Titima Tantikanjana

An alternative transcript of the S locus glycoprotein gene in a class II pollen-recessive self-incompatibility haplotype of Brassica oleracea encodes a membrane-anchored protein.

Recent reports have shown that SLG, one of two genes linked to the S locus of Brassica, encodes a secreted glycoprotein. We have used RNA gel blot analysis, genomic and cDNA clone analysis, expression in transgenic plants, and immunodetection to characterize SLG2, the SLG gene derived from the S2 haplotype. This haplotype belongs to the class II group of S haplotypes that exhibit a weak incompatibility phenotype and are pollen recessive. We showed that SLG2 produces two transcript forms: the expected 1.6-kb transcript that predicts a secreted glycoprotein and an alternative 1.8-kb transcript that predicts a membrane-anchored protein. Stigmas of the S2 haplotype and pistils of transgenic tobacco plants transformed with the SLG2 gene produce a membrane-associated 62-kD protein as well as soluble 57- and 58-kD glycoforms. Because of the sequence similarity between SLG2 and the extracellular domain of the S Locus Receptor Kinase (SRK2) gene, the membrane-anchored form of SLG2 may be viewed as a naturally occurring truncated form of the receptor that lacks the kinase catalytic domain. The occurrence of this protein has potential implications for the activity of the full-length receptor. Furthermore, the underlying structure of the SLG2 gene suggests the evolution of SLG from an ancestral SRK-like gene.

Functional Analysis of the Tandem-Duplicated P450 Genes SPS/BUS/CYP79F1 and CYP79F2 in Glucosinolate Biosynthesis and Plant Development by Ds Transposition-Generated Double Mutants

A significant fraction (approximately 17%) of Arabidopsis genes are members of tandemly repeated families and pose a particular challenge for functional studies. We have used the Ac-Ds transposition system to generate single- and double-knockout mutants of two tandemly duplicated cytochrome P450 genes, SPS/BUS/CYP79F1 and CYP79F2. We have previously described the Arabidopsis supershoot mutants in CYP79F1 that exhibit massive overproliferation of shoots. Here we use a cytokinin-responsive reporter ARR5::uidA and an auxin-responsive reporter DR5::uidA in the sps/cyp79F1 mutant to show that increased levels of cytokinin, but not auxin, correlate well with the expression pattern of the SPS/CYP79F1 gene, supporting the involvement of this gene in cytokinin homeostasis. Further, we isolated Ds gene trap insertions in the CYP79F2 gene, and find these mutants to be defective mainly in the root system, consistent with a root-specific expression pattern. Finally, we generated double mutants in CYP79F1 and CYP79F2 using secondary transpositions, and demonstrate that the phenotypes are additive. Previous biochemical studies have suggested partially redundant functions for SPS/CYP79F1 and CYP79F2 in aliphatic glucosinolate synthesis. Our analysis shows that aliphatic glucosinolate biosynthesis is completely abolished in the double-knockout plants, providing genetic proof for the proposed biochemical functions of these genes. This study also provides further demonstration of how gluconisolate biosynthesis, regarded as secondary metabolism, is intricately linked with hormone homeostatis and hence with plant growth and development.

Complex networks of self-incompatibility signaling in the Brassicaceae.

The self-pollination barrier of self-incompatibility in the Brassicaceae is based on the activity of a polymorphic stigma receptor and its pollen ligand, whose allele-specific interaction triggers a signaling cascade within the stigma epidermal cell that culminates in the inhibition of pollen tube development. Recent analyses have identified signaling intermediates and revealed unexpected cross-talk between self-incompatibility signaling and pistil development. The self-incompatibility response is now thought to be based on a phosphorylation and ubiquitin-mediated degradation pathway that inhibits the secretion of factors required for successful pollination. Because manipulation of the identified signaling intermediates results in only partial disruption of the self-incompatibility reaction, this pathway likely functions in conjunction with other as-yet unidentified signaling pathways to effect complete inhibition of self-pollen.

A Dual Role for the S-Locus Receptor Kinase in Self-Incompatibility and Pistil Development Revealed by an Arabidopsis rdr6 Mutation

The coordinate evolution of self-incompatibility (SI) and stigma-anther separation, two mechanisms that promote cross-pollination in plants, has been a long-standing puzzle in evolution and development. Using a transgenic self-incompatible Arabidopsis thaliana model, we performed screens for mutants exhibiting a modified SI response. A mutation in the RNA-dependent RNA polymerase RDR6, which functions in trans-acting short interfering RNA (ta-siRNA) production, was found that simultaneously enhances SI and causes stigma exsertion, without associated increases in SRK transcript levels. While rdr6 mutants had been previously shown to exhibit stochastic stigma exsertion, our results demonstrate that the S-locus receptor kinase (SRK) gene further enhances pistil elongation and stigma exsertion in this mutant background, a process that requires SRK catalytic activity and correlates with SRK transcript levels. These results suggest that positive regulators or effectors of SI and pistil development are regulated by ta-siRNA(s). By establishing complex connections between SI and stigma exsertion through the sharing of a ta-siRNA–mediated regulatory pathway and the dual role of SRK in SI and pistil development, our study provides a molecular explanation for the coordinate evolution of these processes.

An Inducible Targeted Tagging System for Localized Saturation Mutagenesis in Arabidopsis

We describe a system of inducible insertional mutagenesis based on the Ac-Ds family of transposons for targeted tagging in Arabidopsis (Arabidopsis thaliana). In this system, the Ac and Ds elements are carried within the same T-DNA and a heat shock-inducible transposase fusion is utilized to control the levels of transposase gene expression, generating transpositions that can be subsequently stabilized without requiring crossing or segregation. We have mapped 40 single-copy lines by thermal asymmetric interlaced-PCR, which can be used as potential launch pads for heat shock mutagenesis. Using a starter line selected for detailed analysis, the efficiency of tagging over a 50-kb region in the genome was examined. Hits were obtained in the targeted genes with multiple alleles for most genes, with approximately equal numbers of hits detected in genes on either side of the T-DNA. These results establish the feasibility of our approach for localized saturation mutagenesis in Arabidopsis. This system is very efficient and much less laborious as compared to conventional crossing schemes and may be generally applicable to other plant species for which large-scale T-DNA tagging is not currently feasible.

Non-cell-autonomous regulation of crucifer self-incompatibility by Auxin Response Factor ARF3

In many angiosperms, outcrossing is enforced by genetic self-incompatibility (SI), which allows cells of the pistil to recognize and specifically inhibit “self” pollen. SI is often associated with increased stigma-anther separation, a morphological trait that promotes cross-pollen deposition on the stigma. However, the gene networks responsible for coordinate evolution of these complex outbreeding devices are not known. In self-incompatible members of the Brassicaceae (crucifers), the inhibition of “self”-pollen is triggered within the stigma epidermal cell by allele-specific interaction between two highly polymorphic proteins, the stigma-expressed S-locus receptor kinase (SRK) and its pollen coat-localized ligand, the S-locus cysteine-rich (SCR) protein. Using Arabidopsis thaliana plants that express SI as a result of transformation with a functional SRK–SCR gene pair, we identify Auxin Response Factor 3 (ARF3) as a mediator of cross-talk between SI signaling and pistil development. We show that ARF3, a regulator of pistil development that is expressed in the vascular tissue of the style, acts non-cell-autonomously to enhance the SI response and simultaneously down-regulate auxin responses in stigma epidermal cells, likely by regulating a mobile signal derived from the stylar vasculature. The inverse correlation we observed in stigma epidermal cells between the strength of SI and the levels of auxin inferred from activity of the auxin-responsive reporter DR5::GUS suggests that the dampening of auxin responses in the stigma epidermis promotes inhibition of “self” pollen in crucifer SI.

The Brassica S gene family : molecular characterization of the SLR2 gene

Abstract The S gene family of higher plants includes secreted glycoprotein-encoding genes and transmembrane protein kinase-encoding genes all of which share sequence similarity to the S-locus genes of Brassica. In our continuing effort to understand the structural and functional diversification of this gene family in Brassica, we analyzed the S-locus-related SLR2 gene from B. oleracea and B. campestris. SLR2 is a flower-specific gene that exhibits a high degree of sequence similarity to the S-locus genes derived from the S2 self-incompatibility haplotype. We present data which suggest that the SLR2 gene encodes a secreted protein as predicted from its nucleotide sequence. However, unlike other secreted glycoprotein-encoding members of the S gene family, the gene contains a 591 base-pair intron at or near its 3′ terminus. Based on this structure, we propose that this gene arose prior to speciation by partial duplication of an ancestral gene similar in structure to the S-locus-linked SRK (S Receptor Kinase) gene. The high degree of variability in SLR2 transcript levels noted in different Brassica strains was found to be correlated with the segregation of an SLR2 polymorphism, whereby a mutated SLR2 allele containing a duplication within the promoter region directs low expression. The implications of these findings for SLR2 gene function are discussed.

Site-Specific N- Glycosylation of the S-Locus Receptor Kinase and Its Role in the Self-Incompatibility Response of the Brassicaceae

N-glycosylation of the S-locus receptor kinase ensures its localization to the stigma epidermal cell membrane, where it interacts with its pollen coat-localized ligand to inhibit self-pollination. The S-locus receptor kinase SRK is a highly polymorphic transmembrane kinase of the stigma epidermis. Through allele-specific interaction with its pollen coat-localized ligand, the S-locus cysteine-rich protein SCR, SRK is responsible for recognition and inhibition of self pollen in the self-incompatibility response of the Brassicaceae. The SRK extracellular ligand binding domain contains several potential N-glycosylation sites that exhibit varying degrees of conservation among SRK variants. However, the glycosylation status and functional importance of these sites are currently unclear. We investigated this issue in transgenic Arabidopsis thaliana stigmas that express the Arabidopsis lyrata SRKb variant and exhibit an incompatible response toward SCRb-expressing pollen. Analysis of single- and multiple-glycosylation site mutations of SRKb demonstrated that, although five of six potential N-glycosylation sites in SRKb are glycosylated in stigmas, N-glycosylation is not important for SCRb-dependent activation of SRKb. Rather, N-glycosylation functions primarily to ensure the proper and efficient subcellular trafficking of SRK to the plasma membrane. The study provides insight into the function of a receptor that regulates a critical phase of the plant life cycle and represents a valuable addition to the limited information available on the contribution of N-glycosylation to the subcellular trafficking and function of plant receptor kinases.

Ligand-Mediated cis-Inhibition of Receptor Signaling in the Self-Incompatibility Response of the Brassicaceae

Coexpression of the receptor and ligand that function in crucifer self-incompatibility inhibits receptor signaling and abrogates the ability of stigma epidermal cells to arrest self pollen. The inhibition of self-pollination in self-incompatible Brassicaceae is based on allele-specific trans-activation of the highly polymorphic S-locus receptor kinase (SRK), which is displayed at the surface of stigma epidermal cells, by its even more polymorphic pollen coat-localized ligand, the S-locus cysteine-rich (SCR) protein. In an attempt to achieve constitutive activation of SRK and thus facilitate analysis of self-incompatibility (SI) signaling, we coexpressed an Arabidopsis lyrata SCR variant with its cognate SRK receptor in the stigma epidermal cells of Arabidopsis (Arabidopsis thaliana) plants belonging to the C24 accession, in which expression of SRK and SCR had been shown to exhibit a robust SI response. Contrary to expectation, however, coexpression of SRK and SCR was found to inhibit SRK-mediated signaling and to disrupt the SI response. This phenomenon, called cis-inhibition, is well documented in metazoans but has not as yet been reported for plant receptor kinases. We demonstrate that cis-inhibition of SRK, like its trans-activation, is based on allele-specific interaction between receptor and ligand. We also show that stigma-expressed SCR causes entrapment of its SRK receptor in the endoplasmic reticulum, thus disrupting the proper targeting of SRK to the plasma membrane, where the receptor would be available for productive interaction with its pollen coat-derived SCR ligand. Although based on an artificial cis-inhibition system, the results suggest novel strategies of pollination control for the generation of hybrid cultivars and large-scale seed production from hybrid plants in Brassicaceae seed crops and, more generally, for inhibiting cell surface receptor function and manipulating signaling pathways in plants.

Regulation of the S-Locus Receptor Kinase and Self-Incompatibility in Arabidopsis thaliana

Intraspecific mate selectivity often is enforced by self-incompatibility (SI), a barrier to self-pollination that inhibits productive pollen-pistil interactions. In the Brassicaceae, SI specificity is determined by two highly-polymorphic proteins: the stigmatic S-locus receptor kinase (SRK) and its pollen coat-localized ligand, the S-locus cysteine-rich protein (SCR). Arabidopsis thaliana is self fertile, but several of its accessions can be made to express SI, albeit to various degrees, by transformation with functional SRK-SCR gene pairs isolated from its close self-incompatible relative, Arabidopsis lyrata. Here, we use a newly identified induced mutation that suppresses the SI phenotype in stigmas of SRK-SCR transformants of the Col-0 accession to investigate the regulation of SI and the SRK transgene. This mutation disrupts NRPD1a, a gene that encodes a plant-specific nuclear RNA polymerase required for genomic methylation and production of some types of silencing RNAs. We show that NRPD1a, along with the RNA-dependent RNA polymerase RDR2, is required for SI in some A. thaliana accessions. We also show that Col-0 nrpd1a mutants exhibit decreased accumulation of SRK transcripts in stigmas, which is not, however, responsible for loss of SI in these plants. Together, our analysis of the nrpd1a mutation and of SRK promoter activity in various accessions reveals that the SRK transgene is subject to several levels of regulation, which vary substantially by tissue type and by accession. This study thus helps explain the well-documented differences in expression of SI exhibited by SRK-SCR transformants of different A. thaliana accessions.