae) from Atacama region of Chile

Sporadic rains in the Atacama Desert reveal a high biodiversity of plant species that only occur there. One of these rare species is the “Red añañuca” (Zephyranthes phycelloides), formerly known as Rhodophiala phycelloides. Many species of Zephyranthes in the Atacama Desert are dangerously threatened, due to massive extraction of bulbs and cutting of flowers. Therefore, studies of the biodiversity of these endemic species, which are essential for their conservation, should be conducted sooner rather than later. There are some chloroplast genomes available for Amaryllidaceae species, however there is no complete chloroplast genome available for any of the species of Zephyranthes subgenus Myostemma. The aim of the present work was to characterize and analyze the chloroplast of Z. phycelloides by NGS sequencing. The chloroplast genome of the Z. phycelloides consists of 158,107 bp, with typical quadripartite structures: a large single copy (LSC, 86,129 bp), a small single copy (SSC, 18,352 bp), and two inverted repeats (IR, 26,813 bp). One hundred thirty-seven genes were identified: 87 coding genes, 8 rRNA, 38 tRNA and 4 pseudogenes. The number of SSRs was 64 in Z. phycelloides and a total of 43 repeats were detected. The phylogenetic analysis of Z. phycelloides shows a distinct subclade with respect to Z. mesochloa. The average nucleotide variability (Pi) between Z. phycelloides and Z. mesochloa was of 0.02000, and seven loci with high variability were identified: psbA, trnS^GCU-trnG^UCC, trnD^GUC-trnY^GUA, trnL^UAA-trnF^GAA, rbcL, psbE-petL and ndhG-ndhI. The differences between the species are furthermore confirmed by the high amount of SNPs between these two species. Here, we report for the first time the complete cp genome of one species of the Zephyranthes subgenus Myostemma, which can be used for phylogenetic and population genomic studies.     

The activation and inactivation of the Dunaliella salina chloroplast coupling factor 1 (CF1) in vivo and in situ

Preillumination of intact cells of the eukaryotic, halotolerant, cell-wall-less green alga Dunaliella salina induces a dark ATPase activity the magnitude of which is about 3–5-fold higher than the ATPase activity observed in dark-adapted cells. The light-induced activity arises from the activation and stabilization in vivo of chloroplast coupling factor 1 (CF₁). This activity, 150–300 μmol ATP hydrolyzed/mg Chl per h, rapidly decays (with a half-time of about 6 min at room temperature) in intact cells but only slowly decays (with a half-time of about 45 min at room temperature) if the cells are lysed by osmotic shock immediately after illumination. The activated form of the ATPase in lysed cells is inhibited if the membranes are treated with ferri- but not ferrocyanide, suggesting that the stabilization of the activated form of CF₁ is due to the reduction of the enzyme in vivo in the light.