The Plant Journal

Pool, Martin R.; López‐Huertas, Eduardo; Horng, Jim‐Tong; Baker, Alison

doi: 10.1046/j.1365-313X.1998.00171.xpmid: 9744090

We have studied the import of proteins into glyoxysomes in vitro and show that this process is specifically inhibited by NADPH. NADPH affects both binding and translocation of proteins into glyoxysomes, and inhibition is determined by the ratio of NADP+ to NADPH. The site of action of NADPH is most likely within the glyoxysome because (1) pretreatment of glyoxysomes with NADPH, followed by re‐isolation of the organelles prior to the import assay, resulted in inhibition of import that could be restored by the addition of NADP+; (2) low concentrations of NADPH inhibited binding of proteins to broken glyoxysome membranes. The sensitivity of protein import to inhibition by NADPH declines as glyoxysomes are converted to leaf‐type peroxisomes. A model is proposed that speculates on a possible role for NADPH in regulating protein import into plant peroxisomes.

Kahn, Theodore W.; Lapidot, Moshe; Heinlein, Manfred; Reichel, Christoph; Cooper, Bret; Gafny, Ron; Beachy, Roger N.

doi: 10.1046/j.1365-313X.1998.00172.xpmid: 9744091

To identify and map functionally important regions of the tobacco mosaic virus movement protein, deletions of three amino acids were introduced at intervals of 10 amino acids throughout the protein. Mutations located between amino acids 1 and 160 abolished the capacity of the protein to transport virus from cell to cell, while some of the mutations in the C‐terminal third of the protein permitted function. Despite extensive tests, no examples were found of intermolecular complementation between mutants, suggesting that function requires each movement protein molecule to be fully competent. Many of the mutants were fused to green fluorescent protein, and their subcellular localizations were determined by fluorescence microscopy in infected plants and protoplasts. Most mutants lost the ability to accumulate in one or more of the multiple subcellular sites targeted by wild‐type movement protein, suggesting that specific functional domains were disrupted. The order in which accumulation at subcellular sites occurs during infection does not represent a targeting pathway. Association of the movement protein with microtubules or with plasmodesmata can occur in the absence of other associations. The region of the protein around amino acids 9–11 may be involved in targeting the protein to cortical bodies (probably associated with the endoplasmic reticulum) and to plasmodesmata. The region around residues 49–51 may be involved in co‐alignment of the protein with microtubules. The region around residues 88–101 appears to play a role in targeting to both the cortical bodies and microtubules. Thus, the movement protein contains independently functional domains.

Crombie, Hazel J.; Chengappa, Sumant; Hellyer, Amanda; Reid, J. S. Grant

doi: 10.1046/j.1365-313X.1998.00182.xpmid: 9744092

A ‐D‐glucosidase has been purified to apparent homogeneity from the cotyledons of germinated nasturtium (Tropaeolum majus L.) seedlings during the mobilization of the xyloglucan stored in the cotyledonary cell walls. The purified protein (Mr 76 000; a glycoprotein; pI > 9.5; apparent pH optimum 4.5; temperature optimum 30°C) catalysed the hydrolysis of p‐nitrophenyl‐‐D‐glucopyranoside, cello‐oligosaccharides, ‐linked glucose disaccharides, and certain xyloglucan oligosaccharides. Glucose disaccharides with different linkages were hydrolysed at different rates [(1ν3) > (1ν4) > (1ν2) > (1ν6)] with significant transglycosylation occurring in the early stages of the reaction. Cello‐oligosaccharide hydrolysis was also accompanied by extensive transglycosylation to give transitory accumulations of higher oligosaccharides. At least some of the glycosyl linkages formed during transglycosylation were (1ν6)‐. Xyloglucan oligosaccharides xylose‐substituted at the non‐reducing terminal glucose residue (XXXG, XXLG, XLXG and XLLG, where G is an unsubstituted glucose residue, X is a xylose‐substituted glucose residue, and L is a galactosylxylose‐substituted glucose residue) were not hydrolysed. Some xyloglucan oligosaccharides with an unsubstituted non‐reducing terminal glucose residue (GXXG, GXLG and GXG) were hydrolysed, but others (GLXG and GLLG) were not. This indicated steric hindrance by L but not X substitution at the glucose residue next to the one at the non‐reducing end of the oligosaccharide. Hydrolysis of xyloglucan oligosaccharides was not accompanied by transglycosylation. Natural xyloglucan subunit oligosaccharides (XXXG, XXLG, XLXG, XLLG) were totally degraded to their monosaccharide components when treated with nasturtium ‐D‐galactosidase ( Edwards et al. (1988 ) J. Biol. Chem. 263, 4333–4337), followed by alternations of nasturtium xyloglucan‐specific α‐xylosidase ( Fanutti et al. (1991 ) Planta 184, 137–147) and this enzyme. Several extensively overlapping cDNA clones were obtained by RT–PCR and by screening cDNA libraries. A composite, full‐length DNA had an open reading frame of 1962 bp, encoding a polypeptide of 654 amino acids, including all N‐terminal and internal sequences obtained from the purified ‐glucosidase protein, and a motif resembling plant signal sequences thought to direct proteins to the cell wall. Database searches revealed homology with ‐glucosidases from several sources (plant, bacteria, yeast), notably with glycosylhydrolases of ‘Family 3’, according to the classification of Henrissat ( Henrissat (1991) Biochem. J. 280, 309–316). There was strong sequence homology with a ‐glucan exo‐hydrolase from barley ( Hrmova et al. (1996 ) J. Biol. Chem. 271, 5277–5286). The nasturtium ‐glucosidase is ascribed a role in xyloglucan mobilization, and its interaction with the α‐xylosidase and the ‐galactosidase is modelled.

Girke, Thomas; Schmidt, Hermann; Zähringer, Ulrich; Reski, Ralf; Heinz, Ernst

doi: 10.1046/j.1365-313X.1998.00178.xpmid: 9744093

The moss Physcomitrella patens contains high levels of arachidonic acid. For its synthesis from linoleic acid by desaturation and elongation, novel D5‐ and D6‐ desaturases are required. To isolate one of these, PCR‐based cloning was used, and resulted in the isolation of a full‐length cDNA coding for a putatively new desaturase. The deduced amino acid sequence has three domains: a N‐terminal segment of about 100 amino acids, with no similarity to any sequence in the data banks, followed by a cytochrome b5‐related region and a C‐terminal sequence with low similarity (27% identity) to acyl‐lipid desaturases. To elucidate the function of this protein, we disrupted its gene by transforming P. patens with the corresponding linear genomic sequence, into which a positive selection marker had been inserted. The molecular analysis of five transformed lines showed that the selection cartridge had been inserted into the corresponding genomic locus of all five lines. The gene disruption resulted in a dramatic alteration of the fatty acid pattern in the knockout plants. The large increase in linoleic acid and the concomitant disappearance of γ‐linolenic and arachidonic acid in all knockout lines suggested that the new cDNA coded for a D6‐desaturase. This was confirmed by expression of the cDNA in yeast and analysis of the resultant fatty acids by GC–MS. Only the transformed yeast cells were able to introduce a further double bond into the D6‐position of unsaturated fatty acids. To our knowledge, this is the first report of a successful gene disruption in a multicellular plant resulting in a specific biochemical phenotype.

Zabaleta, ; Heiser, ; Grohmann, ; Brennicke,

doi: 10.1046/j.1365-313X.1998.00177.xpmid: 9744094

Regulatory promoter regions responsible for the enhanced expression in anthers and pollen are defined in detail for three nuclear encoded mitochondrial Complex I (nCI) genes from Arabidopsis thaliana. Specific regulatory elements were found conserved in the 5′ upstream regions between three different genes encoding the 22 kDa (PSST), 55 kDa NADH binding (55 kDa) and 28 kDa (TYKY) subunits, respectively. Northern blot analysis and transgenic Arabidopsis plants carrying progressive deletions of the promoters fused to the β‐glucuronidase (GUS) reporter gene by histochemical and fluorimetric methods showed that all three promoters drive enhanced expression of GUS specifically in anther tissues and in pollen grains. In at least two of these promoters the –200/–100 regions actively convey the pollen/anther‐specific expression in gain of function experiments using CaMV 35S as a minimal promoter. These nCI promoters thus contain a specific regulatory region responding to the physiological demands on mitochondrial function during pollen maturation. Pollen‐specific motifs located in these regions appear to consist of as little as seven nucleotides in the respective promoter context.

Kim, Byung Chul; Soh, Moon Soo; Hong, Sung Hyun; Furuya, Masaki; Nam, Hong Gil

doi: 10.1046/j.1365-313X.1998.00179.xpmid: 9744095

We previously reported a photomorphogenic mutation of Arabidopsis thaliana, shy2–1D, as a dominant suppressor of a hy2 mutation. Here, we report that shy2–1D confers various photo‐responsive phenotypes in darkness and the dark phenotypes of the mutant are affected by phytochrome deficiency. Dark‐grown seedlings of the mutant developed several photomorphogenic characteristics such as short hypocotyls, cotyledon expansion and opening, and partial differentiation of plastids. When grown further in darkness, the mutant plant underwent most of the developmental stages of a light‐grown wild‐type plant, including development of foliar leaves, an inflorescence stem with cauline leaves, and floral organs. In addition, two light‐inducible genes, the nuclear‐encoded CAB and the plastid‐encoded PSBA genes, were highly expressed in the dark‐grown mutant seedlings. Furthermore, reduced gravitropism, a phytochrome‐modulated response, was observed in the mutant hypocotyl in darkness. Thus, shy2–1D is one of the most pleiotropic photomorphogenic mutations identified so far. The results indicate that SHY2 may be a key component regulating photomorphogenesis in Arabidopsis. Surprisingly, double mutants of the shy2–1D mutant with the phytochrome‐deficient mutants hy2, hy3 (phyB‐1) and fre1–1 (phyA‐201) showed reduced photomorphogenic response in darkness with a longer hypocotyl, a longer inflorescence stem, and a lower level expression of the CAB gene than the shy2–1D single mutant. These results showed that phytochromes function in darkness in the shy2–1D mutant background. The implications of these results are discussed.

Chattopadhyay, Sudip; Puente, Pilar; Deng, Xing‐Wang; Wei, Ning

doi: 10.1046/j.1365-313X.1998.00180.xpmid: 9744096

Rhee, Seung Y.; Somerville, Chris R.

doi: 10.1046/j.1365-313X.1998.00183.xpmid: 9744097

The quartet (qrt) mutants of Arabidopsis thaliana produce tetrad pollen in which microspores fail to separate during pollen development. Because the amount of callose deposition between microspores is correlated with tetrad pollen formation in other species, and because pectin is implicated as playing a role in cell adhesion, these cell‐wall components in wild‐type and mutant anthers were visualized by immunofluorescence microscopy at different stages of microsporogenesis. In wild‐type, callose was detected around the pollen mother cell at the onset of meiosis and around the microspores during the tetrad stage. Microspores were released into the anther locule at the stage where callose was no longer detected. Deposition and degradation of callose during tetrad pollen formation in qrt1 and qrt2 mutants were indistinguishable from those in wild‐type. Enzymatic removal of callose from wild‐type microspores at the tetrad stage did not release the microspores, suggesting that callose removal is not sufficient to disperse the microspores in wild‐type. Pectic components were detected in the primary wall of the pollen mother cell. This wall surrounded the callosic wall around the pollen mother cell and the microspores during the tetrad stage. In wild‐type, pectic components of this wall were no longer detectable at the time of microspore release. However, in qrt1 and qrt2 mutants, pectic components of this wall persisted after callose degradation. This result suggests that failure of pectin degradation in the pollen mother cell wall is associated with tetrad pollen formation in qrt mutants, and indicates that QRT1 and QRT2 may be required for cell type‐specific pectin degradation to separate microspores.

Moons, Ann; Valcke, Roland; Van, Marc ; Montagu,

doi: 10.1046/j.1365-313X.1998.00185.xpmid: 9744098

Pyruvate orthophosphate dikinase (PPDK) is known for its role in C4 photosynthesis but has no established function in C3 plants. Abscisic acid, PEG and submergence were found to markedly induce a protein of about 97 kDa, identified by microsequencing as PPDK, in rice roots (C3). The rice genome was found to contain two ppdk loci, osppdka and osppdkb. We isolated osppdka cDNA, which encodes a cytosolic rice PPDK isoform of 96.6 kDa, that corresponded to the ABA‐induced protein from roots. Western blot analysis showed a PPDK induction in roots of rice seedlings during gradual drying, cold, high salt and mannitol treatment, indicating a water deficit response. PPDK was also induced in the roots and sheath of submerged rice seedlings, and in etiolated rice seedlings exposed to an oxygen‐free N2 atmosphere, which indicated a low‐oxygen stress response. None of the stress treatments induced PPDK protein accumulation in the lamina of green rice seedlings. Ppdk transcripts were found to accumulate in roots of submerged seedlings, concomitant with the induction of alcohol dehydrogenase 1. Low‐oxygen stress triggered an increase in PPDK activity in roots and etiolated rice seedlings, accompanied by increases in phosphoenolpyruvate carboxylase and malate dehydrogenase activities. The results indicate that cytosolic PPDK is involved in a metabolic response to water deficit and low‐oxygen stress in rice, an anoxia‐tolerant species.

Cornejo, Juan; Willows, Robert D.; Beale, Samuel I.

doi: 10.1046/j.1365-313X.1998.00186.xpmid: 9744099

The phytobilin chromophores of phycobiliproteins and phytochromes are biosynthesized from heme in a pathway that begins with the opening of the tetrapyrrole macrocycle of protoheme to form biliverdin IXα, in a reaction catalyzed by heme oxygenase. A gene containing an open reading frame with a predicted polypeptide that has a sequence similar to that of a conserved region of animal microsomal heme oxygenases was identified in the published genomic sequence of Synechocystis sp. PCC 6803. This gene, named ho1, was cloned and expressed in Escherichia coli under the control of the lacZ promoter. Cells expressing the gene became green colored due to the accumulation of biliverdin IXα. The size of the expressed protein was equal to the predicted size of the Synechocystis gene product, named HO1. Heme oxygenase activity was assayed in incubations containing extract of transformed E. coli cells. Incubations containing extract of induced cells, but not those containing extract of uninduced cells, had ferredoxin‐dependent heme oxygenase activity. With mesoheme as the substrate, the reaction product was identified as mesobiliverdin IXα by spectrophotometry and reverse‐phase HPLC. Heme oxygenase activity was not sedimented by centrifugation at 100 000 g. Expression of HO1 increased several‐fold during incubation of the cells for 72 h in iron‐deficient medium.