bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional complementation of yeast vma1Δ cells by a plant subunit A homolog rescues the mutant phenotype and partially restores vacuolar H+‐ATPase activity
Authors: Kim Woonbong , Wan Ching‐Yi , Wilkins Thea A. 
Published: March 2003

Summary The ability of a vacuolar H+‐ATPase (V‐ATPase) subunit homolog (subunit A) from plants to rescue the
vma
mutant phenotype of yeast was investigated as a first step towards investigating the structure and function of plant subunits in molecular detail. Heterologous expression of cotton cDNAs encoding near‐identical isoforms of subunit A in mutant
vma1
Δ yeast cells successfully rescued the mutant
vma
phenotype, indicating that subunit A of plants and yeast have retained elements essential to V‐ATPases during the course of evolution. Although vacuoles become acidified, the plant–yeast hybrid holoenzyme only partially restored V‐ATPase activity (approximately 60%) in mutant yeast cells. Domain substitution of divergent N‐or C‐termini only slightly enhanced V‐ATPase activity, whereas swapping both domains acted synergistically, increasing coupled ATP hydrolysis and proton translocation by approximately 22% relative to the native plant subunit. Immunoblot analysis indicated that similar amounts of yeast, plant or plant–yeast chimeric subunits are membrane‐bound. These results suggest that subunit A terminal domains contain structural information that impact V‐ATPase structure and function.
A 13‐bp cis‐regulatory element in the LTR promoter of the tobacco retrotransposon Tto1 is involved in responsiveness to tissue culture, wounding, methyl jasmonate and fungal elicitors
Authors: Takeda Shin , Sugimoto Kazuhiko , Otsuki Hiroshi , Hirochika Hirohiko 
Published: March 2003

SummaryThe tobacco Tto1 is one of the few active LTR‐retrotransposons of plants, and its transposition is activated by tissue culture and is primarily regulated at the transcriptional level. The expression of Tto1 RNA can also be activated by various stresses, including viral infection, wounding, and treatment with jasmonate, a signal molecule of plant defence responses. It is shown here that the Tto1 LTR promoter is responsible for a high level of expression in cultured tissues of transgenic tobacco plants. We demonstrate that a 13‐bp repeated motif (TGGTAGGTGAGAT) in the LTR functions as a cis‐regulatory element, which confers the responsiveness to tissue culture, wounding and methyl jasmonate. Fungal elicitors also activate the promoter containing multiple copies of the 13‐bp motif. Expression mediated by the 13‐bp motif is activated markedly by okadaic acid and moderately by K252a, so that both phosphorylation and dephosphorylation of proteins are possibly involved in the signalling pathways. Interestingly, the 13‐bp motif contains a conserved motif, Box L (also called AC‐I or H‐box like sequence) which has been shown to be involved in the expression of phenylpropanoid synthetic genes. Moreover, extended homologies are found between promoters of Tto1 and an asparagus defence gene, AoPR1, suggesting a possibility that the ancient insertion of an ancestral Tto1‐related retrotransposon has provided some of the promoter/regulatory sequences, including the 13‐bp motif‐related sequence, of the AoPR1 gene. Based on the structural and functional similarity between the two promoters, a possible evolutionary role of the regulatory sequences of LTR‐retrotransposons is discussed.
Non‐invasive quantitative detection and applications of non‐toxic, S65T‐type green fluorescent protein in living plants
Authors: Niwa Yasuo , Hirano Takanori , Yoshimoto Kohki , Shimizu Masanori , Kobayashi Hirokazu 
Published: March 2003

SummaryGreen fluorescent protein (GFP) has emerged as a powerful new tool in a variety of organisms. An engineered sGFP(S65T) sequence containing optimized codons of highly expressed eukaryotic proteins has provided up to 100‐fold brighter fluorescence signals than the original jellyfish GFP sequence in plant and mammalian cells. It would be useful to establish a non‐invasive, quantitative detection system which is optimized for S65T‐type GFP, one of the brightest chromophore mutants among the various GFPs. We demonstrate here that highly fluorescent transgenic Arabidopsis can be generated, and the fluorescence intensity of whole plants can be measured under non‐disruptive, sterile conditions using a quantitative fluorescent imaging system with blue laser excitation. Homozygous plants can be distinguished from heterozygous plants and fully fertile progenies can be obtained from the analyzed plants. In the case of cultured tobacco cells, GFP‐positive cells can be quantitatively distinguished from non‐transformed cells under non‐selective conditions. This system will be useful in applications such as mutant screening, analysis of whole‐body phenomena, including gene silencing and quantitative assessments of colonies from microorganisms to cultured eukaryotic cells. To facilitate the elucidation of protein targeting and organelle biogenesis in planta, we also generated transgenic Arabidopsis that stably express the plastid‐ or mitochondria‐targeted sGFP(S65T). Etioplasts in dark‐grown cotyledons and mitochondria in dry seed embryos could be visualized for the first time in transgenic Arabidopsis plants under normal growing conditions.
Identification of three kinds of mutually related composite elements conferring S phase‐specific transcriptional activation
Authors: Taoka Ken‐ichiro , Kaya Hidetaka , Nakayama Takuya , Araki Takashi , Meshi Tetsuo , Iwabuchi Masaki 
Published: March 2003

SummaryConservation of the Oct motif (CGCGGATC) is a remarkable feature of plant histone gene promoters. Many of the Oct motifs are paired with a distinct motif, Hex, TCA or CCAAT‐box, constituting the type I element (CCACGTCANCGATCCGCG), type II element (TCACGCGGATC) and type III element (GATCCGCG‐N14‐ACCAATCA). To clarify the roles of these Oct‐containing composite elements (OCEs) in cell cycle‐dependent and tissue‐specific expression, we performed gain‐of‐function experiments with transgenic tobacco cell lines and plants harboring a derivative of the 35S core promoter/β‐glucuronidase fusion gene in which three or four copies of an OCE had been placed upstream. Although their activities were slightly different, results showed that each of the three types of OCEs could confer the ability to direct S phase‐specific expression on a heterologous promoter. In transgenic plants, the type I and III elements exhibited a similar activity, directing expression in meristematic tissues, whereas the activity of the type II element appeared to be restricted to young cotyledons and maturating guard cells. Mutational analyses demonstrated that the co‐operation of Oct with another module (Hex, TCA or CCAAT‐box) was absolutely required for both temporal and spatial regulation. Thus, OCEs play a pivotal role in regulation of the expression of plant histone genes.
Decreased expression of sucrose phosphate synthase strongly inhibits the water stress‐induced synthesis of sucrose in growing potato tubers
Authors: Geigenberger Peter , Reimholz Ralph , Deiting Uta , Sonnewald Uwe , Stitt Mark 
Published: March 2003

SummaryWater stress stimulates sucrose synthesis and inhibits starch synthesis in wild‐type tubers. Antisense and co‐suppression potato transformants with decreased expression of sucrose–phosphate synthase (SPS) have been used to analyse the importance of SPS for the regulation of this water‐stress induced change in partitioning. (i) In the absence of water stress, a 70–80% decrease in SPS activity led to a 30–50% inhibition of sucrose synthesis and a slight (10–20%) increase of starch synthesis in tuber discs in short‐term labelling experiments with low concentrations of labelled glucose. Similar changes were seen in short‐term labelling experiments with intact tubers attached to well‐watered plants. Provided plants were grown with ample light and water, transformant tubers had a slightly lower water and sucrose content and a similar or even marginally higher starch content than wild‐type tubers. (ii) When wild‐type tuber slices were incubated with labelled glucose in the presence of mannitol to generate a moderate water deficit (between –0.12 and –0.72 MPa), there was a marked stimulation of sucrose synthesis and inhibition of starch synthesis. A similar stimulation was seen in labelling experiments with wild‐type tubers that were attached to water‐stressed wild‐type plants. These changes were almost completely suppressed in transformants with a 70–80% reduction of SPS activity. (iii) Decreased irrigation led to an increase in the fraction of the dry‐matter allocated to tubers in wild‐type plants. This shift in allocation was prevented in transformants with reduced expression of SPS. (iv) The results show that operation of SPS and the sucrose cycle in growing potato tubers may lead to a marginal decrease in starch accumulation in non‐stressed plants. However, SPS becomes a crucial factor in water‐stressed plants because it is required for adaptive changes in tuber metabolism and whole plant allocation.
A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii†
Authors: Fuhrmann Markus , Oertel Wolfgang , Hegemann Peter 
Published: March 2003

SummaryThe use of Chlamydomonas reinhardtii as a model system has been hindered by difficulties encountered in expressing foreign genes. We have synthesised a gene encoding green fluorescent protein (GFP) adapted to the codon usage of C. reinhardtii (cgfp). After verifying the gene was functional in Escherichia coli, the cgfp was fused in frame to the phleomycin resistance gene ble from Streptoalloteichus hindustanus and expressed in C. reinhardtii under control of the rbcS2 promoter and intron sequences. The GFP‐fluorescence was seen only in the nucleus demonstrating the nuclear accumulation of the Ble‐GFP fusion protein. The cgfp was also fused to the chlamyopsin gene, cop, and expressed in C. reinhardtii under control of the cop promoter. The eyespot became fluorescent indicating that the opsin–GFP fusion protein was correctly directed into the eyespot along with the endogenous unmodified opsin. We conclude that cgfp provides a useful tool to visualize protein synthesis and localisation in vivo in C. reinhardtii and possibly in related green algal species.
The gene encoding tobacco gibberellin 3β‐hydroxylase is expressed at the site of GA action during stem elongation and flower organ development
Authors: Itoh Hironori , Tanaka‐Ueguchi Miyako , Kawaide Hiroshi , Chen Xinbo , Kamiya Yuji , Matsuoka Makoto 
Published: March 2003

SummaryGibberellin 3β‐hydroxylase catalyzes the final step in the biosynthetic pathway leading to the plant hormone gibberellin (GA) and, therefore, the in vivo localization of this enzyme should give a direct indication of the site of synthesis of bioactive GAs in plants. We have isolated a cDNA clone, Nty (Nicotiana tabacum GA 3β‐hydroxylase), which encodes a putative GA 3β‐hydroxylase, by RT‐PCR using RNA from tobacco shoot apices. Functional analysis, using an NTY protein expressed in Escherichia coli, revealed that Nty encoded an active GA 3β‐hydroxylase. A high expression level of Nty was observed in shoot apices, flowers, roots, young internodes but not in leaves or seeds. We performed more detailed expression analyses using in situ hybridization and histochemical analyses of the GUS activity in transgenic tobacco plants carrying an Nty promoter::GUS fusion gene. These studies revealed that expression of Nty was restricted to specific regions, including actively dividing and elongating cells in the various organs; rib meristem and elongation zones of shoot apices, tapetum and pollen grains in developing anthers and root tips, which are consistent with the sites of GA action. It is proposed that GA actions depend on the modulation of endogenous bioactive GA levels through the regulation of GA 3β‐hydroxylase expression in situ.
Molecular mechanisms underlying carotenogenesis in the chromoplast: multilevel regulation of carotenoid‐associated genes
Authors: Vishnevetsky Michael , Ovadis Marianna , Zuker Amir , Vainstein Alexander 
Published: March 2003

SummaryIn nature, carotenoid function and mode of action are highly determined by the neighboring protein and lipid molecules. Therefore an understanding of the proteins’ involvement in carotenoid sequestration would be of great help in elucidating carotenoid function in vivo. Based on a study of the expression of chromoplast‐specific carotenoid‐associated genes from cucumber corolla (CHRC and CHRD), a working model is presented wherein two major regulatory factors control carotenoid sequestration within the chromoplasts: (i) floral tissue‐specific transcriptional regulators of chromoplasto‐ genesis; and (ii) post‐transcriptional regulators related to the amount/type of sequestered carotenoids. This model is supported by the major role transcriptional regulation was found to play in the temporal and spatial expression of the CHRC gene, and by the fact that phytohormones such as gibberellic acid (GA3), abscisic acid and ethylene also acted as transcriptional regulators of CHRC expression. The primary response to GA3 was localized within the CHRC promoter to a 290 bp fragment. Furthermore, we demonstrated strong down‐regulation of CHRC expression at post‐transcriptional and translational/post‐translational levels resulting from inhibition of carotenoid biosynthesis, thus revealing a close link between carotenoid biosynthetic and sequestration machineries.
Chlorophyll breakdown by chlorophyllase: isolation and functional expression of the Chlase1 gene from ethylene‐treated Citrus fruit and its regulation during development
Authors: Jacob‐Wilk Debora , Holland Doron , Goldschmidt Eliezer E. , Riov Joseph , Eyal Yoram 
Published: March 2003

SummaryWe report on the isolation, functional expression and characterization of a cDNA encoding chlorophyllase, the enzyme catalyzing the first step in the chlorophyll breakdown pathway. The Chlase1 cDNA from Valencia Orange (Citrus sinensis cv. Valencia) was obtained by RT–PCR using degenerate primers based on the amino acid sequence of the previously purified protein. Chlase1 encodes a protein of 329 amino acids, including a sequence domain characterizing serine‐lipases and a putative chloroplast‐directing transit peptide. The Chlase1 gene encodes an active chlorophyllase enzyme which catalyzes the dephytylation of chlorophyll as shown by in vitro recombinant enzyme assays. Chlorophyllase expression at the transcript level in Valencia orange peel was found to be low and constitutive during natural fruit development without significant increase towards color‐break and ripening. However, ethylene treatment induced an increase in chlorophyllase transcript at all stages of development. An enhanced response to ethylene treatment was observed during the months of October and November, corresponding to the time of natural color‐break. The senescence‐delaying regulator gibberellin‐A3 (GA3) inhibited the effect of ethylene on chlorophyllase transcript accumulation. The data presented suggest that chlorophyllase may not be the regulator of chlorophyll breakdown during natural fruit ripening but is consistent with the notion that chlorophyll is gradually degraded during ripening due to a negative balance between synthesis and breakdown. According to this model, exogenous application of ethylene accelerates chlorophyll breakdown due to increased de novo synthesis of chlorophyllase. Further experi‐ mentation on the regulation and role of chlorophyllase in planta will be facilitated by the gene tools established in this work.
Over‐expression of a pepper plastid lipid‐associated protein in tobacco leads to changes in plastid ultrastructure and plant development upon stress
Authors: Rey Pascal , Gillet Benjamin , Römer Susanne , Eymery Françoise , Massimino Jacqueline , Peltier Gilles , Kuntz Marcel 
Published: March 2003

SummaryProteins homologous to fibrillin, a pepper plastid lipid‐associated protein involved in carotenoid storage in fruit chromoplasts, have been recently identified in leaf chloroplasts from several species and shown to be induced upon environmental stress. To further investigate the role of the protein, transgenic Nicotiana tabacum plants over‐expressing fibrillin using a constitutive promoter were generated. Transgenics grown under standard light intensities (300 μmol photons m−2 sec−1) were found to contain substantial amounts of fibrillin in flowers and leaves. In leaves, the protein was immunolocalized within chloroplasts in both stromal and thylakoid subfractions. No change was noticed in thylakoid structures from transgenics, but chloroplasts contained an increased number of plastoglobules organized in clusters. In petals, leucoplasts were also found to contain more agglutinated plastoglobules. The effects of environmental factors on fibrillin gene expression and protein localization were studied in tobacco leaves. Less fibrillin was present in plants grown under low light intensities, which can be explained by the involvement of a light‐dependent splicing step in the control of fibrillin gene expression in leaves. Analysis of protein subfractions from plants subjected to drought or high light showed that both stresses resulted in fibrillin association with thylakoids. Whereas no growth difference between wild‐type (WT) and transgenic plants was noticed under low light conditions, transgenics exhibit a longer main stem, enhanced development of lateral stems and accelerated floral development under higher light intensities. These data suggest that fibrillin‐related proteins fulfil an important function in plant development in relation to environmental constraints.
A resistance gene product of the nucleotide binding site –‐ leucine rich repeats class can form a complex with bacterial avirulence proteins in vivo
Authors: Leister R. Todd , Katagiri Fumiaki 
Published: March 2003

SummaryResistance (R) genes in plants mediate gene‐for‐gene disease resistance. The ligand‐receptor model, which explains the gene‐for‐gene specificity, predicts a physical interaction between an elicitor, which is directly or indirectly encoded by an avirulence (avr) gene in the pathogen, and the corresponding R gene product. The nucleotide binding site (NBS) – leucine rich repeats (LRR) class of R genes is the largest known class of R genes. Here we report that an NBS‐LRR R protein and its cognate Avr protein form a complex together in the plant cell. The Arabidopsis thaliana R genes RPS2 and RPM1 confer gene‐for‐gene disease resistance to strains of the phytopathogenic bacterium Pseudomonas syringae carrying the avr genes avrRpt2 and avrB, respectively. Using transient expression of these genes in Arabidopsis leaf mesophyll protoplasts, we first demonstrated that the protoplast system is appropriate for the investigation of the gene‐for‐gene recognition mechanism. Formation of an in vivo complex containing the RPS2 and AvrRpt2 proteins was demonstrated by co‐immunoprecipitation of the proteins following expression of the genes in protoplasts. This complex contained at least one additional plant protein of approximately 75 kDa. Unexpectedly, RPS2 also formed a complex with AvrB. We speculate that complex formation between AvrRpt2 and RPS2 is productive and leads to the elicitation of the resistance response, whilst complex formation between AvrB and RPS2 is unproductive and possibly competes with complex formation between AvrRpt2 and RPS2.
Homologues of a single resistance‐gene cluster in potato confer resistance to distinct pathogens: a virus and a nematode
Authors: Van Der Vossen Edwin A. G. , Van Der Voort Jeroen N. A. M. Rouppe , Kanyuka Konstantin , Bendahmane Abdel , Sandbrink Hans , Baulcombe David C. , Bakker Jaap , Stiekema Willem J. , Klein‐Lankhorst Rene M. 
Published: March 2003

SummaryThe isolation of the nematode‐resistance gene Gpa2 in potato is described, and it is demonstrated that highly homologous resistance genes of a single resistance‐gene cluster can confer resistance to distinct pathogen species. Molecular analysis of the Gpa2 locus resulted in the identification of an R‐gene cluster of four highly homologous genes in a region of approximately 115 kb. At least two of these genes are active: one corresponds to the previously isolated Rx1 gene that confers resistance to potato virus X, while the other corresponds to the Gpa2 gene that confers resistance to the potato cyst nematode Globodera pallida. The proteins encoded by the Gpa2 and the Rx1 genes share an overall homology of over 88% (amino‐acid identity) and belong to the leucine‐zipper, nucleotide‐binding site, leucine‐rich repeat (LZ‐NBS‐LRR)‐containing class of plant resistance genes. From the sequence conservation between Gpa2 and Rx1 it is clear that there is a direct evolutionary relationship between the two proteins. Sequence diversity is concentrated in the LRR region and in the C‐terminus. The putative effector domains are more conserved suggesting that, at least in this case, nematode and virus resistance cascades could share common components. These findings underline the potential of protein breeding for engineering new resistance specificities against plant pathogens in vitro.
Chaperone activity of tobacco HSP18, a small heat‐shock protein, is inhibited by ATP
Authors: Smýkal Petr , Mašín Jiří , Hrdý Ivan , Konopásek Ivo , Žárský Viktor 
Published: March 2003

SummaryNtHSP18P (HSP18), a cytosolic class I small heat‐shock protein from tobacco pollen grains, was expressed in Escherichia coli. The viability of these cells was improved by 50% at 50°C, demonstrating its functionality in vivo. Purified recombinant protein formed 240 kDa HSP18 oligomers, irrespective of temperature. These oligomers interacted with the model substrate citrate synthase (CS) to form large complexes in a temperature‐dependent manner. Furthermore, HSP18 prevented thermally induced aggregation of CS at 45°C. The fluorescence probe bis‐ANS revealed the exposure of HSP18 hydrophobic surfaces at this temperature. Reactivation of chemically denatured CS was also significantly enhanced by HSP18. Surprisingly, HSP18 function was inhibited (in contrast to the related chaperone αβ‐crystallin and plant sHSPs studied so far) by the presence of ATP in a concentration‐dependent manner. The conformational changes of HSP18 imposed by ATP binding were indicated by the difference in the quenching of intrinsic tryptophan fluorescence, and implied more compact structure with ATP. Fluorescence measurements with bis‐ANS showed that the conformational shift of HSP18 is suppressed in the presence of ATP. Decreased chaperone activity of HSP18 in the presence of ATP is caused by the lower affinity of conformationally blocked HSP18 for the substrate, as demonstrated by a higher susceptibility of model substrate, malate dehydrogenase, to proteolytic cleavage. Our results suggest that the chaperone activity of some plant sHSPs could be regulated by the availability of ATP in the cytoplasm, which would provide a mechanism to monitor the cell environment, control biological activity of sHSPs, and coordinate it with other ATP‐dependent chaperones such as HSP70.