Yansheng Zhang

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-S2, encoded by the S2 allele, with the expected features of the Sp gene was identified. AhSLF-S2 is located 9xa0kb downstream of S2 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-S2 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-S2 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.

The F-Box Protein AhSLF-S2 Physically Interacts with S-RNases That May Be Inhibited by the Ubiquitin/26S Proteasome Pathway of Protein Degradation during Compatible Pollination in Antirrhinum

Self-incompatibility S-locus–encoded F-box (SLF) proteins have been identified in Antirrhinum and several Prunus species. Although they appear to play an important role in self-incompatible reaction, functional evidence is lacking. Here, we provide several lines of evidence directly implicating a role of AhSLF-S2 in self-incompatibility in Antirrhinum. First, a nonallelic physical interaction between AhSLF-S2 and S-RNases was demonstrated by both coimmunoprecipitation and yeast two-hybrid assays. Second, AhSLF-S2 interacts with ASK1- and CULLIN1-like proteins in Antirrhinum, and together, they likely form an Skp1/Cullin or CDC53/F-box (SCF) complex. Third, compatible pollination was specifically blocked after the treatment of the proteasomal inhibitors MG115 and MG132, but they had little effect on incompatible pollination both in vitro and in vivo, indicating that the ubiquitin/26S proteasome activity is involved in compatible pollination. Fourth, the ubiquitination level of style proteins was increased substantially after compatible pollination compared with incompatible pollination, and coimmunoprecipitation revealed that S-RNases were ubiquitinated after incubating pollen proteins with compatible but not with incompatible style proteins, suggesting that non-self S-RNases are possibly degraded by the ubiquitin/26S proteasome pathway. Fifth, the S-RNase level appeared to be reduced after 36 h of compatible pollination. Taken together, these results show that AhSLF-S2 interacts with S-RNases likely through a proposed SCFAhSLF-S2 complex that targets S-RNase destruction during compatible rather than incompatible pollination, thus providing a biochemical basis for the inhibition of pollen tube growth as observed in self-incompatible response in Antirrhinum.

The F-Box Protein AhSLF-S2 Controls the Pollen Function of S-RNase–Based Self-Incompatibility

Recently, we have provided evidence that the polymorphic self-incompatibility (S) locus-encoded F-box (SLF) protein AhSLF-S2 plays a role in mediating a selective S-RNase destruction during the self-incompatible response in Antirrhinum hispanicum. To investigate its role further, we first transformed a transformation-competent artificial chromosome clone (TAC26) containing both AhSLF-S2 and AhS2-RNase into a self-incompatible (SI) line of Petunia hybrida. Molecular analyses showed that both genes are correctly expressed in pollen and pistil in four independent transgenic lines of petunia. Pollination tests indicated that all four lines became self-compatible because of the specific loss of the pollen function of SI. This alteration was transmitted stably into the T1 progeny. We then transformed AhSLF-S2 cDNA under the control of a tomato (Lycopersicon esculentum) pollen-specific promoter LAT52 into the self-incompatible petunia line. Molecular studies revealed that AhSLF-S2 is specifically expressed in pollen of five independent transgenic plants. Pollination tests showed that they also had lost the pollen function of SI. Importantly, expression of endogenous SLF or SLF-like genes was not altered in these transgenic plants. These results phenocopy a well-known phenomenon called competitive interaction whereby the presence of two different pollen S alleles within pollen leads to the breakdown of the pollen function of SI in several solanaceaous species. Furthermore, we demonstrated that AhSLF-S2 physically interacts with PhS3-RNase from the P. hybrida line used for transformation. Together with the recent demonstration of PiSLF as the pollen determinant in P. inflata, these results provide direct evidence that the polymorphic SLF including AhSLF-S2 controls the pollen function of S-RNase–based self-incompatibility.

Monitoring of gene expression profiles and isolation of candidate genes involved in pollination and fertilization in rice ( Oryza sativa L.) with a 10K cDNA microarray.

To monitor gene expression profiles during pollination and fertilization in rice at a genome scale, we generated 73 424 high-quality expressed sequence tags (ESTs) derived from the green/etiolated shoot and pistil (0–5 h after pollination, 5hP) of rice, which were subsequently used to construct a cDNA microarray containing ca. 10 000 unique rice genes. This microarray was used to analyze gene expression in pistil unpollinated (UP), 5hP and 5DAP(5 days after pollination), anther, shoot, root, 10-day-old embryo (10EM) and 10-day-old endosperm (10EN). Clustering analysis revealed that the anther has a gene-expression profile more similar to root than to pistil and most pistil-preferentially expressed genes respond to pollination and/or fertilization. There are 253 ESTs exhibiting differential expression (e±2-fold changes) during pollination and fertilization, and about 70% of them can be assigned a putative function. We also recovered 20 genes similar to pollination-related and/or fertility-related genes previously identified as well as genes that were not implicated previously. Microarray and real-time PCR analyses showed that the array sensitivity was estimated at 1–5 copies of mRNA per cell, and the differentially expressed genes showed a high correlation between the two methods. Our results indicated that this cDNA microarray constructed here is reliable and can be used for monitoring gene expression profiles in rice. In addition, the genes that differentially expressed during pollination represent candidate genes for dissecting molecular mechanism of this important biological process in rice.

Structural and transcriptional analysis of S -locus F-box genes in Antirrhinum

A class of ribonucleases termed S-RNases, which control the pistil expression of self-incompatibility, represents the only known functional products encoded by the S locus in species from the Solanaceae, Scrophulariaceae and Rosaceae. Previously, we identified a pollen-specific F-box gene, AhSLF (S locus F-box)-S2, very similar to S2-RNase in Antirrhinum, a member of the Scrophulariaceae. In addition, AhSLF-S2 also detected the presence of its homologous DNA fragments. To identify these fragments, we constructed two genomic DNA libraries from Antirrhinum self-incompatible lines carrying alleles S1S5 and S2S4, respectively, using a transformation-competent artificial chromosome (TAC) vector. With AhSLF-S2-specific primers, TAC clones containing both AhSLF-S2 and its homologs were subsequently identified (S2TAC, S5TACa, S4TAC, and S1TACa). DNA blot hybridization, sequencing and segregation analyses revealed that they are organized as single allelic copies (AhSLF-S2, -S1, -S4 and -S5) tightly linked to the S-RNases. Furthermore, clusters of F-box genes similar to AhSLF-S2 were identified. In total, three F-box genes (AhSLF-S2, -S2A and -S2C) in S2TAC (51xa0kb), three (AhSLF-S4, -S4A and -S4D) in S4TAC (75xa0kb), two (AhSLF-S5 and -S5A) in S5TACa (55xa0kb), and two (AhSLF-S1 and -S1E) in S1TACa (71xa0kb), respectively, were identified. Paralogous copies of these genes show 38–54% identity, with allelic copies sharing 90% amino acid identity. Among these genes, three (AhSLF-S2C, -S4D and -S1E) were specifically expressed in pollen, similar to AhSLF-S2, implying that they likely play important roles in pollen, whereas three AhSLF-SA alleles showed no detectable expression. In addition, several types of retroelements and transposons were identified in the sequenced regions, revealing some detailed information on the structural diversity of the S locus region. Taken together, these results indicate that both single allelic and tandemly duplicated genes are associated with the S locus in Antirrhinum. The implications of these findings in evolution and possible roles of allelic AhSLF-S genes in the self-incompatible reaction are discussed in species like Antirrhinum.

Genome-wide analysis of S-Locus F-box-like genes in Arabidopsis thaliana.

The AntirrhinumS-locus F-box gene, AhSLF-S2, has been shown to determine the pollen function of S-RNase-mediated self-incompatibility (SI). Its initial identification led to the discovery of a large family of plant-specific F-box proteins, named the SLF (S-Locus F-box) family, including members from species with or without S-RNase SI system. To investigate the evolution and function of its family members in Arabidopsis, we first identified 92 Arabidopsis F-box proteins related to AhSLF-S2, referred to as AtSFL (S-locus F-box-like) in this report. Phylogenetic analyses with family members from several plant species revealed that they could be classified into five subgroups, and the SLF genes appeared to have had a monophyletic origin. Yeast two-hybrid analyses showed that most AtSFL proteins could interact with one or more ASK (Arabidopsis Skp1-like) proteins, a component of the SCF (Skp1/Cullin or CDC53/F-box) complex, suggesting that AtSFLs may function in the process of ubiquitin/26S proteasome-mediated proteolysis. Transcript analysis found that most of AtSFL genes are expressed ubiquitously and only three of them (AtSFL61, 79 and 85) displayed a tissue-specific pattern. In consistent, phenotypic observations for T-DNA insertion lines of 37 AtSFL genes revealed that most of them are functionally redundant, but inactivation of two AtSFL genes (AtSFL61 and 70) appears to have caused developmental defects in embryo or female gametophyte. Our results show that a diversified expression and functional pattern are associated with AtSFL genes, indicating that they play important roles in various biological processes in Arabidopsis.

An Auxin-Inducible F-Box Protein CEGENDUO Negatively Regulates Auxin-Mediated Lateral Root Formation in Arabidopsis

Previously, we characterized 92 Arabidopsis genes (AtSFLs) similar to the S-locus F-box genes involved in S-RNase-based self-incompatibility and found that they likely play diverse roles in Arabidopsis. In this study, we investigated the role of one of these genes, CEGENDUO (CEG, AtSFL61), in the lateral root formation. A T-DNA insertion in CEG led to an increased lateral root production, which was complemented by transformation of the wild-type gene. Its downregulation by RNAi also produced more lateral roots in transformed Arabidopsis plants whereas its overexpression generated less lateral roots compared to wild-type, indicating that CEG acts as a negative regulator for the lateral root formation. It was found that CEG was expressed abundantly in vascular tissues of the primary root, but not in newly formed lateral root primordia and the root meristem, and induced by exogenous auxin NAA (α-naphthalene acetic acid). In addition, the ceg mutant was hyposensitive to NAA, IAA (indole-3-acetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid), as well as the auxin transport inhibitor TIBA (3,3,5-triiodobenzoic acid), showing that CEG is an auxin-inducible gene. Taken together, our results show that CEG is a novel F-box protein negatively regulating the auxin-mediated lateral root formation in Arabidopsis.

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.

Expressional profiling of genes related to pollination and fertilization in rice

Pollination and fertilization are key steps leading to seed and fruit formation. To obtain genes involved in pollination and fertilization in rice, an RNA fingerprinting technique, cDNA-AFLP (amplified fragment length polymorphism), was used to generate transcript profiles related to pollination. Of 15,000 cDNA fragments inspected, 2,100 showed altered expression in the pollinated pistil, of which about 1/5 were up-regulated (URP) and the rest down-regulated (DRP), suggesting that gene repression is a predominant mode of gene regulation in the pollinated pistil. Over 200 URP genes were sequenced and databank searches revealed that 70% of them represented previously unnoticed rice genes. DNA blot analysis of 20 URP genes detected no restriction fragment length polymorphisms (RFLP) between two relatively distant rice varieties, suggesting that the URP genes are highly conserved and likely play important roles in pollination and fertilization. Furthermore, two genes, URP47 and URP63, probably encoding an ADP-ribosylation factor and a membrane transporter, respectively, in relation to pollination were discussed.

Isolation of candidate R disease resistance genes from rice

Using a polymerase chain reaction (PCR) based method six distinct candidate disease resistant gene (R) homologs from rice have been isolated. The rice sequences are organized into two phylogenetic groups with contrasting genomic organization patterns. The first group, represented by a single sequence, Osh359-1, is more similar to non-riceR sequences than to rice ones and has a simple genomic organization. The second group, represented by Osh359-3, contains the remaining five rice sequences. Osh359-3 consists of a multi-gene family. The members of Osh359-3 family are further found to be clustered together in the genome.