Transformation of Arabidopsis with a Brassica SLG/SRK region and ARC1 gene is not sufficient to transfer the self-incompatibility phenotype

Self-incompatibility (SI) promotes outbreeding in flowering plants, and in Brassica SI is genetically controlled by the S locus. Self-incompatible Brassica and self-fertile Arabidopsis belong to the same crucifer family. In addition, a comparative analysis reveals a high degree of microsynteny between the B. campestris S locus and its homologous region in Arabidopsis– with the notable exception that the Brassica SI genes, SLG and SRK, are missing. Brassica ARC1 encodes a component of the SRK signal transduction pathway leading to self-pollen rejection, and no closely related ARC1 homolog has been identified in Arabidopsis. The purpose of the research reported here was to introduce Brassica SI components into Arabidopsis in an attempt to compensate for the missing genes and to investigate whether the SI phenotype can be transferred. Inserts of approximately 40 kb from the fosmid clones F20 and F22, which span the B. napus W1 SLG-SRK region, were cloned into the plant transformation vector pBIBAC2. Transgenic plants were generated that expressed the Brassica SI genes in the flower buds. In addition, the endogenous, SLG-like, gene AtS1 was not co-suppressed by the Brassica SLG transgene. No SI phenotype was observed among the T1 BIBAC2-F20 and BIBAC2-F22 transgenic plants. When the ARC1 gene was transformed into BIBAC2-F20 or BIBAC2-F22 plants, the resulting BIBAC2-F20-ARC1 and BIBAC2-F22-ARC1 plants still set seeds normally, and no rejection response was observed when self-incompatible B. napus W1 pollen was placed on BIBAC2-F20-ARC1 or BIBAC2-F22-ARC1 Arabidopsis stigmas. Taken together, our results suggest that complementing Arabidopsis genome with Brassica SLG, SRK and ARC1 genes is unlikely to be sufficient to transfer the SI phenotype. Received: 11 November 1999 / Accepted: 14 February 2000     

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.     

Two large Arabidopsis thaliana gene families are homologous to the Brassica gene superfamily that encodes pollen coat proteins and the male component of the self-incompatibility response

The male component of the self-incompatibility response in Brassica has recently been shown to be encoded by the S locus cysteine-rich gene (SCR). SCR is related, at the sequence level, to the pollen coat protein (PCP) gene family whose members encode small, cysteine-rich proteins located in the proteo-lipidic surface layer (tryphine) of Brassica pollen grains. Here we show that the Arabidopsis genome includes two large gene families with homology to SCR and to the PCP gene family, respectively. These genes are poorly predicted by gene-identification algorithms and, with few exceptions, have been missed in previous annotations. Based on sequence comparison and an analysis of the expression patterns of several members of each family, we discuss the possible functions of these genes. In particular, we consider the possibility that SCR-related genes in Arabidopsis may encode ligands for the S gene family of receptor-like kinases in this species.     

<Emphasis Type="Italic">S</Emphasis> locus-linked F-box genes expressed in anthers of <Emphasis Type="Italic">Hordeum bulbosum</Emphasis>

Diploid Hordeum bulbosum (a wild relative of cultivated barley) exhibits a two-locus self-incompatibility (SI) system gametophytically controlled by the unlinked multiallelic loci S and Z. This unique SI system is observed in the grasses (Poaceae) including the tribe Triticeae. This paper describes the identification and characterization of two F-box genes cosegregating with the S locus in H. bulbosum, named Hordeum S locus-linked F-box 1 (HSLF1) and HSLF2, which were derived from an S ₃ haplotype-specific clone (HAS175) obtained by previous AMF (AFLP-based mRNA fingerprinting) analysis. Sequence analysis showed that both genes encode similar F-box proteins with a C-terminal leucine-rich repeat (LRR) domain, which are distinct from S locus (or S haplotype-specific) F-box protein (SLF/SFB), a class of F-box proteins identified as the pollen S determinant in S-RNase-based gametophytic SI systems. A number of homologous F-box genes with an LRR domain were found in the rice genome, although the functions of the gene family are unknown. One allele of the HSLF1 gene (HSLF1-S ₃) was expressed specifically in mature anthers, whereas no expression was detected from the other two alleles examined. Although the degree of sequence polymorphism among the three HSLF1 alleles was low, a frameshift mutation was found in one of the unexpressed alleles. The HSLF2 gene showed a low level of expression with no tissue specificity as well as little sequence polymorphism among the three alleles. The multiplicity of S locus-linked F-box genes is discussed in comparison with those found in the S-RNase-based SI system. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB511822–AB511825 and AB511859–AB511862.     

Comparative genomic analysis of CIPK gene family in <Emphasis Type="Italic">Arabidopsis</Emphasis> and <Emphasis Type="Italic">Populus</Emphasis>

Calcium serves as a second messenger in various signal transduction pathways in plants. CBL-interacting protein kinases (CIPKs), which have a variety of functions, are involved in calcium signal transduction. Previous, the studies on CIPK family members focused on Arabidopsis and rice. Here, we present a comparative genomic analysis of the CIPK gene family in Arabidopsis and poplar, a model tree species. Twenty-seven potential CIPKs were identified from poplar using genome-wide analysis. Like the CIPK gene family from Arabidopsis, CIPK genes from poplar were also divided into intron-free and intron-harboring groups. In the intron-harboring group, the intron distribution of CIPKs is rather conserved during the genome evolutionary process. Many homologous gene pairs were found in the CIPK gene family, indicating duplication events might contribute to the amplification of this gene family. The phylogenetic comparison of CIPKs in combination with intron distribution analysis revealed that CIPK genes from both Arabidopsis and poplar might have an ancient origin, which formed earlier than the separation of these two eudicot species. Our genomic and bioinformatic analysis will provide an important foundation for further functional dissection of the CBL-CIPK signaling network in poplars. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Structure and expression of AtS1, an Arabidopsis thaliana gene homologous to the S-locus related genes of Brassica

Summary Genetic and molecular analysis of the self-incompatibility locus (S-locus) of the crucifer Brassica has led to the characterization of a multigene family involved in pollen-stigma interactions. While the crucifer Arabidopsis thaliana does not have a self-incompatibility system, S-related sequences were detected in this species by cross-hybridization with Brassica DNA probes. In this paper, we show that an A. thaliana S-related sequence, designated AtS1, is expressed specifically in flower buds. Sequence analysis suggests that AtS1 encodes a secreted glycoprotein that is most similar to the Brassica S-locus related protein SLR1. As has been proposed for SLR1, this gene may be involved in determining some fundamental aspect of pollen-stigma interactions during pollination. The molecular and genetic advantages of the Arabidopsis system will provide many avenues for testing this hypothesis.     

Three members of the S multigene family are linked to the S locus of Brassica

Two self-incompatibility genes in Brassica, SLG and SRK (SLG encodes a glycoprotein; SRK encodes a receptor-like kinase), are included in the S multigene family. Products of members of the S multigene family have an SLG-like domain (S domain) in common, which may function as a receptor. In this study, three clustered members of the S multigene family, BcRK1, BcRL1 and BcSL1, were characterized. BcRK1 is a putative functional receptor kinase gene expressed in leaves, flower buds and stigmas, while BcRL1 and BcSL1 are considered to be pseudogenes because deletions causing frameshifts were identified in these sequences. Sequence and expression pattern of BcRK1 were most similar to those of the Arabidopsis receptor-like kinase gene ARK1, indicating that BcRK1 might have a function similar to that of ARK1, in processes such as cell expansion or plant growth. Interestingly, the region containing BcRK1, BcRL1 and BcSL1 is genetically linked to the S locus and the physical distance between SLG, SRK and the three S-related genes was estimated to be less than 610 kb. Thus the genes associated with self-incompatibility exist within a cluster of S-like genes in the genome of Brassica. Received: 15 April 1997 / Accepted: 13 June 1997     

Molecular chaperone function of Arabidopsis thaliana phloem protein 2-A1, encodes a protein similar to phloem lectin

Although several phloem sap proteins have been identified from protein extracts of heat-treated Arabidopsis seedlings using FPLC gel filtration columns, many of the physiological roles played by these proteins remain to be elucidated. We functionally characterized a phloem protein 2-A1, which encodes a protein similar to phloem lectin. Using a bacterially expressed recombinant protein of AtPP2-A1, we found that it performs dual functions, showing both molecular chaperone activity and antifungal activity. mRNA expression of the AtPP2-1 gene was induced by diverse external stresses such as pathogens, and other signaling molecules, such as ethylene. These results suggest that the AtPP2-A1 molecular chaperone protein plays a critical role in the Arabidopsis defense system against diverse external stresses including fungal pathogenic attack and heat shock.     

Disentangling the effects of breakdown of self-incompatibility and transition to selfing in North American Arabidopsis lyrata

A breakdown of self‐incompatibility (SI) followed by a shift to selfing is commonly observed in the evolution of flowering plants. Both are expected to reduce the levels of heterozygosity and genetic diversity. However, breakdown of SI should most strongly affect the region of the SI locus (S‐locus) because of the relaxation of balancing selection that operates on a functional S‐locus, and a potential selective sweep. In contrast, a transition to selfing should affect the whole genome. We set out to disentangle the effects of breakdown of SI and transition to selfing on the level and distribution of genetic diversity in North American populations of Arabidopsis lyrata. Specifically, we compared sequence diversity of loci linked and unlinked to the S‐locus for populations ranging from complete selfing to fully outcrossing. Regardless of linkage to the S‐locus, heterozygosity and genetic diversity increased with population outcrossing rate. High heterozygosity of self‐compatible individuals in outcrossing populations suggests that SI is not the only factor preventing the evolution of self‐fertilization in those populations. There was a strong loss of diversity in selfing populations, which was more pronounced at the S‐locus. In addition, selfing populations showed an accumulation of derived mutations at the S‐locus. Our results provide evidence that beyond the genome‐wide consequences of the population bottleneck associated with the shift to selfing, the S‐locus of A. lyrata shows a specific signal either reflecting the relaxation of balancing selection or positive selection.     

Electrostatic potentials of the S‐locus F‐box proteins contribute to the pollen S specificity in self‐incompatibility in Petunia hybrida

Self‐incompatibility (SI) is a self/non‐self discrimination system found widely in angiosperms and, in many species, is controlled by a single polymorphic S‐locus. In the Solanaceae, Rosaceae and Plantaginaceae, the S‐locus encodes a single S‐RNase and a cluster of S‐locus F‐box (SLF) proteins to control the pistil and pollen expression of SI, respectively. Previous studies have shown that their cytosolic interactions determine their recognition specificity, but the physical force between their interactions remains unclear. In this study, we show that the electrostatic potentials of SLF contribute to the pollen S specificity through a physical mechanism of ‘like charges repel and unlike charges attract’ between SLFs and S‐RNases in Petunia hybrida. Strikingly, the alteration of a single C‐terminal amino acid of SLF reversed its surface electrostatic potentials and subsequently the pollen S specificity. Collectively, our results reveal that the electrostatic potentials act as a major physical force between cytosolic SLFs and S‐RNases, providing a mechanistic insight into the self/non‐self discrimination between cytosolic proteins in angiosperms.     

Distribution of S-haplotypes and relationship with self-incompatibility in Brassica oleracea. 2. In varieties of broccoli and romanesco

The self-incompatibility (SI) character in Brassica is controlled by the S locus which contains several genes. One of them, the SLG (S Locus Glycoprotein) gene encodes a soluble glycoprotein expressed in the stigma. We used antibodies directed against SLGs and a combination of isoelectric focusing (IEF) and immunoblotting methods to identify S haplotypes, the allelic forms of the S locus, in commercial and open-pollinated varieties of broccoli and romanesco. We found 23 class-I and three class-II S haplotypes among the 199 plants analysed. Nevertheless, for a few plants, SLGs were not detected by the antibodies and these plants, designated Hw for “white pattern” haplotypes, were apparently homozygous at the S locus. Diallel crosses between Hw plants revealed the existence of four different Hw haplotypes. Several hypotheses are discussed to explain the non-recognition of the SLG products in these Hw haplotypes. The data of the present study were compared with those obtained in a previous investigation carried out on cauliflower. As in cauliflower, we observed a high frequency of the sx haplotype and a great variability in the strength of the SI phenotype for sx plants (in the homozygous or heterozygous state). For both broccoli and romanesco, about 50% of the plants presented a SI phenotype strong enough to be exploited for hybrid production. Received: 27 July 1998 / Accepted: 5 August 1998     

Apple MdABCF assists in the transportation of S‐RNase into pollen tubes

Self‐incompatibility (SI) is a reproductive isolation mechanism in flowering plants. Plants in the Solanaceae, Rosaceae and Plantaginaceae belong to the gametophytic self‐incompatibility type. S‐RNase, which is encoded by a female‐specific gene located at the S locus, degrades RNA in the pollen tube and causes SI. Recent studies have provided evidence that S‐RNase is transported non‐selectively into the pollen tube, but have not specified how this transportation is accomplished. We show here that the apple (Malus domestica) MdABCF protein, which belongs to group F of the ABC transporter family, assists in transportation of S‐RNase into the pollen tube. MdABCF is located in the pollen tube membrane and interacts with S‐RNase. S‐RNase was unable to enter the pollen tube when MdABCF was silenced by antisense oligonucleotide transfection. Our results show that MdABCF assists in transportation of either self or non‐self S‐RNase into the pollen tube. Moreover, MdABCF coordinates with the cytoskeleton to transport S‐RNase. Blockage of S‐RNase transport disrupts self‐incompatibility in this system.     

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. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Conservation of S-locus for self-incompatibility in Brassica napus (L.) and Brassica oleracea (L.)

 Self-incompatibility (SI) in Brassica is a sporophytic system, genetically determined by alleles at the S-locus, which prevents self-fertilization and encourages outbreeding. This system occurs naturally in diploid Brassica species but is introduced into amphidiploid Brassica species by interspecific breeding, so that in both cases there is a potential for yield increase due to heterosis and the combination of desirable characteristics from both parental lines. Using a polymerase chain reaction (PCR) based analysis specific for the alleles of the SLG (S-locus glycoprotein gene) located on the S-locus, we genetically mapped the S-locus of B. oleracea for SI using a F₂ population from a cross between a rapid-cycling B. oleracea line (CrGC-85) and a cabbage line (86-16-5). The linkage map contained both RFLP (restriction fragment length polymorphism) and RAPD (random amplified polymorphic DNA) markers. Similarly, the S-loci were mapped in B. napus using two different crosses (91-SN-5263×87-DHS-002; 90-DHW-1855-4×87-DHS-002) where the common male parent was self-compatible, while the S-alleles introgressed in the two different SI female parents had not been characterized. The linkage group with the S-locus in B. oleracea showed remarkable homology to the corresponding linkage group in B. napus except that in the latter there was an additional locus present, which might have been introgressed from B. rapa. The S-allele in the rapid-cycling Brassica was identified as the S₂₉ allele, the S-allele of the cabbage was the S ₅ allele. These same alleles were present in our two B. napus SI lines, but there was evidence that it might not be the active or major SI allele that caused self-incompatibility in these two B. napus crosses. Received: 7 June 1996/Accepted: 6 September 1996     

Pollen-expressed F-box gene family and mechanism of S-RNase-based gametophytic self-incompatibility (GSI) in Rosaceae

Many species of Rosaceae, Solanaceae, and Plantaginaceae exhibit S-RNase-based self-incompatibility (SI) in which pistil-part specificity is controlled by S locus-encoded ribonuclease (S-RNase). Although recent findings revealed that S locus-encoded F-box protein, SLF/SFB, determines pollen-part specificity, how these pistil- and pollen-part S locus products interact in vivo and elicit the SI reaction is largely unclear. Furthermore, genetic studies suggested that pollen S function can differ among species. In Solanaceae and the rosaceous subfamily Maloideae (e.g., apple and pear), the coexistence of two different pollen S alleles in a pollen breaks down SI of the pollen, a phenomenon known as competitive interaction. However, competitive interaction seems not to occur in the subfamily Prunoideae (e.g., cherry and almond) of Rosaceae. Furthermore, the effect of the deletion of pollen S seems to vary among taxa. This review focuses on the potential differences in pollen-part function between subfamilies of Rosaceae, Maloideae, and Prunoideae, and discusses implications for the mechanistic divergence of the S-RNase-based SI.