Chimeric proteins comprising connexins 26, 32, and 43 and aequorin, a chemiluminescent calcium indicator, were made by fusing the amino terminus of aequorin to the carboxyl terminus of connexins. The ...retention of function by the chimeric partners was investigated. Connexin 32-aequorin and connexin 43-aequorin retained chemiluminescent activity whereas that of connexin 26-aequorin was negligible. Immunofluorescent staining of COS-7 cells expressing the chimerae showed they were targeted to the plasma membrane. Gap junction intercellular channel formation by the chimerae alone and in combination with wild-type connexins was investigated. Stable HeLa cells expressing connexin 43-aequorin were functional, as demonstrated by Lucifer yellow transfer. Pairs of Xenopus oocytes expressing connexin 43-aequorin were electrophysiologically coupled, but those expressing chimeric connexin 26 or 32 showed no detectable levels of coupling. The formation of heteromeric channels constructed of chimeric connexin 32 or connexin 43 and the respective wild-type connexins was inferred from the novel voltage gating properties of the junctional conductance. The results show that the preservation of function by each partner of the chimeric protein is dictated mainly by the nature of the connexin, especially the length of the cytoplasmic carboxyl-terminal domain. The aequorin partner of the connexin 43 chimera reported calcium levels in COS-7 cells in at least two different calcium environments.
Flower and fruit development in tomato (Lycopersicon esculentum Mill.) were severely affected when plants were grown at low temperatures, displaying homeotic and meristic transformations and ...alterations in the fusion pattern of the organs. Most of these homeotic transformations modified the identity of stamens and carpels, giving rise to intermediate organs. Complete homeotic transformations were rarely found and always affected organs of the reproductive whorls. Meristic transformations were also commonly observed in the reproductive whorls, which developed with an excessive number of organs. Scanning electron microscopy revealed that meristic transformations take place very early in the development of the flower and are related to a significant increase in the floral meristem size. However, homeotic transformations should occur later during the development of the organ primordia. Steadystate levels of transcripts corresponding to tomato MADS-box genes TM4, TM5, TM6, and TAG1 were greatly increased by low temperatures and could be related to these flower abnormalities. Moreover, in situ hybridization analyses showed that low temperatures also altered the stage-specific expression of TM4
Gap junctions composed of connexin-45 (Cx45) homologs from four species, zebrafish, chicken, mouse, and human, were expressed in pairs of Xenopus oocytes. The macroscopic conductance (gj) of all Cx45 ...junctions was modulated by transjunctional voltage (Vj) and by the inside-outside voltage (Vm), and the modulation was species specific. Although their gating characteristics varied in voltage sensitivity and kinetics, the four Cx45 junctions shared 1) maximum conductance at Vj = 0 and symmetrical gj reduction in response to positive and negative Vj of low amplitude, with little residual conductance; and 2) gj increases in response to simultaneous depolarization of the paired cells. The formation of hybrid channels, comprising Cx45 hemichannels from different species, allowed us to infer that two separate gates exist, one in each hemichannel, and that each Cx45 hemichannel is closed by the negativity of Vj on its cytoplasmic side. Interestingly, the Vm dependence of hybrid channels also suggests the presence of two gates in series, one Vm gate in each hemichannel. Thus the Vj and Vm dependence provides evidence that two independent voltage gates in each Cx45 hemichannel exist, reacting through specific voltage sensors and operating by different mechanisms, properties that have evolved divergently among species.
A tomato (Lycopersicon esculentum Mill.) monogenic semidominant mutation, stamenless (sl), which results in homeotic conversions in two adjacent floral whorls was studied. When grown at standard ...temperature, flowers of sl/sl plants showed sepaloid petals in the second whorl and strong transformation of stamens to carpels in whorl three. These transformed carpels were fused with each other and with the genuine carpels in the fourth whorl to form a unique gynoecium. The mutation is semidominant since heterozygous plants showed a phenotype intermediate between that of the wild type (WT) and that of homozygous mutant plants, with nearly WT petals but with feminized stamens bearing naked ovules on the base of their adaxial face. The initiation and position of organ primordia in sl/sl flowers were not altered when compared with WT primordia although development of organ primordia in the second and third whorls deviated from WT at an early stage as observed by scanning electron microscopy. The mutant phenotype is temperature sensitive and when sl/sl plants were cultured at low temperature, the morphology of some flowers resembled that of the WT. This reversion of the mutant phenotype is also induced by treatment of young sl/sl plants with gibberellic acid, providing evidence that gibberellin synthesis or sensitivity could mediate the effect of low temperature on the mutant phenotype. Southern blot analyses using a Deficiens-homologous gene from Solanum tuberosum as a probe showed a restriction-fragment-length polymorphism (RFLP) linked to the sl mutation. This result indicates that the mutation affects a Deficiens-like gene that controls the identity of petals and stamens.
Chimeric proteins comprising connexins 26, 32, and 43 and aequorin, a chemiluminescent calcium indicator, were made by fusing the amino terminus of aequorin to the carboxyl terminus of connexins. The ...retention of function by the chimeric partners was investigated. Connexin 32-aequorin and connexin 43-aequorin retained chemiluminescent activity whereas that of connexin 26-aequorin was negligible. Immunofluorescent staining of COS-7 cells expressing the chimerae showed they were targeted to the plasma membrane. Gap junction intercellular channel formation by the chimerae alone and in combination with wild-type connexins was investigated. Stable HeLa cells expressing connexin 43-aequorin were functional, as demonstrated by Lucifer yellow transfer. Paris of Xenopus oocytes expressing connexin 43-aequorin were electrophysiologically coupled, but those expressing chimeric connexin 26 or 32 showed no detectable levels of coupling. The formation of heteromeric channels constructed of chimeric connexin 32 or connexin 43 and the respective wild-type connexins was inferred from the novel voltage gating properties of the junctional conductance. The results show that the preservation of function by each partner of the chimeric protein is dictated mainly by the nature of the connexin, especially the length of the cytoplasmic carboxyl-terminal domain. The aequorin partner of the connexin 43 chimera reported calcium levels in COS-7 cells in at least two different calcium environments.
Nuclear recessive mutations at the chloroplast mutator (CHM) locus of Arabidopsis produce a variegated phenotype that is inherited in a non-Mendelian fashion. Molecular analysis of the cytoplasmic ...genomes of variegated plants from two independent chm mutant lines, using specific chloroplast and mitochondrial probes, showed that the chm mutations reproducibly induce the appearance of specific new restriction fragments in the mitochondrial genome. The presence of these restriction fragments cosegregated with the variegated phenotype in the progeny of crosses between mutant and wild-type plants. Sequence analysis of one of the new restriction fragments found in the variegated plants suggested that it was the product of a rearrangement event involving regions of the mitochondrial genome. Thus, it appears that the CHM locus may encode a protein involved in the control of specific mitochondrial DNA reorganization events
We have isolated two Rare Cold-Inducible (RCI1 and RCI2) cDNAs by screening a cDNA library prepared from cold-acclimated etiolated seedlings of Arabidopsis thaliana with a subtracted probe. RNA-blot ...hybridizations revealed that the expression of both RCI1 and RCI2 genes is induced by low temperature independently of the plant organ or the developmental stage considered. However, RCI1 mRNA accumulates faster and at higher levels than the RCI2 one indicating that these genes have differential responsiveness to cold stress. Additionally, when plants are returned to room temperature, RCI1 mRNA decreases faster than RCI2. In contrast to most of the cold-inducible plant genes characterized, the expression of RCI1 and RCI2 is not induced by ABA or water stress. The nucleotide sequences of RCI1 and RCI2 cDNAs predict two acidic polypeptides of 255 and 251 amino acids with molecular weights of 29 and 28 kDa respectively. The alignment of these polypeptides indicates that they have 181 identical amino acids suggesting that the corresponding genes have a common origin. Sequence comparisons reveal no similarities between the RCI proteins and any other cold-regulated plant protein so far described. Instead, they demonstrate that the RCI proteins are highly homologous to a family of proteins, known as 14-3-3 proteins, which are thought to be involved in the regulation of multifunctional protein kinases.
Low temperature treatment of dark-grown seedlings of Arabidopsis thaliana results in a rapid increase in the amount of mRNAs encoding for the major polypeptides of the light-harvesting complex of ...photosystem II (Lhcb1 genes). This increase is transient and seems to be due mainly to the accumulation of Lhcb1*3 transcripts, indicating that low temperature differentially regulates the expression of the Arabidopsis Lhcb1 gene family in the dark. A 1.34 kb fragment of the Lhcb1*3 promoter is sufficient to confer low temperature regulation to a reporter gene in transgenic Arabidopsis etiolated seedlings, suggesting that the regulation is occurring at the transcriptional level. The cold-induced accumulation of Lhcb1*3 mRNA is not part of a general response to stressful conditions since no accumulation is detected in response to water stress, anaerobiosis or salt stress. The amount of Lhcb1*3 mRNA decrease in response to exogenous abscisic acid (ABA) suggesting that this phytohormone acts as a negative regulator. Moreover, the accumulation of Lhcb1*3 mRNAs in cold-treated ABA deficient etiolated seedlings is higher than that of wild-type and ABA insensitive etiolated seedlings, indicating that low temperature regulation of Lhcb1*3 is not mediated by ABA.
PDF
