S-like RNase Gene Research Articles

Expression and function of an S1-type nuclease in the digestive fluid of a sundew, Drosera adelae.

Carnivorous plants trap and digest insects and similar-sized animals. Many studies have examined enzymes in the digestive fluids of these plants and have gradually unveiled the origins and gene expression of these enzymes. However, only a few attempts have been made at characterization of nucleases. This study aimed to reveal gene expression and the structural, functional and evolutionary characteristics of an S1-type nuclease (DAN1) in the digestive fluid of an Australian sundew, Drosera adelae, whose trap organ shows unique gene expression and related epigenetic regulation. Organ-specificity in Dan1 expression was examined using glandular tentacles, laminas, roots and inflorescences, and real-time PCR. The methylation status of the Dan1 promoter in each organ was clarified by bisulphite sequencing. The structural characteristics of DAN1 were studied by a comparison of primary structures of S1-type nucleases of three carnivorous and seven non-carnivorous plants. DAN1 was prepared using a cell-free protein synthesis system. Requirements for metal ions, optimum pH and temperature, and substrate preference were examined using conventional methods. Dan1 is exclusively expressed in the glandular tentacles and its promoter is almost completely unmethylated in all organs. This is in contrast to the S-like RNase gene da-I of Dr. adelae, which shows similar organ-specific expression, but is controlled by a promoter that is specifically unmethylated in the glandular tentacles. Comparison of amino acid sequences of S1-type nucleases identifies seven and three positions where amino acid residues are conserved only among the carnivorous plants and only among the non-carnivorous plants, respectively. DAN1 prefers a substrate RNA over DNA in the presence of Zn2+, Mn2+ or Ca2+ at an optimum pH of 4.0. Uptake of phosphates from prey is suggested to be the main function of DAN1, which is very different from the known functions of S1-type nucleases. Evolution has modified the structure and expression of Dan1 to specifically function in the digestive fluid.

Phosphate-Starvation-Inducible S-Like RNase Genes in Rice Are Involved in Phosphate Source Recycling by RNA Decay.

The fine-tuning of inorganic phosphate (Pi) for enhanced use efficiency has long been a challenging subject in agriculture, particularly in regard to rice as a major crop plant. Among ribonucleases (RNases), the RNase T2 family is broadly distributed across kingdoms, but little has been known on its substrate specificity compared to RNase A and RNase T1 families. Class I and class II of the RNase T2 family are defined as the S-like RNase (RNS) family and have showed the connection to Pi recycling in Arabidopsis. In this study, we first carried out a phylogenetic analysis of eight rice and five Arabidopsis RNS genes and identified mono-specific class I and dicot-specific class I RNS genes, suggesting the possibility of functional diversity between class I RNS family members in monocot and dicot species through evolution. We then compared the in silico expression patterns of all RNS genes in rice and Arabidopsis under normal and Pi-deficient conditions and further confirmed the expression patterns of rice RNS genes via qRT-PCR analysis. Subsequently, we found that most of the OsRNS genes were differentially regulated under Pi-deficient treatment. Association of Pi recycling by RNase activity in rice was confirmed by measuring total RNA concentration and ribonuclease activity of shoot and root samples under Pi-sufficient or Pi-deficient treatment during 21 days. The total RNA concentrations were decreased by < 60% in shoots and < 80% in roots under Pi starvation, respectively, while ribonuclease activity increased correspondingly. We further elucidate the signaling pathway of Pi starvation through upregulation of the OsRNS genes. The 2-kb promoter region of all OsRNS genes with inducible expression patterns under Pi deficiency contains a high frequency of P1BS cis-acting regulatory element (CRE) known as the OsPHR2 binding site, suggesting that the OsRNS family is likely to be controlled by OsPHR2. Finally, the dynamic transcriptional regulation of OsRNS genes by overexpression of OsPHR2, ospho2 mutant, and overexpression of OsPT1 lines involved in Pi signaling pathway suggests the molecular basis of OsRNS family in Pi recycling via RNA decay under Pi starvation.

Organ-specific expression and epigenetic traits of genes encoding digestive enzymes in the lance-leaf sundew (Drosera adelae).

Over the last two decades, extensive studies have been performed at the molecular level to understand the evolution of carnivorous plants. As fruits, the repertoire of protein components in the digestive fluids of several carnivorous plants have gradually become clear. However, the quantitative aspects of these proteins and the expression mechanisms of the genes that encode them are still poorly understood. In this study, using the Australian sundew Drosera adelae, we identified and quantified the digestive fluid proteins. We examined the expression and methylation status of the genes corresponding to major hydrolytic enzymes in various organs; these included thaumatin-like protein, S-like RNase, cysteine protease, class I chitinase, β-1, 3-glucanase, and hevein-like protein. The genes encoding these proteins were exclusively expressed in the glandular tentacles. Furthermore, the promoters of the β-1, 3-glucanase and cysteine protease genes were demethylated only in the glandular tentacles, similar to the previously reported case of the S-like RNase gene da-I. This phenomenon correlated with high expression of the DNA demethylase DEMETER in the glandular tentacles, strongly suggesting that it performs glandular tentacle-specific demethylation of the genes. The current study strengthens and generalizes the relevance of epigenetics to trap organ-specific gene expression in D. adelae. We also suggest similarities between the trap organs of carnivorous plants and the roots of non-carnivorous plants.

Genome-wide identification and functional analysis of S-RNase involved in the self-incompatibility of citrus.

S-RNase-based self-incompatibility is found in Solanaceae, Rosaceae, and Scrophulariaceae, and is the most widespread mechanism that prevents self-fertilization in plants. Although 'Shatian' pummelo (Citrus grandis), a traditional cultivated variety, possesses the self-incompatible trait, the role of S-RNases in the self-incompatibility of 'Shatian' pummelo is poorly understood. To identify genes associated with self-incompatibility in citrus, we identified 16 genes encoding homologs of ribonucleases in the genomes of sweet orange (Citrus sinensis) and clementine mandarin (Citrus clementine). We preliminarily distinguished S-RNases from S-like RNases with a phylogenetic analysis that classified these homologs into three groups, which is consistent with the previous reports. Expression analysis provided evidence that CsRNS1 and CsRNS6 are S-like RNase genes. The expression level of CsRNS1 was increased during fruit development. The expression of CsRNS6 was increased during the formation of embryogenic callus. In contrast, we found that CsRNS3 possessed several common characteristics of the pistil determinant of self-incompatibility: it has an alkaline isoelectric point (pI), harbors only one intron, and is specifically expressed in style. We obtained a cDNA encoding CgRNS3 from 'Shatian' pummelo and found that it is homolog to CsRNS3 and that CgRNS3 exhibited the same expression pattern as CsRNS3. In an in vitro culture system, the CgRNS3 protein significantly inhibited the growth of self-pollen tubes from 'Shatian' pummelo, but after a heat treatment, this protein did not significantly inhibit the elongation of self- or non-self-pollen tubes. In conclusion, an S-RNase gene, CgRNS3, was obtained by searching the genomes of sweet orange and clementine for genes exhibiting sequence similarity to ribonucleases followed by expression analyses. Using this approach, we identified a protein that significantly inhibited the growth of self-pollen tubes, which is the defining property of an S-RNase.

Overexpression of an S-like ribonuclease gene, OsRNS4, confers enhanced tolerance to high salinity and hyposensitivity to phytochrome-mediated light signals in rice

S-like ribonucleases (S-like RNases) are homologous to S-ribonucleases (S-RNases), but are not involved in self-incompatibility. In dicotyledonous plants, S-like RNases play an important role in phosphate recycling during senescence and are induced by inorganic phosphate-starvation and in response to defense and mechanical wounding. However, little information about the functions of the S-like RNase in monocots has been reported. Here, we investigated the expression patterns and roles of an S-like RNase gene, OsRNS4, in abscisic acid (ABA)-mediated responses and phytochrome-mediated light responses as well as salinity tolerance in rice. The OsRNS4 gene was expressed at relatively high levels in leaves although its transcripts were detected in various organs. OsRNS4 expression was regulated by salt, PEG and ABA. The seedlings overexpressing OsRNS4 had longer coleoptiles and first leaves than wild-type seedlings under red light (R) and far-red light (FR), suggesting negative regulation of OsRNS4 in photomorphogenesis in rice seedlings. Moreover, ABA-induced growth inhibition of rice seedlings was significantly increased in the OsRNS4-overexpression (OsRNS4-OX) lines compared with that in WT, suggesting that OsRNS4 probably acts as a positive regulator in ABA responses in rice seedlings. In addition, our results demonstrate that OsRNS4-OX lines have enhanced tolerance to high salinity compared to WT. Our findings supply new evidence on the functions of monocot S-like RNase in regulating photosensitivity and abiotic stress responses.

S-like ribonuclease gene expression in carnivorous plants

Functions of S-like ribonucleases (RNases) differ considerably from those of S-RNases that function in self-incompatibility. Expression of S-like RNases is usually induced by low nutrition, vermin damage or senescence. However, interestingly, an Australian carnivorous plant Drosera adelae (a sundew), which traps prey with a sticky digestive liquid, abundantly secretes an S-like RNase DA-I in the digestive liquid even in ordinary states. Here, using D. adelae, Dionaea muscipula (Venus flytrap) and Cephalotus follicularis (Australian pitcher plant), we show that carnivorous plants use S-like RNases for carnivory: the gene da-I encoding DA-I and its ortholog cf-I of C. follicularis are highly expressed and constitutively active in each trap/digestion organ, while the ortholog dm-I of D. muscipula becomes highly active after trapping insects. The da-I promoter is unmethylated only in its trap/digestion organ, glandular tentacles (which comprise a small percentage of the weight of the whole plant), but methylated in other organs, which explains the glandular tentacles-specific expression of the gene and indicates a very rare gene regulation system. In contrast, the promoters of dm-I, which shows induced expression, and cf-I, which has constitutive expression, were not methylated in any organs examined. Thus, it seems that the regulatory mechanisms of the da-I, dm-I and cf-I genes differ from each other and do not correlate with the phylogenetic relationship. The current study suggests that under environmental pressure in specific habitats carnivorous plants have managed to evolve their S-like RNase genes to function in carnivory.

CgSL2, an S-like RNase gene in ‘Zigui shatian’ pummelo (Citrus grandis Osbeck), is involved in ovary senescence

'Zigui shatian' pummelo (Citrus grandis Osbeck) is one nature mutant from 'Shatian' pummelo, which showed self-compatibility, because self-pollen tubes were not arrested in the style, moreover abnormal post-zygotic development in ovary caused seed abortion in the cultivar. Herein we constructed a cDNA library from flowers of 'Zigui shatian' pummelo and identified one RNase gene fragment. The full length of cDNA sequence of this gene, with an open reading frame of 834bp, was isolated by 5'-RACE method. The gene, named as CgSL2, contained five conserved regions and two histidine residues essential for RNase activity. Phylogenetic analysis indicated that CgSL2 was mostly similar to AhSL28, an S-like RNase from Antirrhinum. Southern hybridization verified CgSL2 existed in the genome as multiple copies. qRT-PCR and RT-PCR analysis showed that the expression of CgSL2 was not tissue-specific. The expression of CgSL2 was down-regulated during senescence of stem, petal, style and stamen, whereas up-regulated during ovary senescence. Further in situ hybridization of CgSL2 in the ovary during the balloon stage to anthesis stage also showed that it dramatically increased in mature flower, consistent with qRT-PCR and RT-PCR results. These findings suggested that CgSL2 might play an important role during ovary senescence.

Structural analysis of the gene encoding Drosera adelae S-like ribonuclease DA-I

Sticky digestive liquid from a carnivorous plant Drosera adelae contains a novel S-like ribonuclease (RNase). In normal plants, S-like RNases corresponding to the D. adelae RNase, which we named DA-I, are induced by phosphate starvation and/or wounding. In this study, the genomic organization of the DA-I gene (da-I) and its 5'-and 3'-flanking sequences were analyzed. The da-I was found to be a gene of single copy in D. adelae. The gene comprises four exons and three introns, with a similarity to the structure of the Class C S-like RNase genes. A nucleotide sequence analysis of the 5'-flanking region revealed that a canonical TATA box sequence (TATAAAT) lies between positions -33 to -27 and a CCAAT box is located between positions -84 to -80 relative to the transcription start site (+1). Although the gene is constitutively transcribed in glandular cells, three wound responsive and two phosphate starvation responsive DNA elements were found within the region between -160 to +2, presenting a riddle regarding the mechanism for the transcriptional regulation of the da-I.

AhSL28, a senescence- and phosphate starvation-induced S-like RNase gene in Antirrhinum

Several species of higher plants have been found to contain S-like ribonucleases (RNases), which are homologous to S-RNases controlling self-incompatibility. No S-like RNase genes have been isolated from self-incompatible Antirrhinum. To investigate the relationship between S- and S-like RNases, we cloned a gene named AhSL28 encoding an S-like RNase in Antirrhinum. Amino acid sequence, genomic structure and phylogenetic analyses indicated that AhSL28 is most similar to RNS2, an S-like RNase from Arabidopsis thaliana and formed a distinct subclass together with several other S-like RNases within the S-RNase superfamily. Unlike S-RNase genes in Antirrhinum, AhSL28 is not only expressed in pistils but also in leaves, petals, sepals and anthers, in particular, showing a strong expression in vascular tissues and transmitting track. Moreover, its RNA transcripts were induced during leaf senescence and phosphate (Pi) starvation but not by wounding, indicating that AhSL28 plays a role in remobilizing Pi and other nutrients, particularly when cells senesce and are under limited Pi conditions in Antirrhinum. Possible evolutionary relations of S- and S-like RNases as well as signal transduction pathways related to S-like RNase action are discussed.

Molecular cloning of the self-incompatibility genes S1 and S3 from almond ( Prunus dulcis cv. Ferragn�s)

Almond (Prunus dulcis) displays gametophytic self-incompatibility. In the work reported here, we cloned two novel S-RNase genes from almond cultivar Ferragnes (genotype S1S3) using PCR. The S1-RNase gene has the same coding region as the Sb gene cloned from almond cultivated in the USA; however, their introns are different in sequence. S1 was cloned and sequenced from six different cultivars originating in Europe. The full-length of the S3-RNase gene was cloned using two primers corresponding to the start and stop codons contexts. Two introns are present in the S3 gene, unique among the S-RNase genes. Sequence-specific PCR was performed to confirm that the two cloned genes co-segregate with the S-locus using progenies of a controlled cross between Tuono (S1Sf) and Ferragnes (S1S3). Based on the structural differences of S- and S-like RNase genes, we discuss the evolutionary relationship between the two groups of RNase genes.

PD1, an S-like RNase gene from a self-incompatible cultivar of almond.

Many flowering plants contain stylar S-RNases that are involved in self-incompatibility and S-like RNases of which the biological function is uncertain. This paper reports the deduced amino acid sequence of an S-like RNase gene (PD1) from the self-incompatible plant Prunus dulcis (almond). The amino acid sequence of PD1, which was derived from cDNA and genomic DNA clones, showed 34-86% identity to acidic plant S-like RNases reported so far, with the highest degree of similarity being to an S-like RNase from Japanese pear (Pyrus pyrifolia). Based on RNA hybridisation experiments it appears that, like for many other S-like RNases, the expression of PD1 is not pistil-specific. Analysis of the genomic structure revealed the presence of three introns, of which one is similar in location to that of the related S-RNase gene from Solanaceae and Rosaceae. At least four bands hybridising to PD1 were found upon Southern hybridisation, suggesting the presence of a multigene family of S-like RNase genes in almond. The putative biological function of PD1 is discussed.

Molecular characterisation of an S-like RNase of Nicotiana alata that is induced by phosphate starvation.

We characterised a cDNA encoding an S-like RNase (RNase NE) from the styles of the self-incompatible plant, Nicotiana alata. RNase NE is 86% identical to an extracellular RNase from tomato cell cultures, RNase LE. DNA hybridisation experiments indicate that there are ca. 5-6 sequences related to RNase NE in the N. alata genome and that RNase NE is not linked to the self-incompatibility (S) locus. RNase NE is expressed in the styles, petals and immature anthers but not in the vegetative tissues of N. alata plants under normal growth conditions. Under phosphate-limited conditions, RNase NE expression is induced in roots but not leaves of N. alata. A transcript hybridising to RNase NE is also induced in N. plumbaginifolia cell cultures in response to phosphate starvation. RNase NE is likely to play a role in the response of N. alata to phosphate limitation, possibly by scavenging phosphate from sources of RNA in the root environment. We also discuss the evolutionary relationships between the S- and S-like RNase genes in plants.