RNase X2, a pistil-specific ribonuclease from Petunia inflata,shares sequence similarity with solanaceous S proteins

Petunia inflata, a species with gametophytic self-incompatibility, has previously been found to contain a large number of ribonucleases in the pistil. The best characterized of the pistil ribonucleases are the products of the S alleles, the S proteins, which are thought to be involved in self-incompatibility interactions. Here we report the characterization of a gene encoding another pistil ribonuclease of P. inflata, RNase X2. Degenerate oligonucleotides, synthesized based on the amino-terminal sequence of RNase X2, were used as probes to isolate cDNA clones, one of which was in turn used as a probe to isolate genomic clones containing the gene for RNase X2, rnx2. The deduced amino acid sequence of RNase X2 shows 42% to 71% identity to the 20 solanaceous S proteins reported so far, with the highest degree of similarity being to S₃ and S₆ proteins of Nicotiana alata. The cDNA sequence predicts a leader peptide of 22 amino acids, suggesting that RNase X2, like S proteins, is an extracellular ribonuclease. Also, similar to the S gene, rnx2 is expressed only in the pistil, and contains a single intron comparable in size and identical in location to that of the S gene. However, rnx2 is not linked to the S locus, and, in contrast to the highly polymorphic S gene, it is monomorphic. The possible biological function of RNase X2 is discussed.     

Features of the extracellular domain of the S-locus receptor kinase from Brassica

An S-receptor kinase (SRK) gene associated with self-incompatibility in a Brassica napus subsp. oleifera line has been characterized. The SRK-A14 cDNA shows the highest levels of homology in the 5 end to the SLG-A14 cDNA present at the same locus. RNA blot analysis shows that the SRK-A14 gene is expressed predominantly in the pistil, and at lower levels in the anthers. The predicted amino acid sequences from the extracellular domain of the SRK-A14 gene and three other SRK genes were compared. The different SRK extracellular domains were for the most part very similar, with the exception of two variable regions containing a high level of amino acid alterations. These extracellular domains also contain a region of similarity to the immunoglobulin domains present in members of the immunoglobulin superfamily. These findings may define regions of the SRK protein that are necessary for interactions between SRK and other proteins.     

Pistil induction by hormones from callus of <Emphasis Type="Italic">Oryza sativa</Emphasis> in vitro

This study describes the successful formation of floral organ pistil from the callus of pistil explants of Oryza sativa L. For induction of floral organs, different explants—including young embryo, lemma, palea and pistil—were used for callus induction with different combinations of N⁶-benzyladenine and 2,4-dichlorophenoxyacetic acid (2,4-D). High frequencies of callus formation from pistil and young embryo explants were achieved. Floral organs were induced after calli from pistils were transferred to medium containing both zeatin and 2,4-D. The morphological characteristics of the pistil-like organs are very similar to those formed in planta though with minor differences. Further histological study revealed that the in vitro pistil contains an ovule within its ovary. Furthermore, a pistil-specific gene, OsMADS3 used as a molecular marker for pistil identity, was expressed in the pistil-like organs as it was in pistils in the flower of the plant.     

Characterization of a predominantly pistil-expressed gene encoding a γ-thionin-like protein of Petunia inflata

We isolated a cDNA clone from a pistil cDNA library of Petunia inflata which encodes a protein, PPT, with sequence similarity to -thionins. Characterization of a genomic clone containing a PPT gene revealed the presence of a single intron. Northern analysis revealed that the PPT gene was predominantly expressed in the pistil during all stages of flower development. Since thionins have been implicated in plant defense against pathogens, PPT may play a role similar to that of other defense-related proteins found in the pistil, defending the pistil against pathogen infection.     

Morphological characterisation and genetic analysis of a bi-pistil mutant (bip) in Medicago truncatula Gaertn.

A floral organ mutant was observed in transgenic Medicago truncatula Gaertn. plants that had two separate stigmas borne on two separate styles that emerged from a single superior carpel primordium. We propose the name bi-pistil, bip for the mutation. We believe this is the first report of such a mutation in this species. Genetic and molecular analyses of the mutant were conducted. The mutant plant was crossed to a mtapetala plant with a wild-type pistil. Expression of the mutant trait in the F₁ and F₂ generations indicates that the bi-pistil trait is under the control of a single recessive gene. Other modifying genes may influence its expression. The mutation was associated with the presence of a T-DNA insert consisting of the Alfalfa mosaic virus (AMV) coat protein gene in antisense orientation and the nptII selectable marker gene. It is suggested that the mutation is due to gene disruption because multiple copies of the T-DNA were observed in the mutant. The bi-pistil gene was found to be independent of the male-sterile gene, tap. This novel mutant may assist in understanding pistil development in legumes.     

Characterization of ethylene effects on sex determination in cucumber plants

Sex differentiation in cucumber plants (Cucumis sativus L.) appears to be determined by the selective arrest of the stamen or pistil primordia. We investigated the influence of an ethylene-releasing agent (ethephon) or an inhibitor of ethylene biosynthesis (aminoethoxyvinyl glycine) on sex differentiation in different developmental stages of flower buds. These treatments influence sex determination only at the stamen primordia differentiation stage in both monoecious and gynoecious cucumbers. To clarify the relationships between the ethylene-producing tissues and the ethylene-perceiving tissues in inducing female flowers in the cucumber, we examined the localization of mRNA accumulation of both the ACC synthase gene (CS-ACS2) and the ethylene-receptor-related genes (CS-ETR1, CS-ETR2, and CS-ERS) in flower buds by in situ hybridization analysis. CS-ACS2 mRNA was detected in the pistil primordia of gynoecious cucumbers, whereas it was located in the tissues just below the pistil primordia and at the adaxial side of the petals in monoecious cucumbers. In flower buds of andromonoecious cucumbers, only CS-ETR1 mRNA was detected, and was located in the pistil primordia. The localization of the mRNAs of the three ethylene-receptor-related genes in the flower buds of monoecious and gynoecious cucumbers overlap but are not identical. We discuss the relationship between the mRNA accumulation patterns and sex expression in cucumber plants.     

An F-box gene linked to the self-incompatibility (S) locus of Antirrhinum is expressed specifically in pollen and tapetum

In many flowering plants, self-fertilization is prevented by an intraspecific reproductive barrier known as self-incompatibility (SI), that, in most cases, is controlled by a single multiallelic S locus. So far, the only known S locus product in self-incompatible species from the Solanaceae, Scrophulariaceae and Rosaceae is a class of ribonucleases called S RNases. Molecular and transgenic analyses have shown that S RNases are responsible for pollen rejection by the pistil but have no role in pollen expression of SI, which appears to be mediated by a gene called the pollen self-incompatibility or Sp gene. To identify possible candidates for this gene, we investigated the genomic structure of the S locus in Antirrhinum, a member of the Scrophulariaceae. A novel F-box gene, AhSLF-S ₂, encoded by the S ₂ allele, with the expected features of the Sp gene was identified. AhSLF-S ₂ is located 9 kb downstream of S ₂ RNase gene and encodes a polypeptide of 376 amino acids with a conserved F-box domain in its amino-terminal part. Hypothetical genes homologous to AhSLF-S ₂ are apparent in the sequenced genomic DNA of Arabidopsis and rice. Together, they define a large gene family, named SLF (S locus F-box) family. AhSLF-S ₂ is highly polymorphic and is specifically expressed in tapetum, microspores and pollen grains in an allele-specific manner. The possibility that Sp encodes an F-box protein and the implications of this for the operation of self-incompatibility are discussed.     

Identification of an estrogenic hormone receptor in Caenorhabditis elegans

Changes in both behavior and gene expression occur in Caenorhabditis elegans following exposure to sex hormones such as estrogen and progesterone, and to bisphenol A (BPA), an estrogenic endocrine-disrupting compound. However, only one steroid hormone receptor has been identified. Of the 284 known nuclear hormone receptors (NHRs) in C. elegans, we selected nhr-14, nhr-69, and nhr-121 for analysis as potential estrogenic hormone receptors, because they share sequence similarity with the human estrogen receptor. First, the genes were cloned and expressed in Escherichia coli, and then the affinity of each protein for estrogen was determined using a surface plasmon resonance (SPR) biosensor. All three NHRs bound estrogen in a dose-dependent fashion. To evaluate the specificity of the binding, we performed a solution competition assay using an SPR biosensor. According to our results, only NHR-14 was able to interact with estrogen. Therefore, we next examined whether nhr-14 regulates estrogen signaling in vivo. To investigate whether these interactions actually control the response of C. elegans to hormones, we investigated the expression of vitellogenin, an estrogen responsive gene, in an nhr-14 mutant. Semi-quantitative RT-PCR showed that vitellogenin expression was significantly reduced in the mutant. This suggests that NHR-14 is a C. elegans estrogenic hormone receptor and that it controls gene expression in response to estrogen.     

Analysis of the S-locus structure in Prunus armeniaca L. Identification of S-haplotype specific S-RNase and F-box genes

The gametophytic self-incompatibility (GSI) system in Rosaceae has been proposed to be controlled by two genes located in the S-locusan S-RNase and a recently described pollen expressed S-haplotype specific F-box gene (SFB). However, in apricot (Prunus armeniaca L.) these genes had not been identified yet. We have sequenced 21kb in total of the S-locus region in 3 different apricot S-haplotypes. These fragments contain genes homologous to the S-RNase and F-box genes found in other Prunusspecies, preserving their basic gene structure features and defined amino acid domains. The physical distance between the F-boxand the S-RNase genes was determined exactly in the S ₂-haplotype (2.9kb) and inferred approximately in the S ₁-haplotype (< 49kb) confirming that these genes are linked. Sequence analysis of the 5 flanking regions indicates the presence of a conserved region upstream of the putative TATA box in the S-RNase gene. The three identified S-RNase alleles (S ₁, S ₂ and S ₄) had a high allelic sequence diversity (75.3 amino acid identity), and the apricot F-box allelic variants (SFB₁, SFB₂ and SFB₄) were also highly haplotype-specific (79.4 amino acid identity). Organ specific-expression was also studied, revealing that S ₁- and S ₂-RNases are expressed in style tissues, but not in pollen or leaves. In contrast, SFB ₁ and SFB ₂ are only expressed in pollen, but not in styles or leaves. Taken together, these results support these genes as candidates for the pistil and pollen S-determinants of GSI in apricot.     

Phenotypic suppression of the Drosophila mitochondrial disease-like mutant tko by duplication of the mutant gene in its natural chromosomal context

A mutation in the Drosophila gene technical knockout (tko^25t), encoding mitoribosomal protein S12, phenocopies human mitochondrial disease. We isolated three spontaneous X-dominant suppressors of tko^25t (designated Weeble), exhibiting almost wild-type phenotype and containing overlapping segmental duplications including the mutant allele, plus a second mitoribosomal protein gene, mRpL14. Ectopic, expressed copies of tko^25t and mRpL14 conferred no phenotypic suppression. When placed over a null allele of tko, Weeble retained the mutant phenotype, even in the presence of additional transgenic copies of tko^25t. Increased mutant gene dosage can thus compensate the mutant phenotype, but only when located in its normal chromosomal context.     

Isolation and Characterization of a Class I SLG Gene from Chinese Cabbage (Brassica campestris)

In Brassica species, self-incompatibility in the recognition reaction between self and non-self pollens is determined by two genes, SLG and SRK, at the S locus. We have cloned and characterized a genomic DNA fragment containing a complete open reading frame of the SLG gene from Chinese cabbage. The genomic clone, named BcSLG2, was found to possess the region that shares a homology of 77% in amino acid identity with the SLG46 gene of Brassica campestris. Northern blot analysis revealed that the BcSLG2 gene expression is restricted to the pistil of Chinese cabbage flower. In situ hybridization showed that in the pistil, the gene is expressed predominantly in the stigmatic tissue. Much lower expression in the tapetum was also detectable at an immature stage of the flower development. Southern blot hybridization with the BcSLG2 DNA probe showed polymorphism in the SLG gene organization of the Chinese cabbage plants. These results will provide valuable information in understanding the S gene complex of the Chinese cabbage plants.     

Different positively selected sites at the gametophytic self-incompatibility pistil S-RNase gene in the Solanaceae and Rosaceae (Prunus, Pyrus, and Malus)

In this work we perform a comparative study on the location of positively selected sites (those likely responsible for defining specificity differences) at the S-RNase gene, the pistil component of the gametophytic self-incompatibility system. For Plantaginaceae and Rosaceae (Prunus and Pyrus/Malus) this is the first study of this kind. A clear sign of positive selection was observed for 13, 17, and 27 amino acid sites in Solanaceae, Prunus, and Pyrus/Malus, respectively, using two different methodologies. In Plantaginaceae no clear positively selected sites were identified. Possible reasons for this result are discussed. Indirect experimental evidence suggests that the identified positively selected amino acid sites play a role in specificity determination. The percentage of positively selected sites is similar in Solanaceae and Rosaceae but the location of those sites is different. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. Reviewing Editor: Dr. Martin Kreitman     

Characterization of PsMPK2, the first C1 subgroup MAP kinase from pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

Mitogen-activated protein kinase (MAPK) cascades play a key role in plant growth and development as well as in biotic and abiotic stress responses. They are classified according to their sequence homology into four major groups (A–D). A large amount of information about MAPKs in groups A and B is available but few data of the C group have been reported. In this study, a C1 subgroup MAP kinase cDNA, PsMPK2, was isolated from Pisum sativum. PsMPK2 is expressed in vegetative (root and leaf) and reproductive (stamen, pistil and fruit) organs. Expression of PsMPK2 in Arabidopsis thaliana shows that mechanical injury and other stress signals as abscisic acid, jasmonic acid and hydrogen peroxide increase its kinase activity, extending previous results indicating that C1 subgroup MAPKs may be involved in the response to stress.