In planta assessment of the role of thioredoxin h proteins in the regulation of S-locus receptor kinase signaling in transgenic Arabidopsis

Abstract

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.

Figure 1.

Phylogenetic and expression analyses of A. thaliana TRX genes. A, Phylogenetic tree of A. thaliana thioredoxin h-type proteins and B. napus THL1 and THL2 proteins. The scale represents the evolutionary distance expressed as the number of substitutions per residue. B, Amino acid sequences of the active sites of thioredoxin h proteins. Note the canonical Trp-Cys-Gly-Pro-Cys (WCGPC) in A. thaliana AtTRX1, AtTRX2, AtTRX7, AtTRX8, and AtTRX9 and the Trp-Cys-Pro-Pro-Cys (WCPPC) sequence in B. napus BnTHL1 and BnTHL2 and A. thaliana AtTRX3, AtTRX4, and AtTRX5. C, Quantitative real-time PCR analysis of AtTRX3, AtTRX4, and AtTRX5 mRNA in C24 wild-type stage 12 stigmas. Relative transcript levels were determined by the standard curve method with the level of AtTRX3 transcripts set at 100. Error bars indicate sds calculated from triplicate experiments. D, Synteny between the A. thaliana AtTRX3 (top), AtTRX4 (middle), and AtTRX5 (bottom) genomic regions and the corresponding genomic regions in B. rapa.

Figure 2.

Analysis of the TM-localized Cys residue in SRK. A, Amino acid sequence alignment of a region encompassing the TM region of SRK protein variants. Bold characters indicate the TM region predicted by TMpred. The box highlights the Cys residue, C463 in AlSRKb, thought to be essential for the interaction of SRK with thioredoxin h proteins. The arrow shows the C463W mutation introduced into SRKb. The underlined amino acids in BnSRK₉₁₀ indicate the residues that were included in the SRK kinase domain fragment used as bait in yeast two-hybrid studies (Bower et al., 1996; Mazzurco et al., 2001). B, Pollination assays of stage 13 stigmas from C24 transformants expressing the SRKb(C463W) mutant. Note the intense incompatibility response (manifested by lack of pollen tube growth) produced by C24 SCRb-expressing pollen and the fully compatible response (manifested by profuse pollen tube growth) produced by C24 wild-type (WT) pollen. Bar = 10 μm.

Figure 3.

Functional analysis of trx3-1 and trx4-1 T-DNA mutants. A, Sites of T-DNA insertion in the AtTRX3 and AtTRX4 genes. Boxes and lines represent exons and introns, respectively. The T-DNA insertion sites as determined by sequencing are indicated by triangles. B, RT-PCR analysis of AtTRX3, AtTRX4, and SRKb in total RNA isolated from stigmas. The left section shows results for Col-0 wild-type (Col), Col-0[SRKb-SCRb] (Col[SRKb]), and trx3-1[SRKb-SCRb] (trx3[SRKb]). The right section shows results for the Col-0 wild type (Col), Col-0[SRKb-SCRb] (Col[SRKb]), trx4-1[SRKb-SCRb] (trx4[SRKb]), and trx3-1 trx4-1[SRKb-SCRb] (trx3 trx4[SRKb]). The Ubiquitin-Conjugating enzyme 21 (UBC21) gene was used as control. C, Pollination phenotypes of the trx3-1 and trx4-1 single and double mutant SRKb-SCRb plants. The images illustrate pollination responses toward untransformed wild-type Col-0 pollen (top row) and Col-0 SCRb-expressing pollen (bottom row). The genotype of stigmas used for pollination is indicated in each section. Note that, irrespective of their genotype at the AtTRX3 and AtTRX4 loci, all stigmas pollinated with wild-type pollen produced profuse pollen tube growth and all stigmas expressing SRKb exhibited an intense incompatibility response toward SCRb-expressing pollen. Bar = 10 μm.