Recombinant holophytochrome in Escherichia coli.

We have successfully co-expressed two genes from the bilin biosynthetic pathway of Synechocystis together with cyanobacterial phytochrome 1 (Cph1) from the same organism to produce holophytochrome in Escherichia coli. Heme oxygenase was used to convert host heme to biliverdin IXalpha which was then reduced to phycocyanobilin via phycocyanobilin:ferredoxin oxidoreductase, presumably with the aid of host ferredoxin. In this host environment Cph1 apophytochrome was able to autoassemble with the phycocyanobilin in vivo to form fully photoreversible holophytochrome. The system can be used as a tool for further genetic studies of phytochrome function and signal transduction as well as providing an excellent source of holophytochrome for physicochemical studies.

Cytosolic heat-stress proteins Hsp17.7 class I and Hsp17.3 class II of tomato act as molecular chaperones in vivo.

Small heat-stress proteins (sHsps) are the most abundant stress-induced proteins with up to 20 different members in higher plants. In the cytoplasm, two different classes can be distinguished. Two cDNA clones from tomato Lycopersicon peruvianum (L.) Mill., each coding for one of the cytoplasmic sHsp subfamilies, were analyzed with respect to their transcript and protein expression, genome organization and chaperone activity. Neither type was present under control conditions but both appeared upon heat stress and in mature fruits. Expression of the class II transcript was found to be induced at slightly lower temperatures than the class I transcript. Protein analysis using class-specific antibodies revealed an identical expression pattern of both corresponding proteins. Transient expression in an Arabidopsis thaliana (L.) Heynh. cell culture showed that, despite the difference in their amino acid sequence, both classes are functionally active as chaperones in vivo, as shown by their ability to prevent thermal inactivation of firefly luciferase in a cellular environment.

A glucosinolate mutant of Arabidopsis is thermosensitive and defective in cytosolic Hsp90 expression after heat stress.

The TU8 mutant of Arabidopsis previously described to be deficient in glucosinolate metabolism and pathogen-induced auxin accumulation was found to be remarkably less tolerant upon exposure to elevated temperatures than wild-type plants. Although moderately increased temperature only affected shoot growth, exposure to severe heat stress led to a dramatic decay of mutant plants. By contrast, wild-type seedlings showed little or no damage under the same conditions. Analysis of different heat stress proteins (Hsps) in TU8 seedlings revealed that only expression of cytoplasmic Hsp90 was affected in these plants. Although Hsp90 was present under control conditions, its level declined in mutant plants at elevated temperatures. Northern-blot analysis indicated that the decrease in Hsp90 protein was accompanied with a reduction of hsp90 transcript levels. Transient expression of Hsp90 in mutant protoplasts increased their survival rate at higher temperatures to near equivalent that of wild-type protoplasts. These data suggest that the reduced level of Hsp90 in TU8 mutants may be the primary cause for the observed reduction in thermostability.

Messenger RNA-binding properties of nonpolysomal ribonucleoproteins from heat-stressed tomato cells

Most cells experiencing heat stress reprogram their translational machinery to favor the synthesis of heat-stress proteins. Translation of other transcripts is almost completely repressed, but most untranslated messengers are not degraded. In contrast to yeast, Drosophila melanogaster, and HeLa cells, plant cells store repressed messengers in cytoplasmic nonpolysomal ribonucleoproteins (RNPs). To follow the fate of untranslated transcripts, we studied protein composition, mRNA content, and RNA-binding properties of nonpolysomal RNPs from heat-stressed tomato (Lycopersicon peruvianum) cells. Contrary to the selective interaction in vivo, RNPs isolated from tomato cells bound both stress-induced and repressed messengers, suggesting that the selection mechanism resides elsewhere. This binding was independent of a cap or a poly(A) tail. The possible role of proteasomes and heat-stress granules (HSGs) in mRNA storage is a topic of debate. We found in vitro messenger-RNA-binding activity in messenger RNP fractions free of C2-subunit-containing proteasomes and HSGs. In addition, mRNAs introduced into tobacco (Nicotiana plumbaginifolia) protoplasts were found in the cytoplasm but were not associated with HSGs.

Stable transformation of an Arabidopsis cell suspension culture with firefly luciferase providing a cellular system for analysis of chaperone activity in vivo.

Using Agrobacterium, we developed a method to transform an Arabidopsis cell suspension culture. A stably transformed cell line expressing high levels of firefly luciferase (Luc) was used for in vivo studies of thermal denaturation and renaturation of the enzyme and the protective role of different chaperones. Luc activity was monitored under heat stress and recovery conditions in control, thermotolerant cells and cells expressing plant chaperones after transient cotransformation with plasmids encoding proteins of the heat shock protein Hsp90, Hsp70, or Hsp20 family. The effects of the expressed proteins were specific. The Hsp17.6 class I protein maintained Luc activity on a level comparable with that observed in thermotolerant cells and improved Luc renaturation. Although transient expression of Hsp90 did not protect Luc from thermal denaturation, it accelerated Luc renaturation during recovery. In contrast to the other chaperones tested, overexpression of Hsp70 alone had no effect on denaturation and renaturation of Luc but enhanced Luc renaturation if coexpressed with Hsp17.6.

Light-independent and light-dependent protochlorophyllide-reducing activities and two distinct NADPH-protochlorophyllide oxidoreductase polypeptides in mountain pine (Pinus mugo).

Lower plants and gymnosperms synthesize chlorophyll and develop photosynthetically competent chloroplasts even when grown in the dark. In cell-free extracts of pine (Pinus mugo, Turra, ssp. mugo) seedlings, light-independent and light-dependent protochlorophyllide-reducing activities are present. Two distinct NADPH-protochlorophyllide-oxidoreductase (POR) polypeptides can be detected immunologically with an antiserum raised against the POR of barley. The subcellular localization and amounts of the two POR polypeptides are differentially affected by light: one of them is predominantly present in prolamellar bodies of etiochloroplasts and its abundance rapidly declines once the pine seedlings are exposed to light; the other is found in thylakoid membranes and its amount does not change during illumination of dark-grown seedlings. Two types of cDNA sequences are identified that encode two distinct POR polypeptides in pine. The relevance of these POR polypeptides for the two chlorophyll biosynthetic pathways active in gymnosperms is discussed.

Evidence for a general light-dependent negative control of NADPH-protochlorophyllide oxidoreductase in angiosperms.

The effect of light on NADPH-protochlorophyllide oxidoreductase and its mRNA has been studied in five different species of dicotyledonous plants, bean (Phaseolus vulgaris L.), pea (Pisum sativum L.), tomato (Lycopersicon esculentum Mill.), sunflower (Helianthus annuns L.) and mustard (Sinapis alba L.), and in two monocotyledonous plant species, maize (Zea mays L.) and barley (Hordeum vulgare L.). In all these species, illumination of etiolated seedlings led to a rapid decline of both the activity and the content of the enzyme protein. These results indicate that there may be a general light-dependent regulation of the enzyme common to higher plants.

Nucleotide sequence of a cDNA coding for the NADPH-protochlorophyllide oxidoreductase (PCR) of barley (Hordeum vulgare L.) and its expression in Escherichia coli.

The primary structure of the NADPH-protochlorophyllide oxidoreductase of barley has been deduced from the nucleotide sequence of a cloned full-length cDNA. This cDNA hybridizes to a 1.7 kb RNA whose steady-state level in dark-grown seedlings is drastically reduced upon illumination. The predicted amino acid sequence (388 residues in length) includes a transit peptide of 74 amino acids whose end point has been delimited by sequencing the N-terminus of the mature protein. Expression of the cDNA in Escherichia coli leads to the synthesis of an enzymatically active precursor of the NADPH-protochlorophyllide oxidoreductase. Activity of this protein in bacterial lysates is completely dependent on the presence of NADPH and protochlorophyllide and requires light.