A small decrease of plastid transketolase activity in antisense tobacco transformants has dramatic effects on photosynthesis and phenylpropanoid metabolism.

Transketolase (TK) catalyzes reactions in the Calvin cycle and the oxidative pentose phosphate pathway (OPPP) and produces erythrose-4-phosphate, which is a precursor for the shikimate pathway leading to phenylpropanoid metabolism. To investigate the consequences of decreased TK expression for primary and secondary metabolism, we transformed tobacco with a construct containing an antisense TK sequence. The results were as follows: (1) a 20 to 40% reduction of TK activity inhibited ribulose-1,5-bisphosphate regeneration and photosynthesis. The inhibition of photosynthesis became greater as irradiance increased across the range experienced in growth conditions (170 to 700 micromol m(-2) sec(-1)). TK almost completely limited the maximum rate of photosynthesis in saturating light and saturating CO(2). (2) Decreased expression of TK led to a preferential decrease of sugars, whereas starch remained high until photosynthesis was strongly inhibited. One of the substrates of TK (fructose-6-phosphate) is the starting point for starch synthesis, and one of the products (erythrose-4-phosphate) inhibits phosphoglucose isomerase, which catalyzes the first reaction leading to starch. (3) A 20 to 50% decrease of TK activity led to decreased levels of aromatic amino acids and decreased levels of the intermediates (caffeic acid and hydroxycinnamic acids) and products (chlorogenic acid, tocopherol, and lignin) of phenylpropanoid metabolism. (4) There was local loss of chlorophyll and carotene on the midrib when TK activity was inhibited by >50%, spreading onto minor veins and lamina in severely affected transformants. (5) OPPP activity was not strongly inhibited by decreased TK activity. These results identify TK activity as an important determinant of photosynthetic and phenylpropanoid metabolism and show that the provision of precursors by primary metabolism colimits flux into the shikimate pathway and phenylpropanoid metabolism.

Mutations in the small subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase increase the formation of the misfire product xylulose-1,5-bisphosphate.

The small subunit (S) increases the catalytic efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) by stabilizing the active sites generated by four large subunit (L) dimers. This stabilization appears to be due to an influence of S on the reaction intermediate 2,3-enediol, which is formed after the abstraction of a proton from the substrate ribulose-1,5-bisphosphate. We tested the functional significance of residues that are conserved among most species in the carboxy-terminal part of S and analyzed their influence on the kinetic parameters of Synechococcus holoenzymes. The replacements in S (F92S, Q99G, and P108L) resulted in catalytic activities ranging from 95 to 43% of wild type. The specificity factors for the three mutant enzymes were little affected (90-96% of wild type), but Km(CO2) values increased 0.5- to 2-fold. Mutant enzymes with replacements Q99G and P108L showed increased mis-protonation, relative to carboxylation, of the 2,3-enediol intermediate, forming 2 to 3 times more xylulose-1,5-bisphosphate per ribulose-1,5-bisphosphate utilized than wild-type or F92S enzymes. The results suggest that specific alterations of the L/S interfaces and of the hydrophobic core of S are transmitted to the active site by long-range interactions. S interactions with L may restrict the flexibility of active-site residues in L.

Composition of photosystem II antenna in light-harvesting complex II antisense tobacco plants at varying irradiances.

Plants with genes coding for chlorophyll a/b-binding proteins of light-harvesting complex II (LHCII) in antisense orientation (Lhcb) that are characterized by severely reduced Lhcb transcript levels (below 10% of wild type) do not show a bleached phenotype due to a specific loss of the polypeptide. To produce such a phenotype, a conceptually different antisense approach was tested with a dual-functional transcript encoding the gene for hygromycin phosphotransferase and the transit sequence of Lhcb1-2 in the antisense orientation. Using increasing concentrations of hygromycin, transformants with Lhcb steady-state levels as low as 9% of wild type were regenerated and grown in a growth chamber. Together with Lhcb antisense plants obtained in an earlier study, these antisense plants were analyzed biochemically for their photosystem II (PSII) antenna composition under varying light conditions. All antisense plants showed a characteristic low-irradiance-induced increase of their PSII antenna size as determined by higher chlorophyll concentrations, an increased content of LHCII, and a constant chlorophyll b-to-lutein ratio in comparison with control plants. One to 5% of the total Lhcb transcript amount was sufficient to allow unrestricted formation of the PSII antenna at low irradiance, suggesting that LHCH biogenesis is not controlled primarily by transcription.

Crystallization and identification of an assembly defect of recombinant antenna complexes produced in transgenic tobacco plants.

A chimeric Lhcb gene encoding light-harvesting chlorophyll a/b-binding protein (LHCII) was expressed in transgenic tobacco plants. To separate native from recombinant LHCII, the protein was extended by six histidines at its C terminus. Recombinant LHCII was isolated by detergent-mediated monomerization of pure trimers followed by affinity-chromatography on Ni(2+)-NTA-agarose (NTA is nitrilotriacetic acid). Elution with imidazole yielded recombinant monomers that formed trimers readily after dilution of the detergent without further in vitro manipulations. LHCII subunits showed the typical chlorophyll a/b ratio at all steps of purification indicating no significant loss of pigments. Transgenic tobacco overexpressed amounts of recombinant protein that corresponded to about 0.7% of total LHCII. This yield suggested that expression in planta might be an alternative to the expression of eukaryotic membrane proteins in yeast. Recombinant LHCII was able to form two-dimensional crystals after addition of digalactolipids, which diffracted electrons to 3.6-A resolution. LHCII carrying a replacement of Arg-21 with Gln accumulated to only 0.004% of total thylakoid proteins. This mutant was monomeric in the photosynthetic membrane probably due to the deletion of the phosphatidylglycerol binding site and was degraded by the plastidic proteolytic system. Exchange of Asn-183 with Leu impaired LHCII biogenesis in a similar way presumably due to the lack of a chlorophyll a binding site.

Accumulation of plant antenna complexes is regulated by post-transcriptional mechanisms in tobacco.

Transgenic tobacco plants expressing antisense RNA directed against the multigene family of the light-harvesting complex of photosystem II (LHCII) were raised and analyzed biochemically and physiologically. A partial 5' terminal sequence with 509 nucleotides complementary to cab (chlorophyll a/b binding protein) genes reduced the amount of transcript to almost undectectable levels. We demonstrated for endogenous genes that a 5' terminal sequence with only 52 to 105 nucleotides complementary to the transit sequence of cab can be equally efficient in gene repression. Chlorophyll content and chlorophyll a-to-chlorophyll b ratios of thylakoid membranes isolated from transgenic plants were unchanged in comparison with the wild type. Photosynthetic oxygen evolution and in vivo-measured chlorophyll fluorescence of the transformants showed that LHCII accumulates to normal levels. The reduced level of cab mRNA did not correlate with the amount of LHCII in thylakoids. This indicates that transcriptional regulation is not the rate-limiting step in the biogenesis of the LHCII apoprotein. The antenna size of photosystem II is therefore modulated by yet undiscovered posttranscriptional mechanisms.

SecY, an integral subunit of the bacterial preprotein translocase, is encoded by a plastid genome.

Although the paradigm for the acquisition of photosynthetic organelles is the endocytosis of cyanobacteria-like progenitors by heterotrophic protists, details of this evolutionary process are unclear. The small organellar chromosomes are remnants of the larger bacterial genomes with most genes from the endosymbiont's DNA having been either relocated to the protist's nucleus or entirely lost. As a result of those gene transfers, differences exist between plastids from different algal phyla and higher plants. We report here on the retention of a secY gene in cyanelle (= plastid) DNA of the eukaryotic protist Cyanophora paradoxa. This cyanelle secY encodes a functional protein homologous to SecY of Escherichia coli, identified as a subunit of the preprotein translocase complex. Similarity of the cyanelle and E. coli SecY topology, predicted from sequence information, has been confirmed experimentally through SecY-PhoA fusion protein analysis in E. coli. Cyanelle SecY, expressed in an E. coli secY mutant, substituted for the defective prokaryotic SecY. A plastid-encoded gene for a membrane protein functioning in protein transport across plastid membranes is unprecedented in higher plants. From these results we infer that a functional homolog of the prokaryotic preprotein translocation machinery is retained in some plastids.

Replacement of a conserved arginine in the assembly domain of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit interferes with holoenzyme formation.

In higher plants the small subunit (S) of ribulose-1,5-bisphosphate carboxylase/oxygenase (ribulose-P2 carboxylase, EC 4.1.1.39) contains a segment of 16 amino acids which is absent from cyanobacterial S. This segment connecting two beta sheets has been shown, by crystallographic analysis, to form a hairpin loop. The quaternary structure of ribulose-P2 carboxylase indicates several S to large subunit (L) interactions. Eleven of 22 residues within the loop form the interface with 20 residues from two different L dimers. Eight of the loop residues are involved in hydrogen bonds, salt links, and hydrophobic interactions. To test the hypothesis, whether this loop had a function in the assembly of L and S into the hexadecameric enzyme, 6 amino acids within the loop were modified by site-directed mutagenesis of the pea rbcS-3A gene. All substituted S were imported by isolated chloroplasts from pea with wild type efficiency. Mutants E54-R, H55-A, P59-A, D63-G, D63-L, and Y66-A were assembly-competent, indicating that changes of side chains at these positions are tolerated. Replacement of arginine 53, whose side chain forms H-bonds with L residues Y226 and G261, with glutamate completely abolished assembly into holoenzyme. We suggest that arginine 53 in S is essential for ribulose-P2 carboxylase quaternary structure in higher plants.

Expression of the E. coli nadB gene and characterization of the gene product L-aspartate oxidase.

Quinolinic acid is synthesized in E. coli by the enzymes L-aspartate oxidase and quinolinate synthase A, the genes of which are named nadB and nadA. In our previous work we cloned and characterized the two genes (Flachmann, R., Kunz, N., Seifert, J., Gütlich, M., Wientjes, F.J., Läufer, A. & Gassen, H.G. (1988) Eur. J. Biochem. 175, 221-228). Here we report on the expression of the nadB gene under control of the inducible left promoter of the bacteriophage lambda. The yield of the active gene product L-aspartate oxidase was enhanced up to 20% of the soluble cell protein. The enzyme was purified to homogeneity in a three-step procedure and the reading frame of the L-aspartate oxidase gene was confirmed by Edman degradation of five cyanogen bromide peptides. L-Aspartate oxidase shows no classical Michaelis-Menten behaviour but is subject to a substrate inactivation. The apparent Km values were different for substrate concentrations below and above 1mM and were determined to 0.5 mM and 4.1mM, respectively. The active form of the enzyme is a monomer of 60,284 Da and contains one molecule of FAD and nine cysteine residues, four of which built up two disulfide bonds. The isoelectric point of the protein was determined to be at pH 5.6. Chemical modifications of the enzyme showed that at least one tyrosine and one histidine residue are essential for enzyme activity. The coenzyme-binding domain is located in the amino-terminal part of the polypeptide chain as revealed by a sequence comparison to other dinucleotide binding enzymes. Furthermore, there is evidence for a relationship to fumarate reductase and succinate dehydrogenase of E. coli.

Molecular biology of pyridine nucleotide biosynthesis in Escherichia coli. Cloning and characterization of quinolinate synthesis genes nadA and nadB.

The two genes, nadA and nadB, responsible for quinolinate biosynthesis from aspartate and dihydroxyacetone phosphate in Escherichia coli were cloned and characterized. Quinolinate (pyridine-2,3-dicarboxylate) is the biosynthetic precursor of the pyridine ring of NAD. Gene nadA was identified by complementation in three different nadA mutant strains. Sequence analysis provided an 840-bp open reading frame coding for a 31,555-Da protein. Gene nadB was identified by complementation in a nadB mutant strain and by the L-aspartate oxidase activity of its gene product. Sequence analysis showed a 1620-bp open reading frame coding for a 60,306-Da protein. For both genes, promoter regions and ribosomal binding sites were assigned by comparison to consensus sequences. The nadB gene product, L-aspartate oxidase, was purified to homogeneity and the N-terminal sequence of 19 amino acids was determined. The enzyme was shown to be specific for L-aspartate. High-copy-number vectors, carrying either gene nadA, nadB or nadA + nadB, increased quinolinate production 1.5-fold, 2.0-fold and 15-fold respectively. Both gene products seem to be equally rate-limiting in quinolinate synthesis.