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R R Klein

Publications and source records attributed to R R Klein.

At least 19 recordsLinked to original sources
PubMed2002

A molecular cytogenetic map of sorghum chromosome 1. Fluorescence in situ hybridization analysis with mapped bacterial artificial chromosomes.

We used structural genomic resources for Sorghum bicolor (L.) Moench to target and develop multiple molecular cytogenetic probes that would provide extensive coverage for a specific chromosome of sorghum. Bacterial artificial chromosome (BAC) clones containing molecular markers mapped across sorghum linkage group A were labeled as probes for fluorescence in situ hybridization (FISH). Signals from single-, dual-, and multiprobe BAC-FISH to spreads of mitotic chromosomes and pachytene bivalents were associated with the largest sorghum chromosome, which bears the nucleolus organizing region (NOR). The order of individual BAC-FISH loci along the chromosome was fully concordant to that of marker loci along the linkage map. In addition, the order of several tightly linked molecular markers was clarified by FISH analysis. The FISH results indicate that markers from the linkage map positions 0.0-81.8 cM reside in the short arm of chromosome 1 whereas markers from 81.8-242.9 cM are located in the long arm of chromosome 1. The centromere and NOR were located in a large heterochromatic region that spans approximately 60% of chromosome 1. In contrast, this region represents only 0.7% of the total genetic map distance of this chromosome. Variation in recombination frequency among euchromatic chromosomal regions also was apparent. The integrated data underscore the value of cytological data, because minor errors and uncertainties in linkage maps can involve huge physical regions. The successful development of multiprobe FISH cocktails suggests that it is feasible to develop chromosome-specific "paints" from genomic resources rather than flow sorting or microdissection and that when applied to pachytene chromatin, such cocktails provide an especially powerful framework for mapping. Such a molecular cytogenetic infrastructure would be inherently cross-linked with other genomic tools and thereby establish a cytogenomics system with extensive utility in development and application of genomic resources, cloning, transgene localization, development of plant "chromonomics," germplasm introgression, and marker-assisted breeding. In combination with previously reported work, the results indicate that a sorghum cytogenomics system would be partially applicable to other gramineous genera.

Chromosome Mapping↗
PubMed2001

Mapping genes on an integrated sorghum genetic and physical map using cDNA selection technology.

Sorghum is an important target of plant genomics. This cereal has unusual tolerance to adverse environments, a small genome (750 Mbp) relative to most other grasses, a diverse germplasm, and utility for comparative genomics with rice, maize and other grasses. In this study, a modified cDNA selection protocol was developed to aid the discovery and mapping of genes across an integrated genetic and physical map of the sorghum genome. BAC DNA from the sorghum genome map was isolated and covalently bound in arrayed tubes for efficient liquid handling. Amplifiable cDNA sequence tags were isolated by hybridization to individual sorghum BACs, cloned and sequenced. Analysis of a fully sequenced sorghum BAC indicated that about 80% of known or predicted genes were detected in the sequence tags, including multiple tags from different regions of individual genes. Data from cDNA selection using the fully sequenced BAC indicate that the occurrence of mislocated cDNA tags is very low. Analysis of 35 BACs (5.25 Mb) from sorghum linkage group B revealed (and therefore mapped) two sorghum genes and 58 sorghum ESTs. Additionally, 31 cDNA tags that had significant homologies to genes from other species were also isolated. The modified cDNA selection procedure described here will be useful for genome-wide gene discovery and EST mapping in sorghum, and for comparative genomics of sorghum, rice, maize and other grasses.

Base Sequence↗
PubMed2000

A high-throughput AFLP-based method for constructing integrated genetic and physical maps: progress toward a sorghum genome map.

Sorghum is an important target for plant genomic mapping because of its adaptation to harsh environments, diverse germplasm collection, and value for comparing the genomes of grass species such as corn and rice. The construction of an integrated genetic and physical map of the sorghum genome (750 Mbp) is a primary goal of our sorghum genome project. To help accomplish this task, we have developed a new high-throughput PCR-based method for building BAC contigs and locating BAC clones on the sorghum genetic map. This task involved pooling 24,576 sorghum BAC clones ( approximately 4x genome equivalents) in six different matrices to create 184 pools of BAC DNA. DNA fragments from each pool were amplified using amplified fragment length polymorphism (AFLP) technology, resolved on a LI-COR dual-dye DNA sequencing system, and analyzed using Bionumerics software. On average, each set of AFLP primers amplified 28 single-copy DNA markers that were useful for identifying overlapping BAC clones. Data from 32 different AFLP primer combinations identified approximately 2400 BACs and ordered approximately 700 BAC contigs. Analysis of a sorghum RIL mapping population using the same primer pairs located approximately 200 of the BAC contigs on the sorghum genetic map. Restriction endonuclease fingerprinting of the entire collection of sorghum BAC clones was applied to test and extend the contigs constructed using this PCR-based methodology. Analysis of the fingerprint data allowed for the identification of 3366 contigs each containing an average of 5 BACs. BACs in approximately 65% of the contigs aligned by AFLP analysis had sufficient overlap to be confirmed by DNA fingerprint analysis. In addition, 30% of the overlapping BACs aligned by AFLP analysis provided information for merging contigs and singletons that could not be joined using fingerprint data alone. Thus, the combination of fingerprinting and AFLP-based contig assembly and mapping provides a reliable, high-throughput method for building an integrated genetic and physical map of the sorghum genome.

Chromosomes, Bacterial↗
PubMed1997

Analysis of expression of an alternative La (SS-B) cDNA and localization of the encoded N- and C-terminal peptides.

A deletion of an (A)-residue was detected in a cDNA encoding for the nuclear autoantigen La/SS-B. The cDNA was recently isolated from a cDNA library made from peripheral blood lymphocytes of a patient with primary Sjögren's Syndrome. The region, where the deletion occurred, represents a hot spot region in the La gene(s). It leads to a frame shift mutation and a premature stop codon eleven amino acids downstream of the deletion site within one of the protease sensitive regions of the La protein. In spite of the frame shift mutation expression of full length La protein occurred efficiently in E. coli. Full length La protein was also made in SF9 cells infected with recombinant baculoviruses, although the efficiency of full length protein production was less. Two major peptides with molecular weights of 29 kDa and 25 kDa were made. The size of these peptides was similar to the known proteolytic degradation products of La protein. The N-terminal 29 kDa fragment containing the RNP consensus sequence located in the cytoplasm. The 25 kDa C-terminal fragment containing the nuclear location signal entered in the nucleus and associated with nuclear speckles. In conclusion, the ability to (i) enter, (ii) remain in the nucleus and (iii) assemble with nuclear speckles resides in the C-terminal domain of La protein and does not depend on the N-terminal RNP-consensus motif.

Amino Acid Sequence↗
PubMed1997

Altered acyl chain length specificity of Rhizopus delemar lipase through mutagenesis and molecular modeling.

The acyl binding site of Rhizopus delemar prolipase and mature lipase was altered through site-directed mutagenesis to improve lipase specificity for short- or medium-chain length fatty acids. Computer-generated structural models of R. delemar lipase were used in mutant protein design and in the interpretation of the catalytic properties of the resulting recombinant enzymes. Molecular dynamics simulations of the double mutant, val209trp + phe112trp, predicted that the introduction of trp112 and trp209 in the acyl binding groove would sterically hinder the docking of fatty acids longer than butyric acid. Assayed against a mixture of triacylglycerol substrates, the val209trp + phe112trp mature lipase mutant showed an 80-fold increase in the hydrolysis of tributyrin relative to the hydrolysis of tricaprylin while no triolein hydrolysis was detected. By comparison, the val94Trp mutant, predicted to pose steric or geometric constraints for docking fatty acids longer than caprylic acid in the acyl binding groove, resulted in a modest 1.4-fold increase in tricaprylin hydrolysis relative to the hydrolysis of tributyrin. Molecular models of the double mutant phe95asp + phe214arg indicated the creation of a salt bridge between asp95 and arg214 across the distal end of the acyl binding groove. When challenged with a mixture of triacylglycerols, the phe95asp + phe214arg substitutions resulted in an enzyme with 3-fold enhanced relative activity for tricaprylin compared to triolein, suggesting that structural determinants for medium-chain length specificity may reside in the distal end of the acyl binding groove. Attempts to introduce a salt bridge within 8 A of the active site by the double mutation leu146lys + ser115asp destroyed catalytic activity entirely. Similarly, the substitution of polar Gln at the rim of the acyl binding groove for phe112 largely eliminated catalytic activity of the lipase.

Caprylates↗
PubMed1996

Coordination of protein and mRNA abundances of stromal enzymes and mRNA abundances of the Clp protease subunits during senescence of Phaseolus vulgaris (L.) leaves.

Our objective was to determine the coordination of transcript and/or protein abundances of stromal enzymes during leaf senescence. First trifolioliate leaves of Phaseolus vulgaris L. plants were sampled beginning at the time of full leaf expansion; at this same time, half of the plants were switched to a nutrient solution lacking N. Total RNA and soluble protein abundances decreased after full leaf expansion whereas chlorophyll abundance remained constant; N stress enhanced the decline in these traits. Abundances of ribulose-1,5-bisposphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39), Rubisco activase and phosphoribulokinase (Ru5P kinase; EC 2.7.1.19) decreased after full leaf expansion in a coordinated manner for both treatments. In contrast, adenosine diphosphate glucose (ADPGlc) pyrophosphorylase (EC 2.7.7.27) abundance was relatively constant during natural senescence but did decline similar to the other enzymes under N stress. Northern analyses indicated that transcript abundances for all enzymes declined markedly on a fresh-weight basis just after full leaf expansion. This rapid decline was particularly strong for the Rubisco small subunit (rbcS) transcript. The decline was enhanced by N stress for rbcS and Rubisco activase (rca), but not for Ru5P kinase (prk) and ADPGlc pyrophosphorylase (agp). Transcripts of the Clp protease subunits clpC and clpP declined in abundance just after full leaf expansion, similar to the other mRNA species. When Northern blots were analyzed using equal RNA loads, rbcS transcripts still declined markedly just after full leaf expansion whereas rca and clpC transcripts increased over time. The results indicated that senescence was initiated near the time of full leaf expansion, was accelerated by N stress, and was characterized by large decline in transcripts of stromal enzymes. The decreased mRNA abundances were in general associated with steadily declining stromal protein abundances, with ADPGlc pyrophosphorylase being the notable exception. Transcript analyses for the Clp subunits supported a recent report (Shanklin et al., 1995, Plant Cell 7: 1713-1722) indicating that the Clp protease subunits were constitutive throughout development and suggested that ClpC and ClpP do not function as a senescence-specific proteolytic system in Phaseolus.

Adenosine Triphosphatases↗
PubMed1995

Characterization of the substrate specificity of sucrose-phosphate synthase protein kinase.

Sucrose-phosphate synthase (SPS; EC 2.4.1.14) is regulated by reversible protein phosphorylation. When the enzyme is phosphorylated it is inactivated and can be reactivated by removal of phosphate. The major regulatory phosphorylation site is known to be Ser158 in the spinach-leaf enzyme, and two protein kinase activities have been resolved chromatographically which phosphorylate SPS at this site in vitro. In this report, we use a set of synthetic peptide substrate analogs based on the phosphorylation site sequence, and a set of Escherichia coli-expressed 26-kDa fragments of spinach SPS which contain the site, to identify the recognition elements that target the two protein kinases to Ser158. The major recognition element consists of basic residues at P-3 and P-6 relative to the phosphorylated serine. Comparison of the spinach enzyme amino-acid sequence with two other plant species show conservation of these amino acids and implies that these signals are also conserved. We also present evidence that glucose-6-phosphate is not only an allosteric activator of SPS but also an inhibitor of SPS-protein kinase per se, thereby allowing it to act at both levels of SPS regulation.

Amino Acid Sequence↗
PubMed1995

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) N-methyltransferase (protein methylase III, Rubisco LSMT, EC 2.1.1.43) catalyzes methylation of the epsilon-amino group of Lys-14 in the LS of Rubisco. With limited internal amino acid sequence information obtained from HPLC-purified peptic polypeptides from Rubisco LSMT, a full-length cDNA clone was isolated utilizing polymerase chain reaction-based technology and conventional bacteriophage library screening. The 1802 bp cDNA of Rubisco LSMT encodes a 489 amino acid polypeptide with a predicted molecular mass of ca. 55 kDa. A derived N-terminal amino acid sequence with features common to chloroplast transit peptides was identified. The deduced sequence of Rubisco LSMT did not exhibit regions of significant homology with other protein methyltransferases. Southern blot analysis of pea genomic DNA indicated a low gene copy number of Rubisco LSMT in pea. Northern analysis revealed a single mRNA species of about 1.8 kb encoding for Rubisco LSMT which was predominately located in leaf tissue. Illumination of etiolated pea seedlings showed that the accumulation of Rubisco LSMT mRNA is light-dependent. Maximum accumulation of Rubisco LSMT transcripts occurred during the initial phase of light-induced leaf development which preceded the maximum accumulation of rbcS and rbcL mRNA. Transcript levels of Rubisco LSMT in mature light-grown tissue were similar to transcript levels in etiolated tissues indicating that the light-dependent accumulation of Rubisco LSMT mRNA is transient. This is the first reported DNA and amino acid sequence for a protein methylase III enzyme.

Amino Acid Sequence↗
PubMed1994

Photoaffinity labeling of the ATP binding domain of Rubisco activase and a separate domain involved in the activation of ribulose-1,5-bisphosphate carboxylase/oxygenase.

Photoaffinity labeling of Rubisco activase with 2- and 8-N3ATP was used to identify the adenine binding domain for ATP. Rubisco activase hydrolyzed both of these analogs of ATP and used their hydrolysis to support a low rate of Rubisco activation. When irradiated with ultraviolet light, these and other azido-substituted adenine nucleotides covalently modified Rubisco activase at two distinct binding sites. Competition binding experiments with ATP and ADP showed that one of the sites was the ATP binding domain. The other site was not a nucleotide binding domain per se but would bind adenine nucleotides if an azido moiety was present on the base. Tryptophan and other indoles prevented azidoadenine nucleotides from labeling this domain but afforded little protection to the ATP binding domain. The ability to selectively protect each of the two binding sites made it possible to localize the adenine binding domain for ATP to the region of Rubisco activase from N68-D74 and the other binding domain to a region near the N-terminus from Q10 to D14. Modification of the region from Q10 to D14 by photoaffinity labeling prevented Rubisco activase from promoting activation of Rubisco without affecting ATP hydrolysis. These data suggest that a specific region of Rubisco activase near the N-terminus may be a site of interaction with Rubisco. Binding of azidoadenine nucleotides in this region appears to be fortuitous and may involve base-stacking with the species-invariant Trp at position 16 and hydrogen bonding of the azido moiety.

Adenosine Triphosphate↗
PubMed1994

Mutational analysis of the tobacco vein mottling virus genome.

We have used a cDNA clone of the potyvirus, tobacco vein mottling virus, to construct 19 mutants bearing 12-nt insertions in the viral genome. These mutants display a variety of phenotypes in inoculated tobacco plants or protoplasts. All mutants with insertions in P3, CI, 6K, NIa, or NIb failed to produce detectable amounts of progeny viral RNA in protoplasts or plants which suggests that they all may be directly involved in replication. Mutants (one in P1 and one in HCpro) presumably affected in polyprotein processing also did not replicate in plants or protoplasts. Seven mutants, with insertions in the 5' noncoding region, P1, HCpro, or CP regions of the genome, were able to infect protoplasts. Three of the 7 mutants (1 in the 5' noncoding region and 2 in HCpro) were able to infect protoplasts but not plants. The remaining 4 mutants replicated in protoplasts and were able to cause systemic infection in plants. The mutation in the CP had no effect on virus accumulation or symptom development in inoculated plants, whereas the other 3 (1 in P1 and 2 in HCpro) induced cyclical patterns of symptom expression. These symptoms ranged from very mild to wild-type-like as new leaves emerged and, as the plants continued to grow, this pattern was repeated. These results support the assignment of roles in replication to five coding regions in the genome and demonstrate that sequence alterations in many parts of other regions of the viral genome may have pronounced effects on replication and the expression of disease symptoms.

Amino Acid Sequence↗
PubMed1994

Site-directed mutagenesis of a reactive lysyl residue (Lys-247) of Rubisco activase.

Chemical modification of tobacco leaf ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase with water-soluble N-hydroxysuccinimide esters identified Lys-247 as a particularly reactive residue necessary for maximal catalytic activity [M.E. Salvucci (1993) Plant Physiol. 103, 501-508]. To further explore the role of Lys-247 in catalysis, this species-invariant residue of Rubisco activase was changed to Arg, Cys, and Gln by mutagenesis of a cDNA clone of the mature form of the tobacco enzyme. Analysis of the purified recombinant proteins showed that all three point mutations reduced the rate of ATP hydrolysis to 2 to 3% of the wild-type enzyme and completely abolished the ability of Rubisco activase to promote activation of decarbamylated Rubisco. Replacement of Lys-247 with Arg, Cys, or Gln had a comparatively minor effect on ATP binding, but eliminated the increase in ATPase-specific activity that normally occurs with increasing concentrations of Rubisco activase protein. In mixing experiments, the K247R mutant enzyme inhibited Rubisco activation by wild-type Rubisco activase, indicating that interactions between Rubisco and Rubisco activase were disrupted by even the most conservative of the substitutions. Chemical elaboration of the K247C mutant by treatment with 2-bromoethylamine converted 39% of the thiols at position 247 to the aminoethyl derivative, but failed to improve the catalytic performance of the mutant enzyme. Our results indicate that the requirement for a lysyl residue at position 247 of Rubisco activase is very stringent, consistent with its proposed role in coordinating precise interactions with gamma-phosphate of ATP.

Adenosine Triphosphatases↗
PubMed1993

Identification of the major regulatory phosphorylation site in sucrose-phosphate synthase.

Sucrose-phosphate synthase (SPS; EC 2.4.1.14) is regulated in part by reversible protein phosphorylation. When dephospho-SPS is partially purified from illuminated spinach leaves and incubated with [gamma-32P]ATP the enzyme is phosphorylated by a copurifying protein kinase. In this report, 32P-phosphopeptides from tryptic digests of in vitro phosphorylated SPS were purified by metal-ion affinity chromatography and reversed-phase high-performance liquid chromatography. Three distinct 32P-phosphopeptides were resolved. Edman sequencing of the major phosphopeptide (which contained > 80% of the total 32P) identified the amino acid sequence as Ile-Ser-Ser(P)-Val-Glu-Met-Met-Asp-Asn-Trp-Ala-Asn-Thr-Phe-Lys. This sequence corresponds to residues 156 to 170 of the deduced amino acid sequence of spinach SPS [Klein, R. R., Crafts-Brandner, S. J., and Salvucci, M. E. (1993) Planta 190, 498-510, and Sonnewald, U., Quick, W. P., MacRae, E., Krause, K.-P., and Stitt, M. (1993) Planta 189, 174-181]. Identification of the phosphoseryl residue was accomplished by manual Edman sequencing. The two other phosphopeptides, which each contained less than 10% of the total 32P, were not sequenced. An Escherichia coli expressed, 26-kDa fragment of SPS which contains the major phosphorylation site was a substrate for the protein kinase which copurifies with SPS. Two-dimensional peptide mapping analysis of this fragment showed the major phosphopeptide was present but not the other site(s), suggesting that other peptides are derived from a site other than Ser158. These results provide additional indirect evidence for the presence of multiple phosphorylation sites in SPS.

Amino Acid Sequence↗
PubMed1993

Cloning and developmental expression of the sucrose-phosphate-synthase gene from spinach.

A 561-base-pair (bp) polymerase-chain-reaction (PCR) product of sucrose-phosphate synthase (SPS) was amplified using degenerate oligonucleotide primers corresponding to tryptic peptides of SPS (EC 2.4.1.14) from spinach (Spinacia oleracea L). Crucial to the primer specificity and the synthesis of the 561-bp product was the use of primer pools in which the number of degenerate primer species was limited. A full-length cDNA was subsequently obtained by screening a cDNA bacteriophage library with the 561-bp product of SPS and 5' PCR-RACE (Rapid Amplification of cDNA Ends). The 3530-bp cDNA of SPS encoded for a 1056-amino-acid polypeptide of predicted molecular mass of 117 kDa. The deduced amino-acid sequence of spinach SPS showed regions of strong homology with SPS from maize (A.C. Worrell et al., 1991, Plant Cell 3, 1121-1130); amino-acid identity was 54% over the entire protein. Western and Northern analyses of root, petiole and spinach leaf tissue showed that SPS was expressed in an organ-specific manner, being predominantly localized in the leaf. The accumulation of SPS protein and mRNA during leaf development coincided with the early rapid phase of leaf expansion and the apparent transition of the leaf from sink to source status. Levels of SPS mRNA and protein were reduced during the acclimation of leaves to low-irradiance conditions. Transfer of low-irradiance-adapted leaves to higher-irradiance conditions resulted in a gradual increase in SPS protein and mRNA. Diurnal changes in irradiance did not alter SPS protein or transcript levels, indicating that short-term regulation of SPS primarily involves a modulation of enzyme activity.

Actins↗
PubMed1993

Identification of the uridine-binding domain of sucrose-phosphate synthase. Expression of a region of the protein that photoaffinity labels with 5-azidouridine diphosphate-glucose.

The uridine diphosphate-glucose (UDP-Glc) binding domain of sucrose-phosphate synthase (SPS) was identified by overexpressing part of the gene from spinach (Spinacia oleracea). Degenerate oligonucleotide primers corresponding to two tryptic peptides common to both the full-length 120-kD SPS subunit and an 82-kD form that photoaffinity labeled with 5-azidouridine diphosphate-glucose (5-N3UDP-Glc) were used in a polymerase chain reaction to isolate a partial cDNA clone. Comparison of the deduced amino acid sequence of spinach SPS with the sequences of potato sucrose synthase showed that the partial cDNA included one region that was highly conserved between the proteins. Expression of the partial cDNA clone of SPS in Escherichia coli produced a 26-kD fusion protein that photoaffinity labeled with 5-N3UDP-Glc. Photoaffinity labeling of the 26-kD fusion protein was specific, indicating that this portion of the SPS protein harbors the UDP-Glc-binding domain. Isolation of a modified peptide from the photolabeled protein provided tentative identification of amino acid residues that make up the uridine-binding domain of SPS.

Amino Acid Sequence↗
PubMed1990

In vitro synthesis of chlorophyll a in the dark triggers accumulation of chlorophyll a apoproteins in barley etioplasts.

An in vitro translation system using lysed etioplasts was developed to test if the accumulation of plastid-encoded chlorophyll a apoproteins is dependent on the de novo synthesis of chlorophyll a. The P700 apoproteins, CP47 and CP43, were not radiolabeled in pulsechase translation assays employing lysed etioplasts in the absence of added chlorophyll precursors. When chlorophyllide a plus phytylpyrophosphate were added to lysed etioplast translation assays in the dark, chlorophyll a was synthesized and radiolabeled P700 apoproteins, CP47 and CP43, and a protein which comigrates with D1 accumulated. Chlorophyllide a or phytylpyrophosphate added separately to the translation assay in darkness did not induce chlorophyll a formation or chlorophyll a apoprotein accumulation. Chlorophyll a formation and chlorophyll a apoprotein accumulation were also induced in the lysed etioplast translation system by the photoreduction of protochlorophyllide to chlorophyllide a in the presence of exogenous phytylpyrophosphate. Accumulation of radiolabeled CP47 was detectable when very low levels of chlorophyll a were synthesized de novo (less than 0.01 nmol/10(7) plastids), and radiolabel increased linearly with increasing de novo chlorophyll a formation. Higher levels of de novo synthesized chlorophyll a were required prior to detection of radiolabel incorporation into the P700 apoproteins and CP43 (greater than 0.01 nmol/10(7) plastids). Radiolabel incorporation into the P700 apoproteins, CP47 and CP43, saturated at a chlorophyll a concentration which corresponds to 50% of the etioplast protochlorophyllide content (0.06 nmol of chlorophyll a/10(7) plastids).

Apoproteins↗
PubMed1990

Light-induced transcription of chloroplast genes. psbA transcription is differentially enhanced in illuminated barley.

Transcription and Northern dot blot analyses were used to determine the relationship between light-induced changes in RNA levels and transcription in barley and maize plastids. Overall plastid transcription activity is near maximum and largely light independent in apical sections of 4.5-day-old dark-grown barley seedings. With continued development in the dark, transcription activity in barley plastids declined precipitously, but transcription could be reactivated if plants were illuminated. Illumination of 8-day-old dark-grown barley seedlings increased the transcription of psbA, rbcL, and 16 S rDNA; however, the stimulation of psbA transcription was 2-4-fold more than rbcL or 16 S rDNA. Differential enhancement of psbA transcription occurred when 4.5- or 8-day-old dark-grown seedlings were illuminated, and the influence persisted in plants illuminated for 72 h. Illumination of 8-day-old dark-grown barley caused an increase in psbA and rbcL mRNA levels, but the increase in psbA mRNA was greater than that of rbcL. Similarly, illumination of 9-day-old dark-grown maize increased plastid transcription activity and the levels of psbA and rbcL mRNA. These results indicate that light can modulate overall plastid transcription activity and differentially stimulate the transcription of genes including psbA.

Blotting, Northern↗
PubMed1990

Chlorophyll regulates accumulation of the plastid-encoded chlorophyll apoproteins CP43 and D1 by increasing apoprotein stability.

Chlorophyll apoprotein accumulation in higher plant chloroplasts is controlled by light-dependent chlorophyll formation. Dark-grown plants lack chlorophyll and chlorophyll apoproteins. However, the plastid genes encoding the chlorophyll apoproteins are transcribed; chlorophyll apoprotein mRNA accumulates and associates with polysomes in plastids of dark-grown plants. Pulse-labeling assays revealed a population of short-lived proteins in plastids of dark-grown plants. One of these transiently labeled proteins was CP43, a chlorophyll apoprotein associated with photosystem II. Pulse-chase assays showed that newly synthesized CP43 was rapidly degraded in plastids of dark-grown plants, which lack chlorophyll. In contrast, CP43 synthesized in plastids from illuminated plants was stable. The synthesis of D1, a chlorophyll apoprotein of the photosystem II reaction center, was also analyzed in plastids of dark-grown and illuminated plants. Radiolabel accumulation into full-length D1 was only detected in plastids of illuminated plants. However, D1 translation intermediates of 15-25 kDa were detected in both plastid populations. Pulse-chase assays showed that the 15- to 25-kDa D1 translation products were precursors of mature D1 in plastids of illuminated plants. In contrast, in plastids of dark-grown plants, the 15- to 25-kDa translation intermediates were converted into a 23-kDa polypeptide previously suggested to be a proteolytic product of D1. These results indicate that chlorophyll produced in illuminated plants stabilizes D1 nascent polypeptides, which allows accumulation of mature D1.

Apoproteins↗