bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differential regulation of the NO3– and NH4+ transporter genes AtNrt2.1 and AtAmt1.1 in Arabidopsis: relation with long‐distance and local controls by N status of the plant
Authors: Gansel Xavier , Muños Stéphane , Tillard Pascal , Gojon Alain 
Published: March 2003

SummaryRegulation of root N uptake by whole‐plant signalling of N status was investigated at the molecular level in Arabidopsis thaliana plants through expression analysis of AtNrt2.1 and AtAmt1.1. These two genes encode starvation‐induced high‐affinity NO3– and NH4+ transporters, respectively. Split‐root experiments indicate that AtNrt2.1 expression is controlled by shoot‐to‐root signals of N demand. Together with 15NO3– influx, the steady‐state transcript level of this gene is increased in NO3–‐fed roots in response to N deprivation of another portion of the root system. Thus AtNrt2.1 is the first identified molecular target of the long‐distance signalling informing the roots of the whole plant's N status. In contrast, AtAmt1.1 expression is predominantly dependent on the local N status of the roots, as it is mostly stimulated in the portion of the root system directly experiencing N starvation. The same behaviour was found for NH4+ influx, suggesting that the NH4+ uptake system is much less efficient than the NO3– uptake system, to compensate for a spatial restriction of N availability. Other major differences were found between the regulations of AtNrt2.1 and AtAmt1.1 expression. AtNrt2.1 is strongly upregulated by moderate level of N limitation, while AtAmt1.1 transcript level is markedly increased only under severe N deficiency. Unlike AtNrt2.1, AtAmt1.1 expression is not stimulated in a nitrate reductase‐deficient mutant after transfer to NO3– as sole N source, indicating that NO3–per se acts as a signal repressing transcription of AtAmt1.1. These results reveal two fundamentally different types of mechanism involved in the feedback regulation of root N acquisition by the N status of the plant.
The shoot apical meristem restores its symplasmic organization during chilling‐induced release from dormancy
Authors: Rinne Päivi L. H. , Kaikuranta Päivi M. , Van Der Schoot Christiaan 
Published: March 2003

SummaryThe shoot apex of overwintering perennials ceases its morphogenetic activity at the end of the growing season and transforms into a bud which is dormant and freezing‐tolerant. In birch (Betula pubescens) these events are triggered by short photoperiod, and involve the production of 1,3‐β‐d‐glucan containing sphincters on the plasmodesmata. As a result, all symplasmic pathways shut down. Here we show that breakage of bud dormancy by chilling involves restoration of the symplasmic organization of the meristem. This restoration is likely to be mediated by 1,3‐β‐d‐glucanase, which was present in small spherosome‐like vacuoles that arose de novo during dormancy induction. During chilling these vacuoles were displaced from the bulk cytoplasm to the cortical cytoplasm where they became aligned with the plasma membrane, often associated with plasmodesmata. At this stage the enzyme also appeared outside the vacuoles. During chilling, 1,3‐β‐d‐glucan disappeared from the plasmodesmal channels and wall sleeves, and the plasmodesmata regained the capacity for cell–cell transport, as demonstrated by microinjection of Lucifer Yellow CH and Fluorescein‐tagged gibberellic acid. Collectively, the present experiments demonstrate that restoration of the symplasmic organization of the meristem is indispensable for the release of buds from dormancy and the assumption of a proliferation‐competent state, and implicate 1,3‐β‐d‐glucanase action at the plasmodesmata. Based on these findings we propose a model for ‘dormancy cycling’ which depicts the meristem as passing through three sequential states of cellular communication with characteristic sensitivities to distinct environmental cues.
Reconstruction of reproductive diversity in Hypericum perforatum L. opens novel strategies to manage apomixis
Authors: Matzk Fritz , Meister Armin , Brutovská Renata , Schubert Ingo 
Published: March 2003

SummaryThe mode of reproduction was characterized for 113 accessions of the tetraploid facultative apomictic species Hypericum perforatum using bulked or single mature seeds in the flow cytometric seed screen (FCSS). This screen discriminates several processes of sexual or asexual reproduction based on DNA contents of embryo and endosperm nuclei. Seed formation in H. perforatum proved to be highly polymorphic. Eleven different routes of reproduction were determined. For the first time, individual seeds were identified that originated from two embryo sacs: the endosperm from an aposporous and the embryo from the legitimate meiotic embryo sac. Moreover, diploid plants were discovered, which apparently reproduce by a hitherto unknown route of seed formation, that is chromosome doubling within aposporous initial cells followed by double fertilization. Although most plants were tetraploid and facultative sexual/apomictic, diploid obligate sexuals and tetraploid obligate apomicts could be selected. Additionally, genotypes were detected which at a high frequency produced embryos either from reduced parthenogenetic or unreduced fertilized egg cells. The endosperm developed most frequently after fertilization of the central cell in aposporous embryo sacs (pseudogamy) but in few cases also autonomously. The genetic control of apomixis appears to be complex in H. perforatum. Basic material was developed for breeding H. perforatum, and strategies are suggested for elucidation of inheritance as well as evolution of apomixis and for molecular approaches of apomixis engineering.
Characterization of the ethanol‐inducible alc gene‐expression system in Arabidopsis thaliana
Authors: Roslan Hairul A. , Salter Michael G. , Wood Chris D. , White Michael R. H. , Croft Kevan P. , Robson Frances , Coupland George , Doonan John , Laufs Patrick , Tomsett A. Brian , Caddick Mark X. 
Published: March 2003

SummaryControlled expression of transgenes in plants is key to the characterization of gene function and the regulated manipulation of growth and development. The alc gene‐expression system, derived from the filamentous fungus Aspergillus nidulans, has previously been used successfully in both tobacco and potato, and has potential for use in agriculture. Its value to fundamental research is largely dependent on its utility in Arabidopsis thaliana. We have undertaken a detailed function analysis of the alc regulon in A. thaliana. By linking the alcA promoter to β‐glucuronidase (GUS), luciferase (LUC) and green fluorescent protein (GFP) genes, we demonstrate that alcR‐mediated expression occurs throughout the plant in a highly responsive manner. Induction occurs within one hour and is dose‐dependent, with negligible activity in the absence of the exogenous inducer for soil‐grown plants. Direct application of ethanol or exposure of whole plants to ethanol vapour are equally effective means of induction. Maximal expression using soil‐grown plants occurred after 5 days of induction. In the majority of transgenics, expression is tightly regulated and reversible. We describe optimal strategies for utilizing the alc system in A. thaliana.
Increasing maize seed methionine by mRNA stability
Authors: Lai Jinsheng , Messing Joachim 
Published: March 2003

SummaryThe amino acid methionine is a common protein building block that is also important in other cellular processes. Plants, unlike animals, synthesize methionine de novo and are thus a dietary source of this nutrient. A new approach for using maize as a source of nutrient methionine is described. Maize seeds, a major component of animal feeds, have variable levels of protein‐bound methionine. This variability is a result of post‐transcriptional regulation of the Dzs10 gene, which encodes a seed‐specific high‐methionine storage protein. Here we eliminate methionine variability by identifying and replacing the cis‐acting site for Dzs10 regulation using transgenic seeds. Interestingly, two different mechanisms affect mRNA accumulation, one dependent on and the other independent of the untranslated regions (UTRs) of Dzs10 RNA. Accumulation of chimeric Dzs10 mRNA was not reduced in hybrid crosses and was uncoupled from genomic imprinting by Dzr1, a regulator of Dzs10. Uniform high levels of Dzs10 protein were maintained over five backcross generations of the transgene. The increased level of methionine in these transgenic seeds allowed the formulation of a useful animal feed ration without the addition of synthetic methionine.
Recognition specificity of self‐incompatibility maintained after the divergence of Brassica oleracea and Brassica rapa
Authors: Kimura Ryo , Sato Keiichi , Fujimoto Ryo , Nishio Takeshi 
Published: March 2003

SummaryThe determinants of recognition specificity of self‐incompatibility in Brassica are SRK in the stigma and SP11/SCR in the pollen, respectively. In the pair of S haplotypes BrS46 (S46 in B. rapa) and BoS7 (S7 in B. oleracea), which have highly similar SRK alleles, the SP11 alleles were found to be similar, with 96.1% identity in the deduced amino acid sequence. Two other pairs of S haplotypes, BrS47 and BoS12, and BrS8 and BoS32, having highly similar SRK and SP11 alleles between the two species were also found. The haplotypes in each pair are considered to have been derived from a single S haplotype in the ancestral species. The allotetraploid produced by interspecific hybridization between homozygotes of BrS46 and BoS15 showed incompatibility with a BoS7 homozygote and compatibility with other B. oleracea S haplotypes in reciprocal crossings. This result indicates that BrS46 and BoS7 have maintained the same recognition specificity after the divergence of the two species and that amino acid substitutions found in such cases in both SRK alleles and SP11 alleles do not alter the recognition specificity. DNA blot analysis of SRK, SP11, SLG and other S‐locus genes showed different DNA fragment sizes between the interspecific pairs of S haplotypes. A much lower level of sequence similarity was observed outside the genes of SRK and SP11 between BrS46 and BoS7. These results suggest that the DNA sequences of the regions intervening between the S‐locus genes were diversified after or at the time of speciation. This is the first report demonstrating the presence of common S haplotypes in different plant species and presenting definite evidence of the trans‐specific evolution of self‐incompatibility genes.
A role for arabinogalactan proteins in gibberellin‐induced α‐amylase production in barley aleurone cells
Authors: Suzuki Yoshihito , Kitagawa Mamiko , Knox J. Paul , Yamaguchi Isomaro 
Published: March 2003

SummaryArabinogalactan proteins (AGPs) are plant proteoglycans that have been implicated in plant growth and development. The possible involvement of AGPs in the action of gibberellin (GA), a class of plant hormones, was examined by applying β‐glucosyl Yariv reagent (β‐Glc)3Y, a synthetic phenyl glycoside that interacts selectively with AGPs, to barley aleurone protoplasts. Gibberellin induces transcription and secretion of α‐amylases in the protoplasts. Induction of α‐amylase was clearly inhibited by (β‐Glc)3Y but not by (α‐Gal)3Y, a negative control of the Yariv reagent that does not interact with AGPs. Transfection analysis, using an α‐amylase promoter–GUS fusion gene in the protoplasts, indicated that the transcriptional activation of the α‐amylase promoter was inhibited specifically by (β‐Glc)3Y. These observations are the first indication of an involvement of AGPs in a plant hormone function. The inhibitory effect of (β‐Glc)3Y was not observed when aleurone layers or half‐seed grains were used. This result, together with the fact that protoplasts do not have cell walls, suggests that the AGPs that function in α‐amylase induction reside at the plasma membrane. An aleurone‐specific AGP was detected by reversed‐phase HPLC, and supported the idea that an AGP may play an important role in aleurone‐specific events. The possible mechanism of AGP function in gibberellin‐induced α‐amylase production is discussed.
EL5, a rice N‐acetylchitooligosaccharide elicitor‐responsive RING‐H2 finger protein, is a ubiquitin ligase which functions in vitro in co‐operation with an elicitor‐responsive ubiquitin‐conjugating enzyme, OsUBC5b
Authors: Takai Ryota , Matsuda Noriyuki , Nakano Akihiko , Hasegawa Koji , Akimoto Chiharu , Shibuya Naoto , Minami Eiichi 
Published: March 2003

Summary EL5, a rice gene responsive to N‐acetylchitooligosaccharide elicitor, encodes a RING‐H2 finger protein with structural features common to the plant‐specific ATL family. We show that the fusion protein of EL5 with maltose binding protein (MBP) was polyubiquitinated by incubation with ubiquitin, ubiquitin‐activating enzyme (E1), and the Ubc4/5 subfamily of the ubiquitin‐conjugating enzyme (E2). EL5 possesses the activity to catalyse the transfer of ubiquitin to the MBP moiety, and the RING‐H2 finger motif of EL5 is necessary for this activity. Thus, we concluded that EL5 represents a ubiquitin ligase (E3). We also show that two rice E2s (OsUBC5a, OsUBC5b) of the Ubc4/5 subfamily function as E2 which catalyses EL5‐mediated ubiquitination, and OsUBC5b was induced by elicitor, as well as EL5. These results strongly suggest that EL5 and OsUBC5b have roles in plant defense response through the turnover of protein(s) via the ubiquitin/proteasome system.
Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast
Authors: Franklin Scott , Ngo Binh , Efuet Ekem , Mayfield Stephen P. 
Published: March 2003

SummaryReporter genes have been successfully used in chloroplasts of higher plants, and high levels of recombinant protein expression have been reported. Reporter genes have also been used in the chloroplast of Chlamydomonas reinhardtii, but in most cases the amounts of protein produced appeared to be very low. We hypothesized that the inability to achieve high levels of recombinant protein expression in the C. reinhardtii chloroplast was due to the codon bias seen in the C. reinhardtii chloroplast genome. To test this hypothesis, we synthesized a gene encoding green fluorescent protein (GFP) de novo, optimizing its codon usage to reflect that of major C. reinhardtii chloroplast‐encoded proteins. We monitored the accumulation of GFP in C. reinhardtii chloroplasts transformed with the codon‐optimized GFP cassette (GFPct), under the control of the C. reinhardtii rbcL 5′‐ and 3′‐UTRs. We compared this expression with the accumulation of GFP in C. reinhardtii transformed with a non‐optimized GFP cassette (GFPncb), also under the control of the rbcL 5′‐ and 3′‐UTRs. We demonstrate that C. reinhardtii chloroplasts transformed with the GFPct cassette accumulate ≈80‐fold more GFP than GFPncb‐transformed strains. We further demonstrate that expression from the GFPct cassette, under control of the rbcL 5′‐ and 3′‐UTRs, is sufficiently robust to report differences in protein synthesis based on subtle changes in environmental conditions, showing the utility of the GFPct gene as a reporter of C. reinhardtii chloroplast gene expression.
A dominant nuclear mutation in Chlamydomonas identifies a factor controlling chloroplast mRNA stability by acting on the coding region of the atpA transcript
Authors: Drapier Dominique , Girard‐Bascou Jacqueline , Stern David B. , Wollman Francis‐André 
Published: March 2003

SummaryWe have characterized a nuclear mutation, mda1‐ncc1, that affects mRNA stability for the atpA gene cluster in the chloroplast of Chlamydomonas. Unlike all nuclear mutations altering chloroplast gene expression described to date, mda1‐ncc1 is a dominant mutation that still allows accumulation of detectable amounts of atpA mRNAs. At variance with the subset of these mutations that affect mRNA stability through the 5′ UTR of a single chloroplast transcript, the mutated version of MDA1 acts on the coding region of the atpA message. We discuss the action of MDA1 in relation to the unusual pattern of expression of atpA that associates particularly short lived‐transcripts with a very high translational efficiency.
Constitutive gain‐of‐function mutants in a nucleotide binding site–leucine rich repeat protein encoded at the Rx locus of potato
Authors: Bendahmane Abdelhafid , Farnham Garry , Moffett Peter , Baulcombe David C. 
Published: March 2003

Summary Rx in potato encodes a protein with a nucleotide binding site (NBS) and leucine‐rich repeats (LRR) that confers resistance against Potato virus X. The NBS and LRR domains in Rx are present in many disease resistance proteins in plants and in regulators of apoptosis in animals. To investigate structure‐function relationships of NBS‐LRR proteins we exploited the potential of Rx to mediate a cell death response. With wild‐type Rx cell death is elicited only in the presence of the viral coat protein. However, following random mutagenesis of Rx, we identified mutants in which cell death is activated in the absence of viral coat protein. Out of 2500 Rx clones tested there were seven constitutive gain‐of‐function mutants carrying eight independent mutations. The mutations encoded changes in the LRR or in conserved RNBS‐D and MHD motifs of the NBS. Based on these findings we propose that there are inhibitory domains in the NBS and LRR. The constitutive gain‐of‐function phenotypes would be due to deletion or modification of these inhibitory domains. However activation of Rx is not simply release of negative regulation by the LRR and adjacent sequence because deleted forms of Rx that lack constitutive gain of function mutations are not active unless the protein is overexpressed.
Micrografting techniques for testing long‐distance signalling in Arabidopsis
Authors: Turnbull Colin G. N. , Booker Jon P. , Leyser H. M. Ottoline 
Published: March 2003

SummaryGrafting in species other than Arabidopsis has generated persuasive evidence for long‐distance signals involved in many plant processes, including regulation of flowering time and shoot branching. Hitherto, such approaches in Arabidopsis have been hampered by the lack of suitable grafting techniques. Here, a range of micrografting methods for young Arabidopsis seedlings are described. The simplest configuration was a single‐hypocotyl graft, constructed with or without a supporting collar, allowing tests of root–shoot communication. More complex two‐shoot grafts were also constructed, enabling tests of shoot–shoot communication. Integrity of grafts and absence of adventitious roots on scions were assessed using plants constitutively expressing a GUS gene as one graft partner. Using the max1 (more axillary growth) and max3 increased branching mutants, it was shown that a wild‐type (WT) rootstock was able to inhibit rosette branching of mutant shoots. In two‐shoot grafts with max1 and WT shoots on a max1 rootstock, the mutant shoot branched profusely, but the WT one did not. In two‐shoot grafts with max1 and WT shoots on a WT rootstock, neither shoot exhibited increased branching. The results mirror those previously demonstrated in equivalent grafting experiments with the ramosus mutants in pea, and are consistent with the concept that a branching signal is capable of moving from root to shoot, but not from shoot to shoot. These grafting procedures will be valuable for revealing genes associated with many other long‐distance signalling pathways, including flowering, systemic resistance and abiotic stress responses.
The role of double‐strand break‐induced allelic homologous recombination in somatic plant cells
Authors: Gisler Brigitte , Salomon Siegfried , Puchta Holger 
Published: March 2003

SummaryDuring meiosis, homologous recombination occurs between allelic sequences. To evaluate the biological significance of such a pathway in somatic cells, we used transgenic tobacco plants with a restriction site for the rare cutting endonuclease I‐SceI within a negative selectable marker gene. These plants were crossed with two tobacco lines containing, in allelic position, either a deletion or an insertion within the marker gene that rendered both marker gene and restriction site inactive. After the double‐strand break induction, we selected for repair events resulting in a loss of marker gene function. This loss was mostly due to deletions. We were also able to detect double strand break‐induced allelic recombination in which the break was repaired by a faithful copying process from the homologue carrying the shortened transgene. The estimated frequency indicates that homologous recombination in somatic cells between allelic sites appears to occur at the same order of magnitude as between ectopic sites, and is thus far too infrequent to act as major repair pathway. As somatic changes can be transferred to the germ line, the prevalence of intrachromatid rearrangements over allelic recombination might be an indirect prerequisite for the enhanced genome plasticity postulated for plants.
Expression of Arabidopsis LINEs from two promoters
Authors: Ohta Yoshizu , Noma Kenichi , Tsuchimoto Suguru , Ohtsubo Eiichi , Ohtsubo Hisako 
Published: March 2003

SummaryMost Arabidopsis long interspersed elements (LINEs, called ATLNs) have two open reading frames, orf1 and orf2. In the 5′ untranslated regions (UTRs) located upstream of orf1, the most proximal segments of tens of base pairs long are not homologous even in two ATLN members with almost identical sequences. In this study, we first show that RT‐PCR products from ATLN39, a member of ATLN, can be detected only in total RNA from the hypomethylation mutant ddm1 or from suspension‐cultured cells treated with a DNA methylation inhibitor 5‐azacytidine, indicating that the expression of ATLN39 is negatively regulated by DNA methylation. We then show that orf1 fused in frame with the luciferase (luc) gene is expressed in suspension‐cultured cells of A. thaliana when the 5′ UTR is present in the region upstream of orf1. Analysis of deletion in the 5′ UTR revealed that the 5′ UTR has two promoters, designated here as P1 and P2. Analysis of transcripts by 5′ RACE showed that their 5′ ends were located at sites immediately upstream of the P1 region or at sites downstream of the P2 region. This observation and the fact that the P1 region contains no TATA sequence indicate that P1 is an internal promoter that initiates transcription from sites upstream of the promoter. A sequence containing GGCGA with a CpG methylatable site is conserved in the P1 regions in members closely related to ATLN39. The P2 region, however, contains the TATA sequence as well as another sequence with a CpG site. The TATA sequence is conserved in members closely related to ATLN39 but not in the other ATLN members, suggesting that P2 is the promoter uniquely present in the ATLN39‐related members. Transcripts from promoter P1 can be used as templates to give new copies proficient in retroposition, but those from promoter P2 cannot because of the lack of the proximal half region of the 5′ UTR sequence. Transcripts from promoter P2, as well as those from promoter P1 can, however, be used for the production of a sufficient amount of proteins for retroposition. Only a short sequence of the non‐homologous region is present at the 5′ ends of transcripts from promoter P1, thus suggesting that the non‐homologous regions seen in the most proximal regions in ATLN elements are not generated in transcription.