A new self-assembled peroxisomal vesicle required for efficient resealing of the plasma membrane.

The Woronin body is a membrane-bound organelle that has been observed in over 50 species of filamentous fungi. However, neither the composition nor the precise function of the Woronin body has yet been determined. Here we purify the Woronin body from Neurospora crassa and isolate Hex1, a new protein containing a consensus sequence known as peroxisome-targeting signal-1 (PTS1). We show that Hex1 is localized to the matrix of the Woronin body by immunoelectron microscopy, and that a green fluorescent protein- (GFP-)Hex1 fusion protein is targeted to yeast peroxisomes in a PTS1- and peroxin-dependent manner. The expression of the HEX1 gene in yeast generates hexagonal vesicles that are morphologically similar to the native Woronin body, implying a Hex1-encoded mechanism of Woronin-body assembly. Deletion of HEX1 in N. crassa eliminates Woronin bodies from the cytoplasm and results in hyphae that exhibit a cytoplasmic-bleeding phenotype in response to cell lysis. Our results show that the Woronin body represents a new category of peroxisome with a function in the maintenance of cellular integrity.

Light signal transduction in plants.

Light signal-transduction pathways are a central component of the mechanisms that regulate plant development. These pathways provide the means by which information from specific wavelengths of light may be amplified and coordinated, resulting in complex physiological and developmental responses. This review focuses upon recent approaches towards establishing the intermediates that transmit signals from photoreceptors, phytochromes in particular, to target elements in the promoters of light-regulated genes.

The sites of synthesis of the principal thylakoid membrane polypeptides in Chlamydomonas reinhardtii.

The sites of synthesis of the major thylakoid membrane polypeptides have been studied in the green alga Chlamydomonas reinhardtii by pulse labeling of cells with [14C]acetate in the presence of inhibitors specific for chloroplast and cytoplasmic protein synthesis. The labeled membrane polypeptides were separated by an improved method of sodium dodecyl sulfate (SDS) gradient gel electrophoresis, and autoradiographs were made of the dried gels. The results demonstrate that of the 33 polypeptides resolved in the gels, at least nine are made on chloroplast ribosomes. Two of these (polypeptides 2 and 6) are associated with the reaction centers of photosystems I and II. Another polypeptide (polypeptide 5) appears from genetic data to be coded by chloroplast DNA. Experiments with a mutant whose chloroplast ribosomes are resistant to spectinomycyn (spr-u-1-6-2) show that polypeptides whose synthesis takes place on chloroplast ribosomes are made in the presence of spectinomycin in the mutant although their synthesis is blocked by this antibiotic in wild type cells.

Isolation of cytoplasmic and chloroplast ribosomes and their dissociation into active subunits from Chlamydomonas reinhardtii.

A mixture of cytoplasmic (80S) and chloroplast (70S) ribosomes from Chlamydomonas reinhardtii was freed of contaminating membranes by sedimentation of the postmitochondrial supernatant through a layer of 1.87 M sucrose. The purified ribosomes were separated into 80S and 70S fractions by centrifugation at a relatively low speed on a 10-40% sucrose gradient containing 25 mM KCl and 5 mM MgCl(2). Both the 80S and 70S ribosomes were dissociated into compact subunits by centrifugations in 5-20% high-salt sucrose gradients. The dissociations of both ribosomal species under these conditions were not affected by the addition of puromycin, indicating that the ribosomes as isolated were devoid of nascent chains. Subunits derived from the 80S ribosomes had apparent sedimentation coefficients of 57S and 37S whereas those from the 70S ribosomes had apparent sedimentation coefficients of 50S and 33S. In the presence of polyuridylic acid and cofactors, the 80S and 70S ribosomes incorporated [(14)C]phenylalanine into material insoluble in hot TCA. The requirements for incorporation were found to be similar to those described for eukaryotic and prokaryotic ribosomes. Experiments with antibiotics showed that the activity of the 80S ribosomes was sensitive to cycloheximide, whereas that of the 70S ribosomes was inhibited by streptomycin. The isolated subunits, when mixed together in an incorporation medium, were also active in the polymerization of phenylalanine in vitro.

A chlorophyll-protein complex lacking in photosystem I mutants of Chlamydomonas reinhardtii.

Sodium dodecyl sulfate gel electrophoresis of unheated, detergent-solubilized thylakoid membranes of Chlamydomonas reinhardtii gives two chlorophyll-protein complexes. Chlorophyll-protein complex I (CP I) is the blue-green in color and can be dissociated by heat into "free" chlorophyll and a constituent polypeptide (polypeptide 2; mol wt 66,000). Similar experiments with spinach and Chinese cabbage show that the higher plant CP I contains an equivalent polypeptide but of slightly lower molecular weight (64,000). Both polypeptide 2 and its counterpart in spinach are soluble in a 2:1 (vol/vol) mixture of chloroform-methanol. Chemical analysis reveals that C. reinhardtii CP I has a chlorophyll a to b weight ratio of about 5 and that it contains approximately 5% of the total chlorophyll and 8-9% of the total protein of the thylakoid membranes. Thus, it can be calculated that each constituent polypeptide chain is associated with eight to nine chlorophyll molecules. Attempts to measure the molecular weight of CP I by calibrated SDS gels were unsuccessul since the complex migrates anomalously in such gels. Two Mendelian mutants of C. reinhardtii, F1 and F14, which lack P700 but have normal photosystem I activity, do not contain CP I or the 66,000-dalton polypeptide in their thylakoid membranes. Our results suggest that CP I is essential for photosystem I reaction center activity and that P700 may be associated with the 66,000-dalton polypeptide.

Synthesis and turnover of ribulose biphosphate carboxylase and of its subunits during the cell cycle of Chlamydomonas reinhardtii.

The chloroplast enzyme ribulose-1,5-bisphosphate (Ru-1,5-P2) carboxylase (EC 4.1 1.39) is made up ot two nonidentical subunits, one synthesized in the chloroplast and the other outside. Both of these subunits of the assembled enzyme are synthesized in a stepwise manner during the synchronous cell cycle of the green alga Chlamydomonas reinhardtii. The activity of this enzyme increases in the light and this increase is due to de novo protein synthesis as shown by the measurement of the amount of protein and by the pulse incorporation of radioactive arginine in the 18S enzyme peak in linear sucrose density gradients. During the dark phase of the cell cycle, there is little change in the enzymatic activity as well as in the amount of this enzyme. Pulse-labeling studies using radioactive arginine indicated that there is a slow but detectable rate of synthesis of the carboxylase and of its subunits in the dark. Ru-1,5-P2 carboxylase, prelabeled with radioactive arginine throughout the entire light period, shows a similarly slow rate of degradation in the following dark period. This slow turnover of the enzyme in the dark accounts for the steady levels of carboxylase protein and of enzymatic activity during this period. A wide variety of inhibitors of protein synthesis by 70S and 80S ribosomes abolished the incorporation of [3H]arginine into total Ru-1,5-P2 carboxylase during short-term incubation. These results suggest a tight-coordinated control of the biosynthesis of the small and large subunits of the enzyme. This stringent control is further substantiated by the finding that both subunits are synthesized in sychrony with each other, that the ratio of radioactivity of the small to the large subunit remains constant throughout the entire light-dark cycle, and that the rates of synthesis and of degradation of both subunits are similar to that of the assembled enzyme.

Periodic variations in the ratio of free to thylakoid-bound chloroplast ribosomes during the cell cycle of Chlamydomonas reinhardtii.

The ratio of free to thylakoid-bound chloroplast ribosomes in Chlamydomonas reinhardtii undergoes periodic changes during the synchronous light-dark cycle. In the light, when there is an increase in the chlorophyll content and synthesis of thylakoid membrane proteins, about 20-30% of the chloroplast ribosomes are bound to the thylakoid membranes. On the other hand, only a few or no bound ribosomes are present in the dark when there is no increase in the chlorophyll content. The ribosome-membrane interaction depends not only on the developmental stage of the cell but also on light. Thus, bound ribosomes were converted to the free variety after cultures at 4 h in the light had been transferred to the dark for 10 min. Conversely, a larger number of chloroplast ribosomes became attached to the membranes after cultures at 4 h in the dark had been illuminated for 10 min. Under normal conditions, when there was slow cooling of the cultures during cell harvesting, chloroplast polysomal runoff occurred in vivo leading to low levels of thylakoid-bound ribosomes. This polysomal runoff could be arrested by either rapid cooling of the cells or the addition of chloramphenicol or erythromycin. Each of these treatments prevented polypeptide chain elongation on chloroplast ribosomes and thus allowed the polyosomes to remain bound to the thylakoids. Addition of lincomycin, an inhibitor of chain initiation on 70S ribosomes, inhibited the assembly of polysome-thylakoid membrane complex in the light. These results support a model in which initiation of mRNA translation begins in the chloroplast stroma, and the polysome subsequently becomes attached to the thylakoid membrane. Upon natural chain termination, the chloroplast ribosomes are released from the membrane into the stroma.

NH2-terminal amino acid sequences of precursor and mature forms of the ribulose-1,5-bisphosphate carboxylase small subunit from Chlamydomonas reinhardtii.

A precursor (pS) to the small subunit (S) of ribulose1-,5-bisphosphate carboxylase is the major product of cell-free protein synthesis directed by poly(A) containing RNA from Chlamydomonas reinhardtii. We present sequence data for in vitro-synthesized pS, for in vitro-synthesized S that in generated from pS by posttranslational incubation with a Chlamydomonas cell extract, and for in vitro-synthesized, mature S. We show that pS contains an NH2-terminal extension of 44 amino acid residues that is removed by cleavage at the correct site when pS is converted to S by an endoprotease present in the Chlamydomonas cell extract.

Rac homologues and compartmentalized phosphatidylinositol 4, 5-bisphosphate act in a common pathway to regulate polar pollen tube growth.

Pollen tube cells elongate based on actin- dependent targeted secretion at the tip. Rho family small GTPases have been implicated in the regulation of related processes in animal and yeast cells. We have functionally characterized Rac type Rho family proteins that are expressed in growing pollen tubes. Expression of dominant negative Rac inhibited pollen tube elongation, whereas expression of constitutive active Rac induced depolarized growth. Pollen tube Rac was found to accumulate at the tip plasma membrane and to physically associate with a phosphatidylinositol monophosphate kinase (PtdIns P-K) activity. Phosphatidylinositol 4, 5-bisphosphate (PtdIns 4, 5-P2), the product of PtdIns P-Ks, showed a similar intracellular localization as Rac. Expression of the pleckstrin homology (PH)-domain of phospholipase C (PLC)-delta1, which binds specifically to PtdIns 4, 5-P2, inhibited pollen tube elongation. These results indicate that Rac and PtdIns 4, 5-P2 act in a common pathway to control polar pollen tube growth and provide direct evidence for a function of PtdIns 4, 5-P2 compartmentalization in the regulation of this process.

Biosynthetic pathways of two polypeptide subunits of the light-harvesting chlorophyll a/b protein complex.

We have used an in vitro reconstitution system, consisting of cell-free translation products and intact chloroplasts, to investigate the pathway from synthesis to assembly of two polypeptide subunits of the light-harvesting chlorophyll-protein complex. These polypeptides, designated 15 and 16, are integral components of the thylakoid membranes, but they are products of cytoplasmic protein synthesis. Double immunodiffusion experiments reveal that the two polypeptides share common antigenic determinants and therefore are structurally related. Nevertheless, they are synthesized in vitro from distinct mRNAs to yield separate precursors, p15 and p16, each of which is 4,000 to 5,000 daltons larger than its mature form. In contrast to the hydrophobic mature polypeptides, the precursors are soluble in aqueous solutions. Along with other cytoplasmically synthesized precursors, p15 and p16 are imported into purified intact chloroplasts by a post-translational mechanism. The imported precursors are processed to the mature membrane polypeptides which are recovered exclusively in the thylakoids. The newly imported polypeptides are assembled correctly in the thylakoid lipid bilayer and they bind chlorophylls. Thus, these soluble membrane polypeptide precursors must move from the cytoplasm through the two chloroplast envelope membranes, the stroma, and finally insert into the thylakoid membranes, where they assemble with chlorophyll to form the light-harvesting chlorophyll protein complex.

Actin depolymerizing factor (ADF/cofilin) enhances the rate of filament turnover: implication in actin-based motility.

Actin-binding proteins of the actin depolymerizing factor (ADF)/cofilin family are thought to control actin-based motile processes. ADF1 from Arabidopsis thaliana appears to be a good model that is functionally similar to other members of the family. The function of ADF in actin dynamics has been examined using a combination of physical-chemical methods and actin-based motility assays, under physiological ionic conditions and at pH 7.8. ADF binds the ADP-bound forms of G- or F-actin with an affinity two orders of magnitude higher than the ATP- or ADP-Pi-bound forms. A major property of ADF is its ability to enhance the in vitro turnover rate (treadmilling) of actin filaments to a value comparable to that observed in vivo in motile lamellipodia. ADF increases the rate of propulsion of Listeria monocytogenes in highly diluted, ADF-limited platelet extracts and shortens the actin tails. These effects are mediated by the participation of ADF in actin filament assembly, which results in a change in the kinetic parameters at the two ends of the actin filament. The kinetic effects of ADF are end specific and cannot be accounted for by filament severing. The main functionally relevant effect is a 25-fold increase in the rate of actin dissociation from the pointed ends, while the rate of dissociation from the barbed ends is unchanged. This large increase in the rate-limiting step of the monomer-polymer cycle at steady state is responsible for the increase in the rate of actin-based motile processes. In conclusion, the function of ADF is not to sequester G-actin. ADF uses ATP hydrolysis in actin assembly to enhance filament dynamics.

GT-1 binding site confers light responsive expression in transgenic tobacco.

Light-dependent expression of rbcS, the gene encoding the small subunit of ribulose-1,5-bisphosphate carboxylase, which is the key enzyme involved in carbon fixation in higher plants, is regulated at the transcriptional level. Sequence analysis of the gene has uncovered a conserved GT motif in the -150 to -100 region of many rbcS promoters. This motif serves as the binding site of a nuclear factor, designated GT-1. Analysis of site-specific mutants of pea rbcS-3A promoter demonstrated that GT-1 binding in vitro is correlated with light-responsive expression of the rbcS promoter in transgenic plants. However, it is not known whether factors other than GT-1 might also be required for activation of transcription by light. A synthetic tetramer of box II (TGTGTGGTTAATATG), the GT-1 binding site located between -152 to -138 of the rbcS-3A promoter, inserted upstream of a truncated cauliflower mosaic virus 35S promoter is sufficient to confer expression in leaves of transgenic tobacco. This expression occurs principally in chloroplast-containing cells, is induced by light, and is correlated with the ability of box II to bind GT-1 in vitro. The data show that the binding site for GT-1 is likely to be a part of the molecular light switch for rbcS activation.

The Cauliflower Mosaic Virus 35S Promoter: Combinatorial Regulation of Transcription in Plants.

Appropriate regulation of transcription in higher plants requires specific cis elements in the regulatory regions of genes and their corresponding trans-acting proteins. Analysis of the cauliflower mosaic virus (CaMV) 35S promoter has contributed to the understanding of transcriptional regulatory mechanisms. The intact 35S promoter confers constitutive expression upon heterologous genes in most plants. Dissection into subdomains that are able to confer tissue-specific gene expression has demonstrated that the promoter has a modular organization. When selected subdomains are combined, they confer expression not detected from the isolated subdomains, suggesting that synergistic interactions occur among cis elements. The expression patterns conferred by specific combinations of 35S subdomains differ in tobacco and petunia. This indicates that a combinatorial code of cisregulatory elements may be interpreted differently in different species.

Regulated genes in transgenic plants.

Transgenic plants are an effective system for the study of regulated gene expression. Developmental control of expression can be monitored by assaying different tissues or by assaying a plant at different developmental stages. Analysis of the petunia 5-enolpyruvylshikimate-3-phosphate synthase gene, which is highly expressed in flowers, allowed identification of an upstream region that confers tissue-specific and developmentally regulated expression. The cell specificity of expression in floral tissues has been defined by histochemical localization. This expression is contrasted to that of the 35S promoter of cauliflower mosaic virus, a nominally constitutive promoter that shows a definite specificity of expression in floral tissues. Moreover, this expression differs in transgenic hosts of different species.

Organ-specific and light-induced expression of plant genes.

Light plays a pivotal role in the development of plants. The photoregulation of plant genes involves recognition of light quality and quantity by phytochrome and other light receptors. Two gene families, rbcS and Cab, which code for abundant proteins active in photosynthesis, the small subunit of ribulose bisphosphate carboxylase and the chlorophyll a/b binding protein, show a 20-to 50-fold increase in transcript abundance in the light. Analyses in calli and transgenic plants of deletions of the rbcS gene and of chimeric constructions has allowed localization of two regions involved in light-induced transcription. One element is confined to a 33-base pair region surrounding the TATA box. In addition, an enhancer-like element contained within a 240-base pair fragment can confer phytochrome-induced transcription and organ specificity on nonregulated promoters.

The regulation of circadian period by phototransduction pathways in Arabidopsis.

Transgenic Arabidopsis plants expressing a luciferase gene fused to a circadian-regulated promoter exhibited robust rhythms in bioluminescence. The cyclic luminescence has a 24.7-hour period in white light but 30- to 36-hour periods under constant darkness. Either red or blue light shortened the period of the wild type to 25 hours. A phytochrome-deficient mutation lengthened the period in continuous red light but had little effect in continuous blue light, whereas seedlings carrying mutations that activate light-dependent pathways in darkness maintained shorter periods in constant darkness. These results suggest that both phytochrome- and blue light-responsive photoreceptor pathways control the period of the circadian clock.

Abscisic acid signaling through cyclic ADP-ribose in plants.

Abscisic acid (ABA) is the primary hormone that mediates plant responses to stresses such as cold, drought, and salinity. Single-cell microinjection experiments in tomato were used to identify possible intermediates involved in ABA signal transduction. Cyclic ADP-ribose (cADPR) was identified as a signaling molecule in the ABA response and was shown to exert its effects by way of calcium. Bioassay experiments showed that the amounts of cADPR in Arabidopsis thaliana plants increased in response to ABA treatment and before ABA-induced gene expression.

Circadian clock mutants in Arabidopsis identified by luciferase imaging.

The cycling bioluminescence of Arabidopsis plants carrying a firefly luciferase fusion construct was used to identify mutant individuals with aberrant cycling patterns. Both long- and short-period mutants were recovered. A semidominant short-period mutation, timing of CAB expression (toc1), was mapped to chromosome 5. The toc1 mutation shortens the period of two distinct circadian rhythms, the expression of chlorophyll a/b-binding protein (CAB) genes and the movements of primary leaves, although toc1 mutants do not show extensive pleiotropy for other phenotypes.

Transcription factors in plant growth and development.

The target DNA sequences of several classes of plant transcription factors, including basic leucine zipper (bZIP) proteins and Myb-related factors, have been characterized in vivo as well as in vitro. The bZIP proteins, for example, act at ACGT elements, the flanking nucleotides determining their binding specificities. Overexpression, co-suppression, and antisense technology studies of factor genes in transgenic plants have uncovered the roles of bZIP, homeodomain, and MADS box factors in plant growth and development; for example, ectopic expression of pMADS1 alone in early Petunia development is sufficient for homeotic conversion of sepals into petaloid organs.

Transcription regulatory proteins in higher plants.

Novel structures and interactions for plant transcription factors have recently been reported. Factors that lack DNA-binding activity, exhibit putative triple-helix DNA-binding motifs, or possess two DNA-binding domains are among the interesting results. Advances in our understanding of protein-protein interactions between plant transcription factors are facilitating the design of new experimental strategies to further define their roles in regulatory pathways.