We have isolated four members of the Arabidopsis cyclophilin (CyP) gene family, designated ROC1 to ROC4 (rotamase CyP). Deduced peptides of ROC1, 2 and 3 are 75% to 91% identical to Brassica napus cytosolic CyP, contain no leader peptides and include a conserved seven amino-acid insertion relative to mammalian cytosolic CyPs. Two other Arabidopsis CyPs, ROC5 (43H1; ATCYP1) and ROC6 (ATCYP2), share these features. ROC1, ROC2, ROC3 and ROC5 are expressed in all tested organs of light-grown plants. ROC2 and ROC5 show elevated expression in flowers. Expression of ROC1, ROC2, and ROC3 decreases in darkness and these genes also exhibit small elevations in expression following wounding. The five Arabidopsis genes encoding putative cytosolic CyPs (ROC1, 2, 3, 5 and 6) contain no introns. In contrast, ROC4, which encodes a chloroplast stromal CyP, is interrupted by six introns. ROC4 is not expressed in roots, and is strongly induced by light. Phylogenetic trees of all known CyPs and CyP-related proteins provide evidence of possible horizontal transfer of CyP genes between prokaryotes and eukaryotes and of a possible polyphyletic origin of these proteins within eukaryotes. These trees also show significant grouping of eukaryotic CyPs on the basis of subcellular localization and structure. Mitochondrial CyPs are closely related to cytosolic CyPs of the source organism, but endoplasmic reticulum CyPs form separate clades. Known plant CyPs fall into three clades, one including the majority of higher plant cytosolic CyPs, one including only ROC2 and a related rice CyP, and one including only chloroplast CyPs.
The INNER NO OUTER (INO) and AINTEGUMENTA (ANT) genes are essential for ovule integument development in Arabidopsis thaliana. Ovules of ino mutants initiate two integument primordia, but the outer integument primordium forms on the opposite side of the ovule from the normal location and undergoes no further development. The inner integument appears to develop normally, resulting in erect, unitegmic ovules which resemble those of gymnosperms. ino plants are partially fertile and produce seeds with altered surface topography demonstrating a lineage dependence in development of the testa. ant mutations affect initiation of both integuments. The strongest of five new ant alleles we have isolated produces ovules that lack integuments and fail to complete megasporogenesis. ant mutations also affect flower development resulting in narrow petals and the absence of one or both lateral stamens. Characterization of double mutants between ant, ino and other mutations affecting ovule development has enabled the construction of a model for genetic control of ovule development. This model proposes parallel independent regulatory pathways for a number of aspects of this process, a dependence on the presence of an inner integument for development of the embryo sac, and the existence of additional genes regulating ovule development.
We have isolated clones of an Arabidopsis gene (ROF1, for rotamase FKBP) encoding a high molecular weight member of the FK506 binding protein (FKBP) family. The deduced amino acid sequence of ROF1 predicts a 551-amino acid, 62 kDa polypeptide which is 44% identical to human FKBP59 - a 59 kDa FKBP which binds to the 90 kDa heat shock protein and is associated with inactive steroid hormone receptors. ROF1 contains three FKBP12-like domains in the N-terminal portion of the protein (in contrast to two domains in mammalian FKBP59), an internal repeat structure associated with protein-protein interactions (tetratricopeptide repeats), and a putative calmodulin binding domain near the C-terminal region of the protein. No sequences associated with protein translocation out of the cytosol were found in ROF1. ROF1 mRNA was found at equivalent low levels in light-grown roots, stems, and flowers and at slightly higher levels in leaves. The abundance of ROF1 mRNA increased several-fold under stress conditions such as wounding or exposure to elevated NaCl levels.
The salt-sensitive phenotype of yeast cells deficient in the phosphoprotein phosphatase, calcineurin, was used to identify genes from the higher plant Arabidopsis thaliana that complement this phenotype. cDNA clones corresponding to two different sequences, designated STO (salt-tolerance) and STZ (salt-tolerance zinc finger), were found to increased tolerance of calcineurin mutants and of wild-type yeast to both Li+ and Na+ ions. STZ is related to Cys2/His2-type zinc-finger proteins found in higher plants, and STO is similar to the Arabidopsis CONSTANS protein in regions which may also be zinc fingers. Although neither protein has sequence similarity to any protein phosphatase, STO was able to at least partially compensate for all tested additional phenotypic effects of calcineurin deficiency, and STZ compensated for a subset of these effects. Salt tolerance produced by STZ appeared to be partially dependent on ENA1/PMR2, a P-type ATPase required for Li+ and Na+ efflux in yeast, whereas the effect of STO on salt tolerance was independent of ENA1/PMR2. STZ and STO were found to be expressed in Arabidopsis roots and leaves whereas only STO message was detectable in flowers. An apparent increase in the level of STZ mRNA was observed in response NaCl exposure in Arabidopsis seedlings, but the level of STO mRNA was not altered by this treatment.
Abstract: The specialized reproductive functions of angiosperm pistils are dependent in part upon the regulated activation of numerous genes expressed predominantly in this organ system. To better understand the nature of these pistil-predominant gene products we have analyzed seven cDNA clones isolated from tomato pistils through differential hybridization screening. Six of the seven cDNAs represent sequences previously undescribed in tomato, each having a unique pistil- and/or floral-predominant expression pattern. The putative protein products encoded by six of the cDNAs have been identified by their similarity to sequences in the database of previously sequenced genes, with a seventh sequence having no significant similarity with any previously reported sequence. Three of the putative proteins appear to be targeted to the endomembrane system and include an endo-beta-1,4-glucanase which is expressed exclusively in pistils at early stages of development, and proteins similar in sequence to gamma-thionin and miraculin which are expressed in immature pistils and stamens, and in either sepals or petals, respectively. Two other clones, similar in sequence to each other, were expressed primarily in immature pistils and stamens and encode distinct proteins with similarity to leucine aminopeptidases. An additional clone, which encodes a protein similar in sequence to the enzyme hyoscyamine 6-beta-hydroxylase and to other members of the family of Fe2+/ascorbate-dependent oxidases, was expressed at high levels in pistils, stamens and sepals, and at detectable levels in some vegetative organs. Together, these observations provide new insight into the nature and possible functional roles of genes expressed during reproductive development.
Arabidopsis plants homozygous for mutations at the SUPERMAN locus (SUP, also referred to as FLORAL MUTANT 10 or FLO10) have previously been shown to have supernumerary stamens and a concomitant decrease in carpel tissue. In a screen for mutations affecting ovule development, a mutant having abnormal ovules and supernumerary stamens was isolated. Subsequent complementation tests confirmed that the newly isolated ovule mutant was allelic to SUP, and is hereafter referred to as sup-5. Detailed analysis revealed that ovules of plants homozygous for sup-5 and for the previously described sup-1 allele were abnormal and indistinguishable from each other, demonstrating that alterations in ovule development are a general feature of sup mutants. Mutations at the SUP locus specifically affect development of the outer integument as the funiculus, inner integument, and embryo sac of sup mutants are normal in appearance at all stages examined. In wild type ovules the outer integument grows extensively on the abaxial side of the developing ovule and shows almost no growth on the adaxial side. This results in formation of the hood-like bilaterally symmetrical outer integument characteristic of Arabidopsis ovules at anthesis. In contrast, the outer integument of sup mutant ovules grows equally on the abaxial and adaxial sides of the ovule, resulting in a nearly radially symmetrical tubular outer integument. Thus, one role of SUP is to specifically suppress growth of the outer integument on the adaxial side of the ovule. Prior studies on sup mutants have shown that SUP acts as a negative regulator of the floral homeotic gene APETALA3 (AP3), and that loss of this regulation is the cause of production of stamens at the expense of carpel tissue in sup flowers. Analysis of sup ap3 double mutants shows, however, that the effects of sup mutations on ovule development are independent of the presence or absence of AP3 activity. Thus, SUP appears to act through different mechanisms in its early role in ensuring proper determination of carpel identity, and in its later role in asymmetric suppression of outer integument growth to produce morphologically normal ovules.
Abstract: Ovules are the developmental precursors of seeds. In angiosperms the ovules are enclosed within the central floral organs, the carpels. We have identified a homeotic mutation in Arabidopsis, "bell" (bel1), which causes transformation of ovule integuments into carpels. In situ hybridization analysis shows that this mutation leads to increased expression of the carpel-determining homeotic gene AGAMOUS (AG) in the mutant ovules. Introduction of a constitutively expressed AG transgene into wild-type plants causes the ovules to resemble those of bel1 mutants. We propose that the BEL1 gene product directs normal integument development, in part by suppressing AG expression in this structure. Our results allow expansion of the current model of floral organ identity to include regulation of ovule integument identity.
Abstract: Cyclophilin (CyP), a protein with peptidyl-prolyl cis-trans isomerase (rotamase) activity, is the specific cellular target of cyclosporin A. We have isolated cDNA clones of two genes (designated ROC1 and ROC4) encoding CyP homologs from Arabidopsis thaliana (L.). The protein products of these genes are distinct from a previously identified Arabidopsis CyP. ROC1 is expressed in all tested plant organs and encodes a protein which is highly similar to previously described cytosolic CyP isoforms of other plants. In contrast, ROC4 is expressed only in photosynthetic organs and encodes a protein which includes an amino-terminal extension with properties of known chloroplast transit peptides. In vitro import experiments using the putative precursor protein to ROC4 showed that the protein is imported into chloroplasts where it is processed to the predicted mature size. Rotamase assays and immunoblot analysis of subcellular fractions indicate the presence of a CyP isoform in the stroma of chloroplasts but not in the thylakoid membranes or thylakoid lumen. Together, these data show that ROC4 is a novel CyP isoform which is located in the stroma of chloroplasts. In vitro chloroplast import of precursors of other chloroplast proteins was unaffected by concentrations of cyclosporin A which completely inhibit rotamase activity of chloroplast stromal CyP. Thus, this activity is not essential for protein import into chloroplasts.
Abstract: Many angiosperms employ self-incompatibility systems to prevent inbreeding. The simple genetics of such systems have made them attractive models of plant cellular communication. Implicit in the single locus genetics is that only one or a few gene products are necessary for recognition and rejection of incompatible pollen. Results in the sporophytic system of the Brassicaceae suggest that different S-locus products are responsible for the pollen and pistil parts of the recognition and rejection response. In solanaeceous plants, which have a gametophytic self-incompatibility system, the S locus product responsible for the pollen portion of the interaction has not been identified, but ribonucleases encoded by the S-locus (S-RNases) are strongly implicated in the style part of the recognition and rejection reaction. In Nicotiana alata, pollen recognition and rejection occur if its S-allele matches either S-allele in the style. The putative stylar S-RNase is abundant in the transmitting tract, and pollen rejection may be related to action of S-RNase on pollen RNAs. Efforts to understand the molecular basis for pollen recognition and rejection have been limited by the lack of a system for manipulating and expressing S-RNases. Here we use the promoter of a style-expressed gene from tomato to obtain high levels of S-RNase expression in transgenic Nicotiana. Recognition and rejection of N. alata pollen S-alleles occur faithfully in the transgenic plants. Our results show that S-RNases alone are sufficient for pollen rejection in this system.
Ovules are complex structures which are present in all seed bearing plants, and are contained within the carpels in flowering plants. Ovules are the site of megasporogenesis and megagametogenesis, and following fertilization, develop into seeds. We are combining genetic methods with anatomical and morphological analyses to dissect ovule development. Here we present the first detailed description of the morphological development of ovules of Arabidopsis thaliana and report on the isolation of two chemically induced mutants, bell (bel1) and short integuments (sin1) with altered ovule development. Phenotypic analyses indicate that bel1 mutants initiate a single integument-like structure which develops aberrantly. sin1 mutants initiate two integuments, but growth of the integuments is disrupted such that cell division continues without normal cell elongation. Both mutants can differentiate archesporial cells, but neither forms a normal embryo sac. Genetic analyses indicate that bel1 segregates as a single recessive mutation and complementation tests show that the two mutants are not allelic. The phenotypes of the mutants indicate that normal morphological development of the integuments and proper embryo sac formation are interdependent or are governed in part by common pathways. The mutants we describe are the first ovule mutants to be reported in Arabidopsis and thus represent novel genetic tools for the study of this stage of reproductive development.
Gasser CS; Gunning DA; Budelier KA; Brown SM. (1990) Structure and expression of cytosolic-cyclophilin/peptidyl- prolyl cis-trans isomerase of higher plants and production of active tomato cyclophilin in Escherichia coli.
Proceedings of the National Academy of Sciences of the United States of America, 1990 Dec, 87(24):9519-23. (UI: 91088549)
Abstract: cDNA clones encoding proteins of approximately 18 kDa in which 83% of the amino acids are conserved relative to the published sequences of mammalian cyclophilin/rotamase (CyP) have been isolated from tomato, maize, and Brassica napus. In correspondence with the mammalian genes, but in contrast with the Neurospora gene and one yeast CyP gene, the plant CyP genes encode only mature proteins lacking transit peptides. RNA blot analyses demonstrate that CyP genes are expressed in all plant organs tested. Southern blots of genomic DNA indicate that there are small families (two to eight members) of CyP-related genes in maize and B. napus. A vector was constructed for expression of the tomato cDNA in E. coli. SDS/polyacrylamide gels show that extracts of appropriately induced cells harboring this vector contain nearly 40% of the protein as a single approximately 18-kDa band. While the majority of this protein is sequestered in insoluble inclusion bodies, the soluble extracts have higher levels of peptidyl-prolyl cis-trans isomerase (rotamase) activity than extracts of wild-type cells. This additional activity is sensitive to inhibition by the cyclic undecapeptide cyclosporin A.
Budelier KA; Smith AG; Gasser CS. Regulation of a stylar transmitting tissue-specific gene in wild-type and transgenic tomato and tobacco.
Molecular and General Genetics, 1990 Nov, 224(2):183-92. (UI: 91117185)
Abstract: We have characterized a gene, 9612, that is expressed predominantly in the styles of tomato pistils according to a tightly regulated temporal program. 9612 RNA levels were maximal in mature pistils from flowers at anthesis, with transcripts undetectable in pistils from flowers collected 5-7 days prior to anthesis. In situ localization of mRNA in tissue sections showed that expression of the gene is confined in the pistil to the outer five cell layers of the strands of transmitting tissue within the upper two-thirds of the style. The maximal levels of 9612 RNA detected in anthers and vegetative organs were more than 50-fold and 250-fold lower than the level in pistils, respectively. A homolog to the 9612 gene was detected in tobacco and was also found to be expressed predominantly in the style. The ability of the 5' flanking region of the tomato gene to appropriately regulate expression of a heterologous coding sequence was examined in transformed tomato and tobacco plants. In contrast to results with previously described regulated genes, the 9612 promoter functions correctly in the pistils of tomato plants, but fails to direct correct expression in tobacco plants. The sequence of the 9612 cDNA includes an open reading frame encoding a polypeptide of 404 amino acids with a highly hydrophobic amino-terminal region that may represent a signal peptide.
Smith AG; Gasser CS; Budelier KA; Fraley RT. Identification and characterization of stamen- and tapetum-specific genes from tomato.
Molecular and General Genetics, 1990 Jun, 222(1):9-16. (UI: 91042433)
Abstract: Differential screening of a tomato cDNA library produced from pre-anthesis stamens resulted in the isolation of 25 cDNA clones that hybridized to probes made from stamen RNA and showed no hybridization to probes made from RNA of vegetative organs. The 25 clones were found to represent 11 noncross-hybridizing classes. The majority of these clones were derived from genes that were single or low copy in the tomato genome. Northern RNA blotting experiments of vegetative and floral organs at several stages of development demonstrated that expression in all 11 classes was confined to floral organs. Of the 11 classes 9 were found to be expressed exclusively in stamens prior to anthesis. Two classes showed expression in immature stamens and in petals, with one of these two additionally being expressed in mature stamens at anthesis. Clones from three of the classes that were expressed exclusively in stamens were used as probes for in situ localization of RNA in floral organs. These experiments demonstrated that expression of the genes corresponding to these clones was confined to the tapetal cells of the anthers. Expression of one of the three genes was found to be limited to a single cell type during the 5-6 day period from late meiosis to immature pollen formation.