In Planta Assessment of the Role of Thioredoxin h Proteins in the Regulation of S-Locus Receptor Kinase Signaling in Transgenic Arabidopsis

The self-incompatibility (SI) response of the Brassicaceae is mediated by allele-specific interaction between the stigma-localized S-locus receptor kinase (SRK) and its ligand, the pollen coat-localized S-locus cysteine-rich protein (SCR). Based on work in Brassica spp., the thioredoxin h-like proteins THL1 and THL2, which interact with SRK, have been proposed to function as oxidoreductases that negatively regulate SRK catalytic activity. By preventing the spontaneous activation of SRK in the absence of SCR ligand, these thioredoxins are thought to be essential for the success of cross pollinations in self-incompatible plants. However, the in planta role of thioredoxins in the regulation of SI signaling has not been conclusively demonstrated. Here, we addressed this issue using Arabidopsis thaliana plants transformed with the SRKb-SCRb gene pair isolated from self-incompatible Arabidopsis lyrata. These plants express an intense SI response, allowing us to exploit the extensive tools and resources available in A. thaliana for analysis of SI signaling. To test the hypothesis that SRK is redox regulated by thioredoxin h, we expressed a mutant form of SRKb lacking a transmembrane-localized cysteine residue thought to be essential for the SRK-thioredoxin h interaction. We also analyzed transfer DNA insertion mutants in the A. thaliana orthologs of THL1 and THL2. In neither case did we observe an effect on the pollination responses of SRKb-expressing stigmas toward incompatible or compatible pollen. Our results are consistent with the conclusion that, contrary to their proposed role, thioredoxin h proteins are not required to prevent the spontaneous activation of SRK in the A. thaliana stigma.Many flowering plants possess self-incompatibility (SI), a genetic system that promotes outcrossing by preventing self-fertilization. In the Brassicaceae family, the SI response is controlled by haplotypes of the S locus, each of which contains two genes that encode highly polymorphic proteins, the S-locus receptor kinase (SRK), which is a plasma membrane resident single-pass transmembrane Ser/Thr receptor kinase displayed at the surface of stigma epidermal cells (Stein et al., 1991; Takasaki et al., 2000), and the S-locus Cys-rich protein (SCR), which is the pollen coat-localized ligand for SRK (Schopfer et al., 1999; Kachroo et al., 2001; Takayama et al., 2001). SRK and SCR exhibit allele-specific interactions, whereby only SRK and SCR encoded by the same S-locus haplotype interact. In a self-pollination, the binding of this “self” pollen-borne SCR to the extracellular domain of SRK activates the SRK kinase, thereby triggering a cellular response in stigma epidermal cells that causes inhibition of pollen germination and tube penetration into the stigma epidermal cell wall (for review, see Tantikanjana et al., 2010).Tight regulation of SRK kinase activity and its signaling cascade is critical for productive pollen-stigma interactions because constitutive (i.e. SCR-independent) activity of the receptor is expected to result in sterile stigmas that reject both compatible and incompatible pollen. In the classical view of ligand-activated receptor kinases, the receptor occurs as catalytically inactive monomers in the absence of ligand and only becomes activated upon ligand-induced dimerization (for review, see Lemmon and Schlessinger, 2010). However, some receptor kinases in both animals (Chan et al., 2000; Ehrlich et al., 2011) and plants (Giranton et al., 2000; Wang et al., 2005, 2008; Shimizu et al., 2010; Bücherl et al., 2013) form catalytically inactive dimers or oligomers in the absence of ligand, with receptor activation presumably resulting from ligand-induced higher order oligomerization or conformational changes (Lemmon and Schlessinger, 2010). Similar to the latter receptors, SRK forms oligomers in unpollinated stigmas, i.e. in the absence of SCR (Giranton et al., 2000), at least partly via ligand-independent dimerization domains located within the SRK extracellular domain (Naithani et al., 2007). It has been proposed that these ligand-independent SRK oligomers are maintained in an inactive state by thioredoxins, the ubiquitous small oxidoreductases that reduce disulfide bridges in proteins (Buchanan and Balmer, 2005). This hypothesis is supported by the following observations: (1) two Brassica napus thioredoxins, the Thioredoxin H-Like proteins THL1 and THL2, were identified as SRK interactors in a yeast (Saccharomyces cerevisiae) two-hybrid screen that used the B. napus SRK₉₁₀ kinase domain as bait (Bower et al., 1996); (2) when purified from pistils or insect cells, the Brassica oleracea SRK₃ variant was found to exhibit constitutive autophosphorylation activity in vitro, and this activity was inhibited by Escherichia coli-expressed THL1 proteins and was restored by addition of pollen coat proteins containing self but not of pollen coat proteins containing nonself SCR (Cabrillac et al., 2001); (3) the catalytic activity of THL1 was required for its inhibition of SRK₃ autophosphorylation activity in vitro (Cabrillac et al., 2001); and (4) antisense suppression of THL1/THL2 gene expression in the stigmas of a self-compatible B. napus strain reportedly produced a low-level constitutive incompatibility (Haffani et al., 2004), as might be expected if the THL1/THL2 proteins prevent the spontaneous activation of SRK-mediated signaling in stigmas.These observations notwithstanding, the in planta role of thioredoxin h proteins as negative regulators of SRK activity has not been conclusively demonstrated. To date, this proposed function has only been evaluated in a self-compatible strain of B. napus (Haffani et al., 2004). Consequently, it is not known if the proposed inhibitory effect of these thioredoxins on SRK catalytic activity is manifested in self-incompatible stigmas and if it applies to all SRK variants, be they derived from Brassica spp. or other self-incompatible species of the Brassicaceae such as Arabidopsis lyrata.In this study, we tested the in planta role of thioredoxin h proteins in the regulation of SI signaling using a transgenic self-incompatible Arabidopsis thaliana model that we generated by transforming A. thaliana with the SRKb-SCRb gene pair isolated from the Sb haplotype of self-incompatible A. lyrata (Kusaba et al., 2001; Nasrallah et al., 2002, 2004). We had previously shown that the stigmas of A. thalianaSRKb-SCRb transformants can exhibit an SI response that is as robust as the SI response observed in naturally self-incompatible A. lyrata, demonstrating that A. thaliana, which harbors nonfunctional S-locus haplotypes (Kusaba et al., 2001; Sherman-Broyles et al., 2007; Shimizu et al., 2008; Boggs et al., 2009c), has nevertheless retained all other factors required for SI. In view of the availability in A. thaliana of a highly efficient transformation method and numerous genetic resources, the A. thalianaSRK-SCR transgenic model has enabled the use of experimental approaches that are difficult or impossible to implement in Brassica species and has thus proven to be an invaluable platform for in planta analysis of SRK and SI signaling (Liu et al., 2007; Boggs et al., 2009a, 2009b; Tantikanjana et al., 2009; Tantikanjana and Nasrallah, 2012).We therefore used this transgenic A. thaliana self-incompatible model to determine if abolishing the proposed SRK-thioredoxin h interaction or eliminating expression of the major thioredoxin h proteins expressed in stigmas would affect the outcome of self- or cross pollination. To this end, we expressed a mutant form of SRKb that lacked the Cys residue previously shown to be required for the interaction of SRK with THLs (Mazzurco et al., 2001), and we analyzed plants carrying knockout insertional mutations in thioredoxin h genes. Our results are inconsistent with the proposed role of thioredoxin h proteins as negative regulators of SRK catalytic activity and SI signaling.