bluuebunny/crossref_metadata_2025 huggingface dataset:
H‐protein of the glycine cleavage system in Flaveria: alternative splicing of the pre‐mRNA occurs exclusively in advanced C4 species of the genus
Authors: Kopriva Stanislav , Chu Chen‐cai , Bauwe Hermann
Published: March 2003

A recent paper reports on the occurrence of alternative splicing of H‐protein pre‐mRNA in the C4 species, Flaveria trinervia, that is organ‐specifically regulated. The analysis of 11 other species of the genus, Fcronquistii and F. pringlei (C3), F. anomala, F. chloraefolia, F. floridana, F. linearis and F. pubescens (C3–C4 intermediate), F. brownii (C4‐like), F. palmeri, F. bidentis and F. australasica (C4), revealed that this post‐transcriptional effect is not specific for F. trinervia. It occurs in all the examined C4 species of the genus Flaveria except the less advanced C4‐like species, F. brownii. Both the position and the direct effect of alternative splicing, the addition of two alanine residues near to the N‐terminus of the derived mature H‐protein, are invariant. A quantification of the relative amounts of both transcripts revealed that, as in F.trinervia, the alternative mRNA strongly dominates in leaves. In sharp contrast, none of the C3,C3–C4 intermediate, or C4‐like species showed alternative splicing. By Western analysis both H‐isoproteins have been detected in F. trinervia and their ratio approximately corresponds to the measured transcript levels. It is concluded that alternative splicing leads to the synthesis of two different H‐proteins of the glycine cleavage system in all advanced Flaveria C4 species.
Identification of a potential redox‐sensitive interdomain disulfide in the sedoheptulose bisphosphatase of Chlamydomonas reinhardtii
Authors: Anderson Louise E. , Huppe Heather C. , Li Alex Dong , Stevens Fred J.
Published: March 2003

In the simulated three‐dimensional structure of the Chlamydomonas reinhardtii sedoheptulose bisphosphatase (EC 3.1.3.37) there are two cysteine residues close enough to one another to form a redox‐sensitive disulfide bond which would cross‐link the nucleotide and carbon substrate domains. Examination of the redox modulation of this sedoheptulose bisphosphatase confirms that it resembles the higher plant enzyme in being activated by reduction. In the wheat and Arabidopsis enzymes, for which there is sequence information and which, like the Chlamydomonas enzyme, can be modeled, both redox‐sensitive Cys residues appear to be located on the regulatory nucleotide‐binding domain. Apparently different Cys residues are involved in modulation in the algal and higher plant sedoheptulose bisphosphatases.
Maturation‐specific translational enhancement mediated by the 5′‐UTR of a late pollen transcript
Authors: Bate N. , Spurr C. , Foster G.D. , Twell D.
Published: March 2003

The tomato lat52 gene encodes an abundant protein specifically expressed in the vegetative cell of the pollen grain during pollen maturation which is essential for normal pollen tube growth. Multiple upstream cis‐regulatory elements controlling the level and specificity of lat52 transcription have previously been identified. This research investigated the role of the 5′‐untranslated region (5′‐UTR) in controlling lat52 expression. In transient expression assays, gene fusion constructs containing the lat52 5′‐UTR were expressed in pollen at levels 13‐ to 60‐fold above those in which synthetic polylinker sequences replaced the lat52 5′‐UTR. This enhancement was shown to be independent of both the promoter sequences, the linked reporter gene and the 3′‐UTR. Analysis of RNA and protein levels in transgenic plants containing such gene fusions demonstrated that the lat52 5′‐UTR conferred a dramatically increased translational yield to heterologous transcripts in a pollen‐specific and strictly developmentally regulated manner during the final stages of pollen maturation. These results represent a novel example of translational enhancement in plants in that translational yield is regulated developmentally in a cell‐specific manner via sequences located within the 5′‐UTR.
Nuclear localization activity of phytochrome B
Authors: Sakamoto Koji , Nagatani Akira
Published: March 2003

Phytochromes are soluble red/far‐red‐light photoreceptor proteins which mediate various photomorphogenic responses of plants. Despite much effort, the signal transduction mechanism of phytochrome has remained obscure. Phytochromes are encoded by a small multigene family in Arabidopsis. Among the members of the family, phytochrome A (phyA) and B (phyB) are the best characterized. PhyB contains putative nuclear localization signals within its C‐terminal region. Transgenic Arabidopsis plants were produced which expressed a fusion protein consisting of GUS and C‐terminal fragments of phyB. GUS staining from the fusion protein in these transgenic plants was observed in the nucleus, which suggests that the nuclear localization signal of the fragment is functional. Next, it was examined whether the endogenous phyB was detected in the nucleus. Nuclei were isolated from the light‐grown wild‐type Arabidopsis leaves and subjected to the immunoblot analysis. The result indicated that a substantial fraction of total phyB was recovered in the isolated nuclei. This result was further confirmed by the immunocytochemical analysis of the protoplasts. Finally, the effects of light treatments on the levels of phyB in the isolated nuclei were examined. Dark adaptation of the plants before the nuclear isolation reduced the levels of phyB. The reduction was accelerated by irradiation of plants with far‐red light before the transfer to darkness. Thus, nuclear localization of phyB was suggested to be light‐dependent.
Calcium and cGMP target distinct phytochrome‐responsive elements
Authors: Wu Yan , Hiratsuka Kazuyuki , Neuhaus Gunther , Chua Nam‐Hai
Published: March 2003

Previous work using microinjection into single cells of the tomato aurea mutant demonstrated that phytochrome A‐dependent activation of rbcS and chs genes was mediated by calcium and cGMP, respectively. This work sought to identify promoter cis‐elements that respond to these two small molecules. Box II and Unit I, derived from rbcS‐3A and chs promoters, respectively, were previously shown to function as light‐responsive cis‐elements. Eleven copies of Box II and four copies of Unit I were linked 5′ to the −90 and −46 35 S promoters, respectively, and, both constructs were fused to the β‐glucuronidase (GUS) reporter gene. GUS activities were obtained upon co‐injection of either Box II/‐90GUS or Unit I/‐46GUS with oat phytochrome A (phyA) and GTPγS, an activator of heterotrimeric G proteins. The activation of Box II/‐90GUS by phyA was insensitive to the cGMP antagonist, Rp‐cGMPS, although anthocyanin accumulation, but not chloroplast development, was totally blocked in the injected cells. Consistent with this result, calcium, but not cGMP, induced Box II/‐90GUS activity. In contrast to Box II/‐90GUS, phyA‐dependent activation of Unit I/‐46GUS activity was blocked by Rp‐cGMPS. Moreover, cGMP, not calcium, induced Unit I/‐46GUS activity. Control experiments showed that −90 GUS and −46 GUS were inactive in the presence of calcium and cGMP, respectively. These results provide evidence that Box II and Unit I are targets of the calcium and cGMP pathways, respectively. Interestingly, calcium activation of Box II/‐90GUS was repressed by a high concentration of cGMP and cGMP induction of Unit I/‐46GUS was blocked by a high concentration of calcium/CaM. Thus, these two small cis‐elements can also serve as targets of the reciprocal control mechanisms that operate to regulate the activities of the two phyA signaling branches.
PRK1, a receptor‐like kinase of Petunia inflata, is essential for postmeiotic development of pollen
Authors: Lee Hyun‐Sook , Karunanandaa Balasulojini , McCubbin Andrew , Gilroy Simon , Kao Teh‐hui
Published: March 2003

A pollen‐expressed gene of Petunia inflata that encodes a receptor‐like kinase named PRK1 was previously identified. The extracellular domain of PRK1 contains leucine‐rich repeats which have been implicated in protein—protein interactions, and the cytoplasmic domain was found to autophosphorylate on serine and possibly tyrosine. To investigate the function of PRK1 in pollen development, P. inflata plants were transformed with a construct containing the promoter of a pollen‐expressed gene of tomato, LAT52, fused to an antisense PRK1 cDNA corresponding to part of the extracellular domain of PRK1. Three transgenic plants were found to each produce approximately equal amounts of normal and aborted pollen. Analysis of the inheritance of the transgene inserts in two of the transgenic plants, ASRK‐13 and ASRK‐20, to their progeny revealed that certain transgene inserts co‐segregated with the pollen abortion phenotype. Microscopic examination of the aborted pollen grains showed that their outer wall, the exine, was essentially normal, but that their cytoplasm contained only starch‐like granules. Staining of the nuclei of the microspores at different stages of anther development revealed that the microspores of the transgenic plants developed normally until the uninucleate stage. However, in subsequent stages half of the microspores completed mitosis and developed into normal binucleate pollen, but the other half initially remained uninucleate and subsequently lost their nuclei. Analysis of the amounts of PRK1 mRNA and the antisense PRK1 transcript suggested that the pollen abortion phenotype most likely resulted from downregulation of the PRK1 gene by the antisense PRK1 transgene. These results suggest that PRK1 plays an essential role in a signal transduction pathway that mediates postmeiotic development of microspores.
AtDMC1, the Arabidopsis homologue of the yeast DMC1 gene: characterization, transposon‐induced allelic variation and meiosis‐associated expression
Authors: Klimyuk Victor I. , Jones Jonathan D.G.
Published: March 2003

Based on homologies between the yeast DMC1 and the lily LIM15 meiosis‐specific genes, degenerate PCR primers were designed that amplified the Arabidopsis DMC1 gene (AtDMC1). AtDMC1 genomic DNA (8 kb) was sequenced, and the transcript was characterized by reverse transcriptase‐polymerase chain reaction (RT‐PCR) and by 5′ and 3′ RACE (rapid amplification of cDNA ends). The AtDMC1 gene contains 15 exons and 14 introns. RNA in situ hybridization analysis showed that expression of the AtDMC1 is restricted to pollen mother cells in anthers and to megaspore mother cells in ovules. The AtDMC1 promoter was fused to the GUS reporter gene, and conferred meiosis‐associated expression in both male and female floral lineages. Comparison of AtDMC1 isolated from Landsberg erecta ecotype to its Columbia allele ArLIM15, revealed the presence of a 1874 bp transposon‐like element within the promoter region of ArLIM15. RT‐PCR analysis showed that the expression levels of AtDMC1 and ArLIM15 are similar. Possible uses for the AtDMC1 promoter are discussed.
Salicylic acid and the plant pathogen Erwinia carotovora induce defense genes via antagonistic pathways
Authors: Vidal Sabina , de León Inés Ponce , Denecke Jürgen , Palva E. Tapio
Published: March 2003

Infection of tobacco plants with the plant pathogenic bacterium Erwinia carotovora subsp. carotovora or treatment of plants with Erwinia‐derived elicitor preparations leads to the induction of a number of genes thought to play a role in plant defense response to pathogens. In order to determine the role of salicylic acid (SA) in the induction of the Erwinia responsive genes, the accumulation of mRNAs for these and other genes encoding pathogenesis‐related proteins (PR genes) in response to both Erwinia elicitors and SA was determined. PR genes were identified which were preferentially induced by Erwinia elicitor preparations, one gene was induced by SA but not by Erwinia, and another gene was induced by both type of treatments. The differential expression of these genes and the timing of induction suggest that SA is not the signal molecule leading to the early response of plants to Erwinia. This was demonstrated by experiments using transgenic NahG plants that overproduce a salicylate hydroxylase inactivating SA. The elicitation of PR genes by Erwinia was similar in NahG and wild‐type plants. Therefore, induction of plant defense genes by Erwinia and SA seems to be by two distinct pathways leading to expression of separate sets of genes. Furthermore, we could demonstrate that Erwinia elicitors antagonize the SA‐mediated induction of PR genes. Similarly, SA appeared to inhibit the induction of PR genes elicited by Erwinia. The observed antagonism between the two signal transduction pathways indicates the presence of a common regulatory element in both pathways that acts downstream of SA in the SA‐mediated response.
Ds elements on all five Arabidopsis chromosomes and assessment of their utility for transposon tagging
Authors: Long Deborah , Goodrich Justin , Wilson Kate , Sundberg Eva , Martin Marta , Puangsomlee Preeya , Coupland George
Published: March 2003

The maize transposons Activator (Ac) and Dissociation (Ds) tend to transpose to sites close to their original position and can be efficiently used to transposon‐tag genetically linked genes. To facilitate this approach, we describe the locations of seven T‐DNAs carrying Ds elements, including at least one on each of the five chromosomes. For five of these T‐DNAs, we have confirmed that the Ds element transposes preferentially to genetically linked sites. A large‐scale transposon‐tagging experiment was performed by activating Ds from eight chromosomal locations that included at least one on each of the five chromosomes. These experiments produced a total of 1132 F3 families that were predicted to carry around 870 independent Ds insertions. In these populations, 33 independently isolated mutants that were visibly different from wild‐type were identified. Twenty‐nine of these mutants were studied genetically, and 14 were not tagged with Ds because the element could be separated from the mutation by recombination. The remaining 15 mutations were possibly tagged because the transposon and the mutation were not separated by recombination. These experiments provide tools for transposon‐tagging on each chromosome, and indicate that approximately 50% of identified mutations are likely to be tagged, thereby enabling cloning of the affected genes.
FISH physical mapping with barley BAC clones
Authors: Lapitan Nora L.V. , Brown Susan E. , Kennard Wayne , Stephens Janice L. , Knudson Dennis L.
Published: March 2003

Fluorescence in situ hybridization (FISH) is a useful technique for physical mapping of genes, markers, and other single‐ or low‐copy sequences. Since clones containing less than 10 kb of single‐copy DNA do not reliably produce detectable signals with current FISH techniques in plants, a bacterial artificial chromosome (BAC) partial library of barley was constructed and a FISH protocol for detecting unique sequences in barley BAC clones was developed. The library has a 95 kb average barley insert, representing about 20% of a barley genome. Two BAC clones containing hordein gene sequences were identified and partially characterized. FISH using these two BAC clones as probes showed specific hybridization signals near the end of the short arm of one pair of chromosomes. Restriction digests of these two BAC clones were compared with restriction patterns of genomic DNA; all fragments contained in the BAC clones corresponded to bands present in the genomic DNA, and the two BAC clones were not identical. The barley inserts contained in these two BAC clones were faithful copies of the genomic DNA. FISH with four BAC clones with inserts varying from 20 to 150 kb, showed distinct signals on paired chromatids. Physical mapping of single‐ or low‐copy sequences in BAC clones by FISH will help to correlate the genetic and physical maps. FISH with BAC clones also provide an additional approach for saturating regions of interest with markers and for constructing contigs spanning those regions.
Programmed cell death in the root cortex of soybean root necrosis mutants
Authors: Kosslak Renee M. , Chamberlin Mark A. , Palmer Reid G. , Bowen Benjamin A.
Published: March 2003

The soybean root necrosis (rn) mutation causes a progressive browning of the root soon after germination that is associated with accumulation of phytoalexins and pathogenesis‐related proteins and an increased tolerance to root‐borne infection by the fungal pathogen, Phytophthora sojae. Grafting and decapitation experiments indicate that the rn phenotype is root‐autonomous at the macroscopic level. However, the onset and severity of browning was modulated in intact plants by exposure to light, as was the extent of lateral root formation, suggesting that both lateral roots and the rn phenotype could be directly or indirectly controlled by similar shoot‐derived factors. Browning first occurs in differentiated inner cortical cells adjacent to the stele and is preceded by a wave of autofluorescence that emanates from cortical cells opposite the xylem poles and spreads across the cortex. Before any visible changes in autofluorescence or browning, fragmented DNA was detected by TUNEL (Terminal deoxynucleotidyl transferase‐mediated dUTP‐digoxigenin nick end labeling) in small clusters of inner cortical cells that subsequently could be distinguished cytologically from neighboring cells throughout rn root development. Inner cortical cells overlying lateral root primordia in either Rn or rn plants also were stained by TUNEL. Features commonly observed in animal cell apoptosis were confirmed by electron microscopy but, surprisingly, cells with a necrotic morphology were detected alongside apoptotic cells in the cortex of rn roots when TUNEL‐positive cells were first observed. The two morphologies may represent different stages of a common pathway for programmed cell death (pcd) in plant roots, or two separate pathways of pcd could be involved. The phenotype of rn plants suggests that the Rn gene could either negatively regulate cortical cell death or be required for cortical cell survival. The possibility of a mechanistic link between cortical cell death in rn plants and during lateral root emergence is discussed.
Spatial expression dynamics of Men‐9 delineate the third floral whorl in male and female flowers of dioecious Silene latifolia
Authors: Robertson Shona E. , Li Yi , Scutt Charles P. , Willis Malcolm E. , Gilmartin Philip M.
Published: March 2003

Sex determination in Silene latifolia is controlled by heteromorphic sex chromosomes. Female flowers have five fused carpels and ten arrested stamen primordia. The male‐determining Y chromosome overrides female development to suppress carpel formation and promote stamen development. The isolation and characterization of two S. latifoliaMale enhanced cDNAs, Men‐9a and Men‐9b, which probably represent different alleles of a novel gene are reported here. Men‐9a and Men‐9b share 91.8% coding sequence nucleotide identity, yet only 85.4% amino acid identity. The Men‐9 cDNAs are related to the previously reported MROS3 cDNA from S. latifolia. However, MROS3 is not present in the S. latifolia population used in these studies and the expression dynamics of Men‐9a and Men‐9b contrast dramatically with those reported for MROS3. Men‐9 cDNAs are expressed primarily in anthers of young male flowers, with highest expression in 1–2 mm buds. Men‐9 expression is also observed at a low level in female flowers. In situ hybridization analysis reveals two phases of Men‐9 expression. The first phase is during a common stage of early stamen development in male and female flowers prior to stamen arrest in female flowers. The second phase of Men‐9 expression is maximal in the epidermis and endothecium of Y chromosome‐ and Ustilago violacea‐induced stamens; expression in male and female flowers extends to the epidermis of the staminal nectaries with strict boundaries at the second and fourth whorls. Men‐9 gene expression therefore delineates the boundaries of the third floral whorl in S. latifolia flowers.
A nuclear gene, erd1, encoding a chloroplast‐targeted Clp protease regulatory subunit homolog is not only induced by water stress but also developmentally up‐regulated during senescence in Arabidopsis thaliana
Authors: Nakashima Kazuo , Kiyosue Tomohiro , Yamaguchi‐Shinozaki Kazuko , Shinozaki Kazuo
Published: March 2003

A cDNA, ERD1, isolated from one‐hour‐dehydrated plants of Arabidopsis thaliana L. encodes a putative protein that is similar to the regulatory ATPase subunit (ClpA) of the Clp protease and contains a putative chloroplast‐targeting transit‐peptide at the N‐terminus. A chimeric gene with the putative plastid‐targeting sequence of the erd1 gene fused to the synthetic green‐fluorescent protein (sGFP) gene was constructed and introduced into Arabidopsis protoplasts. The N‐terminal region of the ERD1 protein directed the sGFP protein into the plastids of the protoplasts, and functioned as a transit peptide. Northern blot analysis indicated that expression of the erd1 gene was induced not only by water stress, such as dehydration and high salinity, but also by natural senescence and dark‐induced etiolation. The erd1 gene was not strongly induced by exogenous abscisic acid. A chimeric gene with the 0.9 kb promoter region of the erd1 gene fused to the β‐glucuronidase (GUS) reporter gene was constructed, and tobacco plants transformed with the construct. The GUS reporter gene driven by the erd1 promoter was induced by dehydration and high salt stress at significant levels in the transgenic plants. The GUS gene was strongly expressed in older leaves without dehydration, and was induced by dark‐induced etiolation. Furthermore, GUS activity was reduced by cytokinin treatment during dark‐induced etiolation. These results indicate that expression of the erd1 gene is developmentally up‐regulated by senescence as well as by water stress.
Mg‐chelatase of tobacco: identification of a Chl D cDNA sequence encoding a third subunit, analysis of the interaction of the three subunits with the yeast two‐hybrid system, and reconstitution of the enzyme activity by co‐expression of recombinant CHL D, CHL H and CHL I
Authors: Papenbrock Jutta , Gräfe Susanna , Kruse Elisabeth , Hänel Frank , Grimm Bernhard
Published: March 2003

Mg‐protoporphyrin IX chelatase catalyzes insertion of the magnesium ion into protoporphyrin IX, the last common intermediate precursor in chlorophyll and heme biosynthesis, to form Mg‐protoporphyrin IX. In Rhodobacter sphaeroides, and Synechocystis, the three open reading frames bchD/chlD, bchH/chlH and bchl/chll encode proteins which are required for in vitro Mg‐chelatase activity. In higher plants also, three proteins are necessary for the Mg chelation, and genes homologous to bchH and bchl have been isolated previously. In this study, a novel tobacco cDNA sequence homologous to bchD is isolated and initially characterized. Together with the tobacco clones encoding the other two subunits, full‐length cDNAs are now available for the first time for all three subunits of one plant species. The CHL D polypeptide deduced from the open reading frame encodes a protein of 758 aa (82.9 kDa) with an amino terminal extension that resembles a plastid transit peptide. Sequence comparison of tobacco CHL D revealed similarities to the D subunit of Rhodobacter and Synechocystis of 44% and 75%. The amino terminal half of CHL D shows significant similarity (46%) to the entire CHL I peptide sequence, indicating a gene duplication from an ancestral gene. The carboxy terminal half seemed to be unique. Both parts of CHL D are linked with a glutamine/asparagine/proline‐rich region flanked by a highly acid‐rich segment. Protein‐protein interaction among the three subunits CHL D, H and I was studied using the yeast two‐hybrid system. Physical interaction was demonstrated between CHL D and CHL I indicating that CHL D is part of the Mg‐chelatase. Heterodimer formation of CHL H with CHL I or CHL D could not be demonstrated by transactivation of the lacZ reporter gene. Homodimerization of the CHL D subunit was indicated in the more sensitive assay on X‐Gal‐containing agar plates. In vitro Mg2+ insertion into protoporphyrin IX was demonstrated in protein extracts of yeast strains expressing the three subunits of tobacco Mg‐chelatase. The reconstitution of the recombinant enzyme activity required additional ATP.
Compromising early salicylic acid accumulation delays the hypersensitive response and increases viral dispersal during lesion establishment in TMV‐infected tobacco
Authors: Mur Luis A.J. , Bi Yong‐Mei , Darby Robert M. , Firek Simon , Draper John
Published: March 2003

To investigate the role of salicylic acid (SA) in the hypersensitive response (HR) its accumulation was compromised during different phases of lesion development by differential expression of a salicylate hydroxylase gene (SH‐L). Constitutive suppression of SA accumulation was achieved by expression of a gene fusion between the CaMV35S promoter (35S) and SH‐L. Using the H2O2‐responsive AoPR1 promoter to drive SH‐L SA accumulation could be compromised at an early stage, on lesion formation and possibly prior to visible necrosis, whilst use of the salicylate‐responsive PR1a promoter reduced SA accumulation at a later stage as lesions expand. TMV infection of 35S‐SH‐L and AoPR1‐SH‐L, but not PR1a‐SH‐L, tobacco resulted in significantly greater rates of lesion growth than in wild‐type tobacco. TMV was detected in asymptomatic tissue surrounding lesions only in 35S‐SH‐L and AoPR1‐SH‐L lines; subsequently these transgenic lines exhibited a ‘spreading‐necrosis’ originating from the lesion which entered the stem and eventually other leaves, a phenotype which could be correlated with the presence of TMV particles. Analysis of TMV‐infected and ‘temperature‐shifted’ tobacco indicated that both 35S‐SH‐L and AoPR1‐SH‐L, but not PR1a‐SH‐L, transgenics exhibited delayed cell‐death compared to wild‐type infections. We propose that the SH‐L phenotypes indicate that early SA accumulation is a major factor in preventing viral escape, via mechanism(s) which may include influencing the rate of host‐cell death and, possibly, an effect on viral function.
Overexpressed phytochrome C has similar photosensory specificity to phytochrome B but a distinctive capacity to enhance primary leaf expansion
Authors: Qin Minmin , Kuhn Robert , Moran Sharon , Quail Peter H.
Published: March 2003

Phytochrome C (phyC) is a low‐abundance member of the five‐membered phytochrome family of photoreceptors in Arabidopsis. Towards developing an understanding of the photosensory and physiological functions of phyC, transgenic Arabidopsis plants were generated that overexpress cDNA‐encoded phyC and seedling responses to continuous white, red, or far‐red light (Wc, Rc or FRc, respectively) were examined. Transgenic seedlings overexpressing phyC displayed enhanced inhibition of hypocotyl elongation in Rc, but were unchanged in responsiveness to FRc relative to wild‐type. These data indicate that phyC has photosensory specificity that is similar to that of phyB and thus distinct from that of phyA. phyC overexpressors with levels only 3 to 4 times the level of endogenous phyC exhibited enhanced primary leaf expansion in Wc. This is in contrast to phyA or phyB overexpressors which respectively have levels that are 500‐and 100‐fold that of overexpressed phyC but showed no enhancement of primary leaf expansion. Therefore, phyC may have some physiological roles that are different to those of phyA and phyB in the control of seedling responses to light signals.
In vitro and in vivo evidence for actin association of the naphthylphthalamic acid‐binding protein from zucchini hypocotyls
Authors: Butler John H. , Hu Shiquan , Brady Shari R. , Dixon Michael W. , Muday Gloria K.
Published: March 2003

The N‐1‐naphthylphthalamic acid (NPA)‐binding protein is part of the auxin efflux carrier, the protein complex that controls polar auxin transport in plant tissues. This study tested the hypothesis that the NPA‐binding protein (NBP) is associated with the actin cytoskeleton in vitro and that an intact actin cytoskeleton is required for polar auxin transport in vivo. Cytoskeletal polymerization was altered in extracts of zucchini hypocotyls with reagents that stabilized either the polymeric or monomeric forms of actin or tubulin. Phalloidin treatment altered actin polymerization, as demonstrated by immunoblot analyses following native and denaturing electrophoresis. Phalloidin increased both filamentous actin (F‐actin) and NPA‐binding activity, while cytochalasin D and Tris decreased both F‐actin and NPA‐binding activity in cytoskeletal pellets. The microtubule stabilizing drug taxol increased pelletable tubulin, but did not alter either the amount of pelletable actin or NPA‐binding activity. Treatment of etiolated zucchini hypocotyls with cytochalasin D decreased the amount of auxin transport and its regulation by NPA. These experimental results are consistent with an in vitro actin cytoskeletal association of the NPA‐binding protein and with the requirement of an intact actin cytoskeleton for maximal polar auxin transport in vivo.
Dual regulation of a heat shock promoter during embryogenesis: stage‐dependent role of heat shock elements
Authors: Almoguera Concepción , Prieto‐Dapena Pilar , Jordano Juan
Published: March 2003

Transgenic tobacco expression was analysed of chimeric genes with point mutations in the heat shock element (HSE) arrays of a small heat shock protein (sHSP) gene from sunflower: Ha hsp17.7 G4. The promoter was developmentally regulated during zygotic embryogenesis and responded to heat stress in vegetative tissues. Mutations in the HSE affected nucleotides crucial for human heat shock transcription factor 1 (HSF1) binding. They abolished the heat shock response of Ha hsp17.7 G4 and produced expression changes that demonstrated dual regulation of this promoter during embryogenesis. Thus, whereas activation of the chimeric genes during early maturation stages did not require intact HSE, expression at later desiccation stages was reduced by mutations in both the proximal (–57 to –89) and distal (–99 to –121) HSE. In contrast, two point mutations in the proximal HSE that did not severely affect gene expression during zygotic embryogenesis, eliminated the heat shock response of the same chimeric gene in vegetative organs. Therefore, by site‐directed mutagenesis, it was possible to separate the heat shock response of Ha hsp17.7 G4 from its developmental regulation. The results indicate the co‐existence, in a single promoter, of HSF‐dependent and ‐independent regulation mechanisms that would control sHSP gene expression at different stages during plant embryogenesis.
Domains of the TMV movement protein involved in subcellular localization
Authors: Kahn Theodore W. , Lapidot Moshe , Heinlein Manfred , Reichel Christoph , Cooper Bret , Gafny Ron , Beachy Roger N.
Published: March 2003

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.
Photomorphogenic development of the Arabidopsisshy2–1D mutation and its interaction with phytochromes in darkness
Authors: Kim Byung Chul , Soh Moon Soo , Hong Sung Hyun , Furuya Masaki , Nam Hong Gil
Published: March 2003

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.
Germination‐related genes encoding proteolytic enzymes are expressed in the nucellus of developing wheat grains
Authors: Domínguez Fernando , Cejudo Francisco J.
Published: March 2003

During wheat (Triticum aestivum) grain germination the aleurone layer shows a high expression of genes encoding hydrolytic enzymes. In this study we analyzed the spatio‐temporal pattern of expression of two of these genes, a carboxypeptidase III and a thiol protease, in developing grains. Our results show that transcripts of both genes accumulate at early stages of grain development (5–15 dpa) in the nucellus, a maternal tissue that degenerates during wheat grain development. In addition, the thiol protease gene is expressed in the nucellar projections and the modified aleurone cells, the cellular pathway of photosynthate transfer in the developing wheat grain. These results show that genes related to the germination programme are also expressed in the development programme where they may be implicated in the degenerative process that takes place in the nucellar tissue and the cytoplasm of nucellar projections cells.
Cytoplasmic polyamines block the fast‐activating vacuolar cation channel
Authors: Brüggemann Lioubov I. , Pottosin Igor I. , Schönknecht Gerald
Published: March 2003

The fast‐activating vacuolar (FV) channel dominates the electrical characteristics of the tonoplast at physiological free Ca2+ concentrations. Since polyamines are known to increase in plant cells in response to stress, the regulation of FV channels by polyamines was investigated. Patch‐clamp measurements were performed on whole barley (

Hordeum vulgare

) mesophyll vacuoles and on excised tonoplast patches. The trivalent polyamine spermidine and the tetravalent polyamine spermine blocked FV channels with Kd≈ 100 μM and Kd≈ 5 μM, respectively. Increasing cytosolic and vacuolar Ca2+ had no effect on putrescine and spermidine binding to FV channels but slightly decreased the affinity for spermine. The inhibition of FV channels by all three polyamines was not voltage‐dependent. This points to a different mode of binding compared to inward rectifier K+ channels and Ca2+‐permeable glutamate receptor channels from animal cells, which show rectification due to a voltage‐dependent block by polyamines. In plant cells, the common polyamines (putrescine, spermidine and spermine) are likely to mediate a salt stress‐induced decrease of ion flux across the vacuolar membrane by blocking FV channels.
A 100 kDa polypeptide associates with the Vo membrane sector but not with the active oat vacuolar H+‐ATPase, suggesting a role in assembly
Authors: Li Xuhang , Sze Heven
Published: March 2003

The vacuolar H+‐ATPase (V‐ATPase) is responsible for acidifying endomembrane compartments in eukaryotic cells. Although a 100 kDa subunit is common to many V‐ATPases, it is not detected in a purified and active pump from oat (Ward J.M. & Sze H. (1992)

Plant Physiol

. 99, 925–931). A 100 kDa subunit of the yeast V‐ATPase is encoded by

VPH1

. Immunostaining revealed a Vph1p‐related polypeptide in oat membranes, thus the role of this polypeptide was investigated. Membrane proteins were detergent‐solubilized and size‐fractionated, and V‐ATPase subunits were identified by immunostaining. A 100 kDa polypeptide was not associated with the fully assembled ATPase; however, it was part of an approximately 250 kDa Vo complex including subunits of 36 and 16 kDa. Immunostaining with an affinity‐purified antibody against the oat 100 kDa protein confirmed that the polypeptide was part of a 250 kDa complex and that it had not degraded in the ≈≈ 670 kDa holoenzyme. Co‐immunoprecipitation with a monoclonal antibody against A subunit indicated that peripheral subunits exist as assembled V1 subcomplexes in the cytosol. The free V1 subcomplex became attached to the detergent‐solubilized Vo sector after mixing, as subunits of both sectors were co‐precipitated by an antibody against subunit A. The absence of this polypeptide from the active enzyme suggests that, unlike the yeast Vph1p, the 100 kDa polypeptide in oat is not required for activity. Its association with the free Vo subcomplex would support a role of this protein in V‐ATPase assembly and perhaps in sorting.
Inter‐organ signaling in plants: regulation of ATP sulfurylase and sulfate transporter genes expression in roots mediated by phloem‐translocated compound
Authors: Lappartient Anne G. , Vidmar J. John , Leustek Thomas , Glass Anthony D. M. , Touraine Bruno
Published: March 2003

Sulfate uptake and ATP sulfurylase activity in the roots of Arabidopsis thaliana and Brassica napus were enhanced by S deprivation and reduced following resupply of SO42–. Similar responses occurred in split‐root experiments where only a portion of the root system was S‐deprived, suggesting that the regulation involves inter‐organ signaling. Phloem‐translocated glutathione (GSH) was identified as the likely transducing molecule responsible for regulating SO42– uptake rate and ATP sulfurylase activity in roots. The regulatory role of GSH was confirmed by the finding that ATP sulfurylase activity was inhibited by supplying Cys except in the presence of buthionine sulfoximine, an inhibitor of GSH synthesis. In direct and remote (split‐root) exposures, levels of protein detected by antibodies against the Arabidopsis APS3 ATP sulfurylase increased in the roots of A. thaliana and B. napus during S starvation, decreased after SO42– restoration, and declined after feeding GSH. RNA blot analysis revealed that the transcript level of APS1, which codes for ATP sulfurylase, was reduced by direct and remote GSH treatments. The abundance of AST68 (a gene encoding an SO42– transporter) was similarly affected by altered sulfur status. This report presents the first evidence for the regulation of root genes involved in nutrient acquisition and assimilation by a signal that is translocated from shoot to root.
Molecular cloning and functional expression of codeinone reductase: the penultimate enzyme in morphine biosynthesis in the opium poppy Papaver somniferum
Authors: Unterlinner Bernhard , Lenz Rainer , Kutchan Toni M.
Published: March 2003

The narcotic analgesic morphine is the major alkaloid of the opium poppy Papaver somniferum
. Its biosynthetic precursor codeine is currently the most widely used and effective antitussive agent. Along the morphine biosynthetic pathway in opium poppy, codeinone reductase catalyzes the NADPH‐dependent reduction of codeinone to codeine. In this study, we have isolated and characterized four cDNAs encoding codeinone reductase isoforms and have functionally expressed them in Escherichia coli
. Heterologously expressed codeinone reductase‐calmodulin‐binding peptide fusion protein was purified from E. coli using calmodulin affinity column chromatography in a yield of 10 mg enzyme l

‐1

. These four isoforms demonstrated very similar physical properties and substrate specificity. As least six alleles appear to be present in the poppy genome. A comparison of the translations of the nucleotide sequences indicate that the codeinone reductase isoforms are 53% identical to 6′‐deoxychalcone synthase from soybean suggesting an evolutionary although not a functional link between enzymes of phenylpropanoid and alkaloid biosynthesis. By sequence comparison, both codeinone reductase and 6′‐deoxy‐ chalcone synthase belong to the aldo/keto reductase family, a group of structurally and functionally related NADPH‐dependent oxidoreductases, and thereby possibly arise from primary metabolism.
Transcriptional activation by Arabidopsis GT‐1 may be through interaction with TFIIA–TBP–TATA complex
Authors: Le Gourrierec José , Li You‐Fang , Zhou Dao‐Xiu
Published: March 2003

GT‐1 belongs to the class of trihelix DNA‐binding proteins and binds to a promoter sequence found in many different genes. Data presented in this report show that GT‐1 contains a trans‐activation function in yeast and in plant cells. However, in tobacco BY‐2 protoplasts, this activity functions only when an internal region containing the DNA‐binding domain is deleted. Gel‐shift and co‐immunoprecipitation assays have revealed that GT‐1 can interact with and stabilize the TFIIA–TBP–TATA complex. These results suggest that GT‐1 may activate transcription through direct inter‐ action with the transcriptional pre‐initiation complex.
Cloning and characterisation of PGA1 and PGA2: two G protein α‐subunits from pea that promote growth in the yeast Saccharomyces cerevisiae
Authors: John F. Marsh III  , Kaufman Lon S.
Published: March 2003

We report here on the cloning and characterization of two G protein α‐subunits from pea: PGA1 and PGA2. Based on DNA gel blot analysis, PGA1 and PGA2 are the only Gα homologous sequences in pea. RT–PCR analysis reveals that PGA1 and PGA2 transcripts are present in a variety of adult pea tissues. However, PGA2 mRNA is consistently detected at a lower level than PGA1 and demonstrates some degree of tissue specificity relative to PGA1. In the apical bud of pea seedlings, PGA1 and PGA2 transcripts decrease in response to 24 h of white light following growth for 6 days in darkness. The G protein mediated, yeast mating pathway was used to analyse the function of PGA1 and PGA2 in vivo. PGA1 downregulates the mating pathway, but through a mechanism that is independent of Gβγ sequestration. Unexpectedly, both PGA1 and PGA2 promote growth through a mating pathway independent mechanism.
Diterpene synthesis in Stevia rebaudiana: recruitment and up‐regulation of key enzymes from the gibberellin biosynthetic pathway
Authors: Richman Alex S. , Gijzen Mark , Starratt Al N. , Yang Zhiyi , Brandle Jim E.
Published: March 2003

Stevia rebaudiana Bertoni leaves accumulate a mixture of at least eight different glycosides derived from the tetracyclic diterpene steviol. These natural products taste intensely sweet and have similar biosynthetic origins to those of gibberellic acid (GA). The initial steps leading to the formation of GA result from the two‐step cyclization of geranylgeranyl diphosphate (GGDP) to (–)‐kaurene via the action of two terpene cyclases (–)‐copalyl diphosphate synthase (CPS) and (–)‐kaurene synthase (KS). Steviol biosynthesis probably uses the same mechanism although the genes and enzymes from S. rebaudiana that are involved in the cyclization of GGDP have not been characterized. We have isolated both the CPS and KS genes from S. rebaudiana and found that recombinant CPS and KS were catalytically active, suggesting that the CPS and KS genes participate in steviol biosynthesis. The genes coding for CPS and KS are usually present in single copies in most plant species and their expression is normally low and limited to rapidly growing tissues. The KS gene has been duplicated in the S. rebaudiana genome and both the KS and CPS genes are highly expressed in mature leaves, a pattern opposite to that found with GA biosynthesis. This pattern may, at least in part, lead to temporal and spatial separation of GA and steviol biosynthesis and probably helps to prevent over‐expression from interfering with normal GA metabolism. Our results show that CPS and KS are part of the steviol glycoside biosynthetic pathway and that Stevia rebaudiana has recruited two genes to secondary metabolism from a highly regulated pathway involved in hormone biosynthesis.
The DIF1 gene of Arabidopsis is required for meiotic chromosome segregation and belongs to the REC8/RAD21 cohesin gene family
Authors: Bhatt Anuj M. , Lister Clare , Page Tania , Fransz Paul , Findlay Kim , Jones Gareth H. , Dickinson Hugh G. , Dean Caroline
Published: March 2003

Cohesins are a group of conserved proteins responsible for cohesion between replicated sister chromatids during mitosis and meiosis and which are implicated in double‐strand break repair and meiotic recombination. We describe here the identification and characterisation of an Arabidopsis gene –DETERMINATE, INFERTILE1 (DIF1), which is a homolog of the Schizosaccharomyces pombe REC8/RAD21 cohesin genes, and is essential for meiotic chromosome segregation. Five independent alleles of the DIF1 gene were isolated by transposon mutagenesis, and the mutants show complete male and female sterility. Pollen mother cells (PMCs) of dif1 mutants show multiple meiotic defects which are represented by univalent chromosomes and chromosome fragmentation at metaphase I, and acentric fragments and chromatin bridges in meiosis I and II. Consequently, chromosome segregation is strongly affected, resulting in meiotic products of uneven size, shape and of variable ploidy. The similarities in phenotype, and the sequence homology between DIF1 and the REC8/RAD21 cohesins suggests that cohesin function is largely conserved between eukaryotes and highlights the essential role cohesins play in plant meiosis.