bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cold‐activation of Brassica napus BN115 promoter is mediated by structural changes in membranes and cytoskeleton, and requires Ca2+ influx
Authors: Sangwan Veena , Foulds Inge , Singh Jas , Dhindsa Rajinder S. 
Published: March 2003

SummaryPrevious studies on cold‐triggered events leading to Ca2+ influx during cold acclimatization have been conducted on either unicellular cyanobacterium Synechocystis or plant cell suspensions, and used transcript levels of cold‐induced genes as end‐point markers. Whether the results of these studies are valid for intact plants or their organs is not known. Here we examine cold signaling in transgenic Brassica napus seedlings carrying, in addition to the endogenous cold‐inducible BN115 gene, the β‐glucuronidase (GUS) gene placed under control of the BN115 promoter. The activity of BN115 promoter was monitored at the transcriptional and translational levels by determining accumulation of BN115 transcripts and by histochemical assay of GUS activity. Cold‐activation of BN115 was strongly inhibited by the membrane fluidizer benzyl alcohol, but mimicked at 25°C by the membrane rigidifier dimethylsulfoxide (DMSO). The cold induction of BN115 was also inhibited by stabilizers of microtubules and actin microfilaments, taxol and jasplakinolide, respectively, but was mimicked at 25°C by microtubule destabilizer oryzalin or colchicine, or by microfilament destabilizer latrunculin B. Gd3+ or ruthenium red prevented the cold activation of BN115, but Ca2+ ionophore A23187 or cyclic ADP‐ribose activated it at 25°C. Inhibitors of tyrosine kinases, protein kinase C and phosphoinositide kinases prevented the cold activation of BN115, but inhibitors of protein phosphatases (PP) 1 and 2 A activated BN115 at 25°C. Constitutively expressed GUS activity in another transgenic line of the same cultivar of B. napus, was not affected by cold or any of the chemical treatments used in the experimentation. Activation of BN115 at 25°C by DMSO, Ca2+ ionophore, cADPR, and by inhibitors of PP1 and 2A was accompanied by an increased freezing tolerance. It was concluded that the cold‐activation of BN115 requires membrane rigidification, cytoskeleton reorganization, Ca2+ influx and action of several types of protein kinases.
Partial complementation of embryo defective mutations: a general strategy to elucidate gene function
Authors: Despres Barbara , Delseny Michel , Devic Martine 
Published: March 2003

SummaryThe EMB 506 gene has been characterised as essential for embryo development. To provide insights into the role of EMB 506, which is hidden by the embryo defective phenotype, the ABI3 promoter was fused to the EMB 506 cDNA. The expression of such a transgene should provide sufficient protein during embryogenesis to ensure normal embryo development in homozygous emb 506 seeds. We show that homozygous emb 506 seedlings, partially complemented with the ABI3::EMB 506 transgene, can be obtained. Most of the rescued emb 506 plants are able to flower and to set normal seeds, but show mild to severe depigmentation of rosette leaves and/or inflorescences. This effect on chloroplast development indicated a putative chloroplast localisation of the EMB 506 protein, which was demonstrated by GFP‐protein fusion. However, EMB 506 cannot be considered as a chloroplast housekeeping protein only, since EMB 506 is not present in all photosynthetic tissues. This study demonstrates the power of this simple strategy, which could be widely applied to other emb mutants and which may reveal similar or additional roles for EMB genes at vegetative stages of the life cycle.
Localization and targeting of the VP14 epoxy‐carotenoid dioxygenase to chloroplast membranes
Authors: Tan Bao‐Cai , Cline Kenneth , McCarty Donald R. 
Published: March 2003

SummaryAbscisic acid (ABA) is a key regulator of seed dormancy and plant responses to environmental challenges. ABA is synthesized via an oxidative cleavage of 9‐cis epoxy‐carotenoids, the first committed and key regulatory step in the ABA biosynthetic pathway. Vp14 of maize encodes an epoxy‐carotenoid dioxygenase that is soluble when expressed in E. coli. An important goal has been to determine how the soluble VP14 protein is targeted to epoxy‐carotenoid substrates that are located in the thylakoid and envelope membranes of chloroplasts and other plastids. Using an in vitro chloroplast import assay, we have shown that VP14 is imported into chloroplasts with cleavage of a short stroma‐targeting domain. The mature VP14 exists in two forms, one which is soluble in stroma and the other bound to thylakoid membranes. Analysis of a series of truncated VP14 mutants mapped the membrane targeting signal to the 160 amino acid N‐terminal sequence. A putative amphipathic α‐helix within this region is essential, but not sufficient, for the membrane targeting. Either deletion of or insertion of helix breaking residues into this region abolished the membrane binding, whereas a chimeric protein carrying just the amphipathic region fused with bacterial glutathione S‐transferase failed to associate with the thylakoid membrane. The membrane‐bound VP14 was partially resistant to chaotropic washes such as 0.1 m Na2CO3 (pH 11.5) and 6 m urea. Unlabelled recombinant VP14 inhibited the tight binding of imported VP14, suggesting that VP14 is associated with specific components of the thylakoid membrane.
The Arabidopsis MALE STERILITY1 (MS1) gene is a transcriptional regulator of male gametogenesis, with homology to the PHD‐finger family of transcription factors
Authors: Wilson Zoe A. , Morroll Shaun M. , Dawson Janet , Swarup Ranjan , Tighe Paddy J. 
Published: March 2003

SummaryWe report here the molecular characterisation of the Arabidopsis MALE STERILITY1 gene, which is a critical sporophytic controlling factor for anther and pollen development. Homozygous ms1 mutants do not produce viable pollen, but are otherwise phenotypically normal. Degeneration of pollen occurs soon after microspore release from the tetrads, at which time the tapetum also appears abnormally vacuolated. The MS1 gene is expressed at low levels in anthers from closed buds, with expression in the tapetum at the stage of microspore release. No expression is seen in open flowers. The deduced MS1 protein sequence shows strong homology to the PHD‐finger motif found in known transcription factors from humans, yeast and higher plants. Six alleles of ms1 have been identified; all result in premature termination of the MS1 protein and loss of the PHD‐finger motif. MS1 is likely to play a key role in regulating transcription during specific stages of male gametogenesis and anther development. As such, MS1 provides a valuable tool for the manipulation of male sterility in higher plants.
Sites and homeostatic control of auxin biosynthesis in Arabidopsis during vegetative growth
Authors: Ljung Karin , Bhalerao Rishikesh P. , Sandberg Göran 
Published: March 2003

SummaryThe distribution and biosynthesis of indole‐3‐acetic acid (IAA) was investigated during early plant development in Arabidopsis. The youngest leaves analysed, less than 0.5 mm in length, contained 250 pg mg−1 of IAA and also exhibited the highest relative capacity to synthesize this hormone. A decrease of nearly one hundred‐fold in IAA content occurred as the young leaves expanded to their full size, and this was accompanied by a clear shift in both pool size and IAA synthesis capacity. The correlation between high IAA content and intense cell division was further verified in tobacco leaves, where a detailed analysis revealed that dividing mesophyll tissue contained ten‐fold higher IAA levels than tissue growing solely by elongation. We demonstrated that all parts of the young Arabidopsis plant can potentially contribute to the auxin needed for growth and development, as not only young leaves, but also all other parts of the plant such as cotyledons, expanding leaves and root tissues have the capacity to synthesize IAA de novo. We also observed that naphthylphthalamic acid (NPA) treatment induced tissue‐dependent feedback inhibition of IAA biosynthesis in expanding leaves and cotyledons, but intriguingly not in young leaves or in the root system. This observation supports the hypothesis that there is a sophisticated tissue‐specific regulatory mechanism for auxin biosynthesis. Finally, a strict requirement for maintaining the pool sizes of IAA was revealed as reductions in leaf expansion followed both decreases and increases in the IAA levels in developing leaves. This indicates that leaves are not only important sources for IAA synthesis, but that normal leaf expansion depends on rigorous control of IAA homeostasis.
Cytochrome b6f mutation specifically affects thermal dissipation of absorbed light energy in Arabidopsis
Authors: Munekage Yuri , Takeda Satomi , Endo Tsuyoshi , Jahns Peter , Hashimoto Takashi , Shikanai Toshiharu 
Published: March 2003

SummaryLight‐induced lumenal acidification controls the efficiency of light harvesting by inducing thermal dissipation of excess absorbed light energy in photosystem II. We isolated an Arabidopsis mutant, pgr1 (proton gradient regulation), entirely lacking thermal dissipation, which was observed as little non‐photochemical quenching of chlorophyll fluorescence. Map‐based cloning showed that pgr1 had a point mutation in petC encoding the Rieske subunit of the cytochrome b6f complex. Although the electron transport rate was not affected at low light intensity, it was significantly restricted at high light intensity in pgr1, indicating that the lumenal acidification was not sufficient to induce thermal dissipation. This view was supported by (i) slow de‐epoxidation of violaXanthin, which is closely related to lumenal acidification, and (ii) reduced 9‐aminoacridine fluorescence quenching. Although lumenal acidification was insufficient to induce thermal dissipation, growth rate was not affected under low light growth conditions in pgr1. These results suggest that thermal dissipation is precisely regulated by lumenal pH to maintain maximum photosynthetic activity. We showed that pgr1 was sensitive to changes in light conditions, demonstrating that maximum activity of the cytochrome b6f complex is indispensable for short‐term acclimation.
Overexpression of chlorophyllide a oxygenase (CAO) enlarges the antenna size of photosystem II in Arabidopsis thaliana
Authors: Tanaka Ryouichi , Koshino Yoshihiro , Sawa Shinichiro , Ishiguro Sumie , Okada Kiyotaka , Tanaka Ayumi 
Published: March 2003

SummaryThe light‐harvesting efficiency of a photosystem is thought to be largely dependent on its photosynthetic antenna size. It has been suggested that antenna size is controlled by the biosynthesis of chlorophyll b. To verify this hypothesis, we overexpressed the enzyme for chlorophyll b biosynthesis, chlorophyllide a oxygenase (CAO), in Arabidopsis thaliana by transforming the plant with cDNA for CAO under the control of the 35S cauliflower mosaic virus promoter. In the early de‐etiolation phase, when the intrinsic CAO expression is very low, the chlorophyll a : b ratio was drastically decreased from 28 to 7.3, indicating that enhancement of chlorophyll b biosynthesis had been successfully achieved. We made the following observations in full‐green rosette leaves of transgenic plants. (1) The chlorophyll a : b ratio was reduced from 2.85 to 2.65. (2) The ratio of the peripheral light‐harvesting complexes (LHCII) to the core antenna complex (CPa) resolved with the green‐gel system increased by 20%. (3) The ratio of the light‐harvesting complex II apoproteins (LHCP) to 47‐kDa chlorophyll a protein (CP47), which was estimated by the results of immunoblotting, increased by 40%. These results indicated that the antenna size increased by at least 10–20% in transgenic plants, suggesting that chlorophyll b biosynthesis controls antenna size. To the best of our knowledge, this is the first report on enlargement of the antenna size by genetic manipulations.
Brassinosteroid functions to protect the translational machinery and heat‐shock protein synthesis following thermal stress
Authors: Dhaubhadel Sangeeta , Browning Karen S. , Gallie Daniel R. , Krishna Priti 
Published: March 2003

SummaryIn addition to their essential role in plant development, brassinosteroids have the ability to protect plants from various environmental stresses. Currently it is not understood how brassinosteroids control plant stress responses at the molecular level. We have begun an investigation into the molecular mechanisms underlying 24‐epibrassinolide (EBR)‐mediated stress resistance. Earlier we found that treatment of Brassica napus seedlings with EBR leads to a significant increase in their basic thermotolerance, and results in higher accumulation of four major classes of heat‐shock proteins (hsps) as compared to untreated seedlings. Surprisingly, previous studies have shown that while hsp levels were significantly higher in treated seedlings during the recovery period, transcripts corresponding to these hsps were present at higher levels in untreated seedlings. To understand mechanisms controlling hsp synthesis in EBR‐treated and untreated seedlings, we studied protein synthesis in vivo as well as in vitro, and assessed the levels of components of the translational machinery in these seedlings. We report here that increased accumulation of hsps in EBR‐treated seedlings results from higher hsp synthesis, even when the mRNA levels are lower than in untreated seedlings, and that several translation initiation and elongation factors are present at significantly higher levels in EBR‐treated seedlings as compared to untreated seedlings. These results suggest that EBR treatment limits the loss of some of the components of the translational apparatus during prolonged heat stress, and increases the level of expression of some of the components of the translational machinery during recovery, which correlates with a more rapid resumption of cellular protein synthesis following heat stress and a higher survival rate.
A role for nitrilase 3 in the regulation of root morphology in sulphur‐starving Arabidopsis thaliana
Authors: Kutz Alexandra , Müller Axel , Hennig Peter , Kaiser Werner M. , Piotrowski Markus , Weiler Elmar W. 
Published: March 2003

Summary Arabidopsis thaliana expresses four nitrilases, three of which (NIT1, NIT2 and NIT3) are able to convert indole‐3‐acetonitrile to indole‐3‐acetic acid (IAA), the plant growth hormone, while the isozyme NIT4 is a β‐cyano‐l‐alanine hydratase/nitrilase. NIT3 promoter activity is marginal in leaves or roots of vegetative plants and undetectable in bolting and flowering plants, but its level increases strongly when plants experience sulphur deprivation. No other nitrilase genes respond to sulphur supply/deficiency. Neither N‐ nor P‐deprivation cause detectable changes in NIT3 promoter activity. In transgenic plants expressing uidA under the control of the NIT3 promoter (NIT3p::uidA), sulphate deprivation leads to the appearance of β‐glucuronidase activity in shoots and particularly in roots, most strongly in the conductive tissues and lateral root primordia. Deletion analysis allowed localization of the sulphur‐responsive element to a 317 bp segment of the NIT3 promoter encompassing nt −2151 to −1834 upstream of the transcriptional start point. Both nitrilase polypeptide and nitrilase activity were also induced by sulphur starvation. NIT3 promoter activity was strongly induced by O‐acetylserine, suggesting that, as is the case with enzymes of sulphate assimilation, sulphate deficiency may be communicated to NIT3 via an increase in the level of the cysteine precursor, O‐acetylserine. During sulphur deprivation, a preferential depletion of the pool of the indole‐3‐acetonitrile precursor glucobrassicin compared with that of total glucosinolates was noticed. In the absence of an external sulphate supply, plants developed longer roots with a higher number of lateral roots. The increased growth of the root system occurred at the expense of shoot growth which was retarded under conditions of sulphur starvation. Taken together, these results suggest that a regulatory loop appears to exist by which sulphate deficiency, through an increase in glucobrassicin turnover and nitrilase 3 accumulation, initiates the production of extra auxin leading to increased root growth and branching, thus allowing the root system to penetrate new areas of soil effectively to gain access to fresh supplies of sulphur.
The IRE gene encodes a protein kinase homologue and modulates root hair growth in Arabidopsis
Authors: Oyama Tokitaka , Shimura Yoshiro , Okada Kiyotaka 
Published: March 2003

SummaryWe identified an Arabidopsis mutant, incomplete root hair elongation (ire), whose root hairs are about 40% shorter than the wild type owing to the early cessation of their growth. In contrast, the hy5 mutant has root hairs about twice as long as the wild type, possibly because of delayed cessation of growth. Thus IRE and HY5 are likely to modify the duration of growth of root hairs. We cloned the IRE locus using the T‐DNA tagged line. The IRE locus encodes a protein that includes a serine/threonine protein kinase domain. The primary structure of this kinase domain shows significant similarity to a group of protein kinases among various eukaryotes. In the Arabidopsis genome there are at least three genes that are closely related to IRE. The IRE transcript was detected in every organ examined. However, the IRE promoter–GUS fusion gene was strongly expressed in the elongating root hair cells, suggesting the cell‐autonomous function of IRE in root hairs. GUS activity was also detected in pollen grains, which develop by tip growth, suggesting that IRE has a common role in the tip growth of plant cells.
Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N‐mediated resistance to tobacco mosaic virus
Authors: Liu Yule , Schiff Michael , Marathe Rajendra , Dinesh‐Kumar S. P. 
Published: March 2003

SummaryThe tobacco N gene confers resistance to tobacco mosaic virus (TMV) and encodes a Toll‐interleukin‐1 receptor/nucleotide binding site/leucine‐rich repeat (TIR‐NBS‐LRR) class protein. We have developed and used a tobacco rattle virus (TRV) based virus induced gene silencing (VIGS) system to investigate the role of tobacco candidate genes in the N‐mediated signalling pathway. To accomplish this we generated transgenic Nicotiana benthamiana containing the tobacco N gene. The transgenic lines exhibit hypersensitive response (HR) to TMV and restrict virus spread to the inoculated site. This demonstrates that the tobacco N gene can confer resistance to TMV in heterologous N. benthamiana. We have used this line to study the role of tobacco Rar1‐, EDS1‐, and NPR1/NIM1‐ like genes in N‐mediated resistance to TMV using a TRV based VIGS approach. Our VIGS analysis suggests that these genes are required for N function. EDS1‐like gene requirement for the N function suggests that EDS1 could be a common component of bacterial, fungal and viral resistance signalling mediated by the TIR‐NBS‐LRR class of resistance proteins. Requirement of Rar1‐ like gene for N‐mediated resistance to TMV and some powdery mildew resistance genes in barley provide the first example of converging points in the disease resistance signalling pathways mediated by TIR‐NBS‐LRR and CC‐NBS‐LRR proteins. The TRV based VIGS approach as described here to study N‐mediated resistance signalling will be useful for the analysis of not only disease resistance signalling pathways but also of other signalling pathways in genetically intractable plant systems.
On the origin of class B floral homeotic genes: functional substitution and dominant inhibition in Arabidopsis by expression of an orthologue from the gymnosperm Gnetum
Authors: Winter Kai‐Uwe , Saedler Heinz , Theißen Günter 
Published: March 2003

SummaryClass B floral homeotic genes are involved in specifying stamen and petal identity in angiosperms (flowering plants). Here we report that gymnosperms, the closest relatives of the angiosperms, contain at least two different clades representing putative orthologues of class B genes, termed GGM2‐like and DAL12‐like genes. To obtain information about the functional conservation of the class B genes in seed plants, the representative of one of these clades from Gnetum, termed GGM2, was expressed under the control of the CaMV 35S promoter in Arabidopsis wild‐type plants and in different class B mutants. In wild‐type plants and in a conditional mutant grown at a permissive temperature, gain‐of‐function phenotypes were obtained in whorls 1 and 4, where class B genes are usually not expressed. In contrast, loss‐of‐function phenotypes were observed in whorls 2 and 3, where class B genes are expressed. In different class B gene null mutants of Arabidopsis, and in the conditional B mutant grown at the non‐permissive temperature, a partial complementation of the mutant phenotype was obtained. In situ hybridization studies and class B gene promoter test fusion experiments demonstrated that the gain‐of‐function phenotypes are not due to an upregulation of the endogenous B genes from Arabidopsis, and hence probably involve interactions between GGM2 protein homodimers and class B protein target genes other than the Arabidopsis class B genes itself. To our knowledge, this is the first time that partial complementation of a homeotic mutant by an orthologous gene from a distantly related species has been reported. These data suggest that GGM2 has a function in the gymnosperm Gnetum which is related to that of class B floral organ identity genes of angiosperms. That function may be in the specification of male reproductive organ identity, and in distinguishing male from female reproductive organs.
Light regulated modulation of Z‐box containing promoters by photoreceptors and downstream regulatory components, COP1 and HY5, in Arabidopsis
Authors: Yadav Vandana , Kundu Snehangshu , Chattopadhyay Debasis , Negi Prem , Wei Ning , Deng Xing‐Wang , Chattopadhyay Sudip 
Published: March 2003

SummaryThe Z‐box is one of the light‐responsive elements (LREs) found in the promoters of light inducible genes. We have studied the light responsive characteristics of Z‐box containing synthetic as well as native promoters. We show that promoters with Z‐box as a single LRE or paired with another LRE can respond to a broad spectrum of light. The response is primarily mediated by phyA, phyB and CRY1 photoreceptors at their respective wavelengths of light. We have demonstrated that CAB1 and Z‐GATA containing promoters are down‐regulated in hy5 mutants in the light. On the other hand, a promoter with Z‐box alone is down‐regulated in hy5 mutants both in dark and in light conditions, suggesting involvement of a similar regulatory system in the regulation of the promoter in two distinct developmental pathways: skotomorphogenesis and photomorphogenesis. Furthermore, similar to the CAB1 promoter, a Z‐GATA containing promoter is derepressed in cop1 mutants in the dark. DNA–protein interaction studies reveal the presence of a DNA‐binding activity that is specific to Z‐box. These results provide insights into the regulation of the Z‐box LRE mediated by various light signaling components.