ISOLATION AND PRELIMINARY CHARACTERIZATION OF THREE CHLAMYDOMONAS STRAINS INTERFERTILE WITH CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

Three new strains of the unicellular green alga Chlamydomonas reinhardtii Dangeard were isolated from soil. The isolates differed from one another and from standard laboratory strains of C. reinhardtii in a number of traits, including heavy metal resistance, protein composition, and mitochondrial DNA length. The new isolates also exhibited distinctive restriction fragment length polymorphisms in their nuclear, chloroplast, and mitochondrial genomes. The new isolates were interfertile with the standard laboratory strains and appeared to transfer chloroplast and mitochondrial genomes in a similar manner, that is, predominantly from the material (mt⁺) and paternal (mt^?) parents, respectively.     

IN VIVO SPECIFIC LABELING OF CHLAMYDOMONAS CHLOROPLAST DNA

When Chlamydomonas reinhardi is supplied with (methyl-³H)-thymidine, radioactivity is incorporated specifically into chloroplast DNA Chromatographic analysis of the products of enzymatic hydrolysis of the DNA reveals that only thymidine monophosphate has been labeled. Use of thymidine-6-³H yields an identical result. If thymidine-³H monophosphate is supplied, a small amount of radioactivity is incorporated into both nuclear and chloroplast DNA in proportion to the abundance of these DNA components. These observations are consistent with earlier suggestions that algae lack cytoplasmic thymidine kinase, but that the enzyme is present within their chloroplasts.     

EFFECT OF LIGHT HISTORY ON THE ULTRASTRUCTURE AND PHYSIOLOGY OF PROROCENTRUM MARIAE-LEBOURIAE (DINOPHYCEAE)1

Ultrastructural and physiological responses of Prorocentrum mariae-lebouriae (Parke & Ballantine) Faust are reported for cultures maintained at growth irradiances (I_g) ranging from 20.6 to 0.3 E m^?2.d^?1 and following downward shifts in light intensity. We tested the hypothesis that Prorocentrum grown under light regimes that elicit different responses in photosynthesis and pigmentation exhibit distinctive cell ultrastructures. Prorocentrum from high-light conditions had high saturation intensities for photosynthesis (I_k) and low levels of Chl a, Chl c and peridinin-cell^?1 These cultures were morphologically distinguished by a large starch volume fraction (V_v), small chloroplast V_v and fewer thylakoids lamella^?1. I_k values were lower and pigment concentrations higher in low-light treatments, and cells showed reduced starch V_v, large chloroplast V_v, and higher numbers of thylakoids · lamella ^?1. Cells grown under extremely low-light conditions appeared stressed as indicated by the absence of starch reserves and the presence of large vacuoles within the cytoplasm. Results for presence of large vacuoles within the cytoplasm. Results for quantiative electron microscopy, photosynthesis-irradiance (P-I) relations and cell pigmentation indicate that photoadaptation in P. mariae-lebouriae involves a strategy that encompasses changes in both the “size” and “number” of photosynthetic units.     

Effect of N6-benzyladenine and indole-3-butyric acid on photosynthetic apparatus of Orthosiphon stamineus plants grown in vitro

The leaf structure and chloroplast ultrastructure of kidney tea (Orthosiphon stamineus Benth.) was studied in in vitro culture on standard MS medium supplemented with or without plant growth regulators (PGRs). The cytokinin N⁶-benzyladenine (BA) negatively affected the structure of the palisade parenchyma and chloroplast ultrastructure and increased the stomatal frequency of the adaxial epidermis. The auxin indole-3-butyric acid (IBA) did not modify the morphology of regenerated leaf tissues as well as the chloroplast ultrastructure. The effect of both PGRs applied in combination was manifested in well-differentiated mesophyll parenchyma, typical chloroplast ultrastructure and increased stomatal frequency on both leaf surfaces. This protocol can be suggested for further ex vitro propagation.     

Effects of lanthanum and calcium on photoelectron transport activity and the related protein complexes in chloroplast of cucumber leaves

The effects of lanthanum and calcium ions on electron transport, dichlorephenol indophenol (DCIP) photoreduction, and oxygen evolution activities in chloroplast from cucumber (Cucumis satives L.) were determined. The lanthanum inhibited the whole electron chain-transport activity of chloroplast. DCIP photoreduction and oxygen evolution activities of the photosystem I (PSII) also decrease after treatment with La³⁺. But the diminished activities of PSII and chloroplast caused by La³⁺ could be reversed by Ca²⁺ and even became higher than the control level. The concentration analysis of related protein complexes to photoelectron transport in chloroplast included that La³⁺ induced the concentration of chlorophyll protein complexes increasing but caused some nonchlorophyll protein complexes to decompose partially. This increasing effect of La³⁺ on chlorophyll protein complexes results in the improvement of chlorophyll content, which will improve the absorption of photoelectron and energy transport in the process of photosynthesis.     

LOSS OF HYBRID LETHALITY DURING BACKCROSS PROGRAMS INVOLVING CHLAMYDOMONAS EUGAMETOS AND CHLAMYDOMONAS MOEWUSII (CHLOROPHYCEAE)1

Wild-type strains of the interfertile species Chlamydomonas eugametos (UTEX 9 and 10) and Chlamydomonas moewusii (UTEX 96 and 97) male readily and reciprocally; however, considerable lethality occurs among F₁ hybrid meiotic products. We prepared two hybrid backcross lineages using C. eugametos and C. moewusii. One lineage began with the cross C. eugametos mating-type-plus (mt⁺) × C. moewusii mating-type-minus (mt^?). An F₁ mt⁺ hybrid from this cross was back-crossed to C. moewusii mt^?, and a B₁ mt⁺ hybrid was recovered. The B₁ hybrid was again backcrossed to C. moewusii mt^?, and this process was repeated through the fifth backcross. The other backcross lineage began with the reciprocal cross C. moewusii mt⁺× C. eugametos mt^? and employed C. eugametos as the recurring mt^? parent. This lineage also was continued through the fifth backcross. Meiotic product survival in the reciprocal interspecific crosses was less than 10%. In successive back-cross generations associated with both lineages, this value increased progressively to a maximum of 85–90%, the level observed for the intraspecific crosses. These results are consistent with the hypothesis that multiple genetic differences exist between C. eugametos and C. moewusii and that these are the major source of meiotic product lethality associated with the interspecific crosses. The inheritance of chloroplast genetic markers for resistance to streptomycin (sr-2) and for resistance to erythromycin (er-nM1) was also scored w the interspecific crosses and in the backcrosses. Most hybrid zygospores transmitted the resistance markers of the mt⁺ parent only, or of both parents, with the former zygospore type being more common. Although the intraspecific C. eugametos and C. moewusii crosses differ conspicuously with respect to the fraction of zygospores which transmit chloroplast genetic markers of both parents, the inheritance of chloroplast genetic markers in the interspecific crosses and backcrosses at' scribed here failed to clarify the genetic basis for this difference.     

Unlike in other monocotyledonous plants such as wheat and rice, the region containing the tRNA (UCC), tRNA (UCU) and CF1 ATPase α-subunit genes has not undergone recombination in the leek chloroplast DNA

The nucleotide sequence of a 1.1 kbp BamHI fragment of the leek chloroplast DNA (Allium porrum., fam. Liliaceae) has been determined. The fragment contains the 3' part of the tRNA^Gly (UCC) gene and the tRNA^Arg (UCU) gene on the same strand, and the 3' end of the atpA gene encoding the CF₁ ATPase α-subunit which is located on the opposite strand. The gene arrangement and nucleotide sequence of this fragment are similar to those of the corresponding region in the tobacco chloroplast DNA but differ significantly from what has been observed in other monocotyledonous plants such as wheat and rice, in which the region containing these genes has undergone intensive rearrangement.     

Localization and nucleotide sequences of the tRNA GCC Gly,tRNA GUC Asp and tRNA GCA Cys genes from wheat chloroplast

The location on the wheat chloroplast DNA map and the nucleotide sequences of the genes coding for tRNA _GCC ^Gly (trnG-GCC), tRNA _GUC ^Asp (trnD-GUC) and tRNA _GCA ^Cys (trnC-GCA) have been determined. These three genes are located in the large single copy region of the chloroplast genome, about half-way between one of the inverted repeats and the gene for the α subunit of ATP synthase. They are located on two Bam H1 fragments, called B6 and B18 by Bowmanet al. (1), which are separated by about 450 bp and which were cloned in our laboratory to allow sequencing. ThetrnD-GUC andtrnC-GCA sequences show 98.6 and 89% homology, respectively, with the corresponding spinach chloroplast tRNA genes sequences (2), which are the only other higher plant chloroplasttrnD-GUC andtrnC-GCA sequenced so far, while no othertrnG-GCC sequence has been published. ThetrnG-GCC sequence shows only 58% homology with the corresponding gene sequence inEuglena chloroplasts (3).     

RELATIONSHIPS BETWEEN PHOTOSYNTHETIC CAPACITY AND LEAF STRUCTURE IN SEVERAL SHADE PLANTS

Leaf structure, photosynthetic characteristics and related physiological parameters have been studied in three ornamental shade species: Fatsia japonica, Cissus rhombifolia (relatively light-tolerant plants), and Philodendron scandens (obligate shade plant). Species were grown in a shadehouse. Maximum photosynthetic photon flux density was 470 μmol m^-2 s^-1. Net rate of CO₂ uptake at light saturation (maximum Pn) in Fatsia was 6.90 ± 1.27 μmol m^-2 s^-1. In Cissus and Philodendron values were about 30% and 63% less respectively, than those measured in Fatsia. The nitrogen content, relative dry wt, specific leaf dry wt (SLDW), chlorophyll a/b ratio, and nitrogen to chlorophyll ratio were lower in Philodendron. However, leaf thickness in Philodendron (296 ± 17 μm) was about 54% and 160% higher, respectively, than in Fatsia and Cissus, and the ratio between mesophyll cell area and leaf surface area (A^mes/A) was nearly similar in the three species. However Philodendron exhibited a percentage of palisade parenchyma about three times lower than that observed in the two other species. The chloroplast number per mm of cell wall in transverse sections (chloroplast density) in the palisade parenchyma was fairly constant (about 65), irrespective of species. The “chloroplast density” in the spongy parenchyma of Philodendron was about 53% and 63%, respectively, of Fatsia and Cissus values. In Fatsia and Cissus chloroplast ultrastructure seems to change gradually and continuously from sun to shade type with the depth from the adaxial to abaxial surface. Special emphasis was given in order to determine the structural parameters best correlated with maximum Pn between the different species. In this way chloroplast number in transverse sections (chloroplast number) and the ratio between chloroplast area and leaf surface area (A^chl/A) were the parameters best correlated with maximum Pn, and stomatal frequency was also a good determinant of maximum Pn. However, leaf thickness, SLDW, and even A^mes/A ratio were weakly correlated with maximum Pn.     

Catabolism of indole-3-acetic acid in chloroplast fractions from light-grown Pisum sativum L. seedlings

Abstract The catabolism of indole-3-acetic acid was investigated in chloroplast preparations and a crude enzyme fraction derived from chloroplasts of Pisum sativum seedlings. Data obtained with both systems indicate that indole-3-acetic acid undergoes decarboxylative oxidation in pea chloroplast preparations. An enhanced rate of decarboxylation of [1′-¹C]indole-3-acetic acid was obtained when chloroplast preparations were incubated in the light rather than in darkness. Results from control experiments discounted the possibility of this being due to light-induced breakdown of indole-3-acetic acid. High performance liquid chromatography analysis of [2′-¹⁴C]indole-3-acetic acid-fed incubates showed that indole-3-methanol was the major catabolite in both the chloroplast and the crude enzyme preparations. The identification of this reaction product was confirmed by gas chromatography-mass spectrometry when [²H₅]indole-3-methanol was detected in a purified extract derived from the incubation of an enzyme preparation with ³²H₅]indole-3-acetic acid.     

Chloroplast DNA synthesis in light and dark grown cultured Nicotiana tabacum cells as determined by molecular hybridization

A simple method using molecular hybridization was devised to quantitatively measure chloroplast DNA synthesis in vivo. Total cellular DNA isolated from Nicotiana tabacum suspension cells, labeled with ³H-thymidine, was hybridized to nitrocellulose membrane-bound cloned chloroplast DNA (ct DNA) fragments. Colorless, dark grown N. tabacum cells were found to contain approximately 3300–4800 chloroplast genome copies per cell, whereas light grown, green cells contain about 9500–12000 chloroplast genomes per cell. This difference in ct DNA levels suggests that the chloroplast genome is somewhat amplified during growth of the cells in the light. The hybridization technique was also used to measure the efficiency of hybridization between cloned spinach ct DNA and tobacco ct DNA. The two DNAs were found to cross-hybridize with an efficiency of 69–75%.     

Electrophoretic and immunological comparisons of chloroplast and prokaryotic ribosomal proteins reveal that certain families of large subunit proteins are evolutionarily conserved

Summary Antibodies to individual chloroplast ribosomal (r-)proteins ofChlamydomonas reinhardtii synthesized in either the chloroplast or the cytoplasm were used to examine the relatedness ofChlamydomonas r-proteins to r-proteins from the spinach (Spinacia oleracea) chloroplast,Escherichia coli, and the cyanobacteriumAnabaena 7120. In addition,³⁵S-labeled chloroplast r-proteins from large and small subunits ofC. reinhardtii were coelectrophoresed on 2-D gels with unlabeled r-proteins from similar subunits of spinach chloroplasts,E. coli, andAnabaena to compare their size and net charge. Comigrating protein pairs were not always immunologically related, whereas immunologically related r-protein pairs often did not comigrate but differed only slightly in charge and molecular weight. In constrast, when³⁵S-labeled chloroplast r-proteins from large and small subunits of a closely related speciesC. smithii were coelectrophoresed with unlabeledC. reinhardtii chloroplast r-proteins, only one pair of proteins from each subunit showed a net displacement in mobility.Analysis of immunoblots of one-dimensional SDS and two-dimensional urea/SDS gels of large and small subunit r-proteins from these species revealed more antigenic conservation among the four species of large subunit r-proteins than small subunit r-proteins.Anabaena r-proteins showed the greatest immunological similarity toC. reinhardtii chloroplast r-proteins. In general, antisera made against chloroplast-synthesized r-proteins inC. reinhardtii showed much higher levels of cross-reactivity with r-proteins fromAnabaena, spinach, andE. coli than did antisera to cytoplasmically synthesized r-proteins. All spinach r-proteins that cross-reacted with antisera to chloroplast-synthesized r-proteins ofC. reinhardtii are known to be made in the chloroplast (Dorne et al. 1984b). FourE. coli r-proteins encoded by the S10 operon (L2, S3, L16, and L23) were found to be conserved immunologically among the four species. Two of the large subunit r-proteins, L2 and L16, are essential for peptidyltransferase activity. The third (L23) and two otherE. coli large subunit r-proteins (L5 and L27) that have immunological equivalents among the four species are functionally related to but not essential for peptidyltransferase activity.     

INTERSTRAIN VARIABILITY IN PHYSIOLOGY AND GENETICS OF HETEROSIGMA AKASHIWO (RAPHIDOPHYCEAE) FROM THE WEST COAST OF NORTH AMERICA1

High levels of intraspecific variability are often associated with HAB species, and this variability is likely an important factor in their competitive success. Heterosigma akashiwo (Hada) Hada ex Y. Hara et M. Chihara is an ichthyotoxic raphidophyte capable of forming dense surface‐water blooms in temperate coastal regions throughout the world. We isolated four strains of H. akashiwo from fish‐killing northern Puget Sound blooms in 2006 and 2007. By assessing numerous aspects of biochemistry, physiology, and toxicity, we were able to describe distinct ecotypes that may be related to isolation location, source population, or bloom timing. Contrasting elements among strains were cell size, maximum growth and photosynthesis rates, tolerance of low salinities, amino acid use, and toxicity to the ciliate grazer Strombidinopsis acuminatum (Fauré‐Fremiet). In addition, the rDNA sequences and chloroplast genome of each isolate were examined, and while all rDNA sequences were identical, the chloroplast genome identified differences among the strains that tracked differences in ecotype. H. akashiwo strain 07A, which was isolated from an unusual spring bloom, had a significantly higher maximum potential photosynthesis rate (28.7 pg C · cell^?1 · h^?1) and consistently exhibited the highest growth rates. Strains 06A and 06B were not genetically distinct from one another and were able to grow on the amino acids glutamine and alanine, while the other two strains could not. Strain 07B, which is genetically distinct from the other three strains, exhibited the only nontoxic effect. Thus, molecular tools may support identification, tracking, and prediction of strains and/or ecotypes using distinctive chloroplast gene signatures.     

PEROXIREDOXIN Q stimulates the activity of the chloroplast 16:1Δ3trans FATTY ACID DESATURASE4

Thylakoid membrane lipids, comprised of glycolipids and the phospholipid phosphatidylglycerol (PG), are essential for normal plant growth and development. Unlike other lipid classes, chloroplast PG in nearly all plants contains a substantial fraction of the unusual trans fatty acid 16:1^Δ3trans or 16:1t. We determined that, in Arabidopsis thaliana, 16:1t biosynthesis requires both FATTY ACID DESATURASE4 (FAD4) and a thylakoid‐associated redox protein, PEROXIREDOXIN Q (PRXQ), to produce wild‐type levels of 16:1t. The FAD4–PRXQ biochemical relationship appears to be very specific in planta, as other fatty acids (FA) desaturases do not require peroxiredoxins for their activity, nor does FAD4 require other chloroplast peroxiredoxins under standard growth conditions. Although most of chloroplast PG assembly occurs at the inner envelope membrane, FAD4 was primarily associated with the thylakoid membranes facing the stroma. Furthermore, co‐production of PRXQ with FAD4 was required to produce Δ3‐desaturated FAs in yeast. Alteration of the redox state of FAD4 or PRXQ through site‐directed mutagenesis of conserved cysteine residues impaired Δ3 FA production. However, these mutations did not appear to directly alter disulfide status of FAD4. These results collectively demonstrate that the production of 16:1t is linked to the redox status of the chloroplast through PRXQ associated with the thylakoids.     

Biosynthesis of indole-3-acetic acid in protoplasts,chloroplasts and a cytoplasmic fraction from barley (Hordeum vulgare L.)

Protoplast preparations from barley (Hordeum vulgare L.) enzymatically converted [5-³H]tryptophan to [³H]indole-3-acetic acid (IAA). Both a chloroplast and a crude cytoplasmic fraction, isolated from protoplasts that had previously been fed [5-³H]tryptophan, contained [³H]IAA. Chloroplast and cytoplasmic preparations, isolated from protoplasts and thereafter incubated with [5-³H]tryptophan, also synthesized [³H]IAA, although, in both instances the pool size was less than 50% of that detected in the in-vivo feeds. There were no significant differences in the amounts of [³H]IAA that accumulated in protoplast and chloroplast preparations incubated in light and darkness.Abbreviations HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - RC radiocounting