Allele-specific effects of PDF2 on floral morphology in Arabidopsis thaliana

The class IV homeodomain-leucine zipper (HD-ZIP IV) proteins represent plant-specific transcription factors, most of which are expressed predominantly in the epidermis.Citation^1,Citation² They are suggested to form homo- and/or hetero-dimers via the HD-zipper-loop-zipper (ZLZ) domain and bind to a cis-regulatory element termed the L1 box of epidermis-specific genes.Citation^1,Citation³ The HD-ZLZ domain is followed by the steroidogenic acute regulatory protein-related lipid transfer (START) domain, which is conserved both in HD-ZIP III and IV proteins.Citation⁴ While the mammalian START domain proteins bind lipids or sterols,Citation⁵ the START domain of a cotton HD-ZIP IV protein has been shown to interact with a MYB transcription factor, suggesting formation of a protein complex that specifies target genes.Citation⁶ The START-adjacent (SAD) domain follows the START domain in HD-ZIP IV proteinsCitation⁴ but its function remains unknown. Molecular and genetic studies of the HD-ZIP IV family in Arabidopsis thaliana, which consists of 16 genes, have revealed some of their roles in plant development: GLABRA2 (GL2) is required for trichome and root hair differentiation,Citation^7,Citation⁸ ANTHOCYANINLESS2 (ANL2) for anthocyanin accumulation in the epidermis,Citation⁹ and HOMEODOMAIN GLABROUS11 (HDG11, pronounced “hedge”11) and HDG2 for normal trichome development.Citation^1,Citation¹⁰ ARABIDOPSIS THALIANA MERISTEM LAYER1 (ATML1) and PROTODERMAL FACTOR2 (PDF2) are a pair of paralogous genes and play a critical role in the differentiation and maintenance of shoot epidermis.Citation³

In our recent study, we generated double mutants between pdf2 and several mutants of the HDG genes expressed in the shoot epidermis and found that, in addition to the occasional lack of short stamens observed in pdf2, hdg1, hdg2, and hdg12 single mutant alleles, double mutant combinations of pdf2–1 with hdg1–1, hdg2–2, hdg2–3, hdg5–1 or hdg12–2 had abnormal floral organs with homeotic conversion: petals were transformed into sepaloid organs and stamens into infertile and/or carpelloid organs.Citation¹¹ However, double mutant combinations between another pdf2 allele, pdf2–2, and hdg alleles showed only reduction in stamen number without homeotic conversions of floral organs.Citation¹¹ These results indicate that PDF2 and these HDG genes are cooperatively involved in stamen development and in specifying petal and stamen identity, while suggesting an allele-specific effect of pdf2–1

To further investigate the allele-specific effect of pdf2 mutations on the phenotype, we obtained 2 additional pdf2 alleles, pdf2–3 (GK-201 A01–014488) and pdf2–4 (SAIL 70G06), from ABRC and generated double mutants with hdg1 and hdg2 alleles (Fig. 1). In the pdf2–3 single mutant, which has a T-DNA insertion in the middle of the START domain (Fig. 1A), about 20% of flowers lacked 1 or 2 short stamens, and the flowers of the double mutants between pdf2–3 and hdg1–1, hdg1–2, or hdg2–3 had defective stamens infrequently besides a reduced number of stamens (Fig. 2). On the other hand, in the pdf2–4 mutant, which contains a T-DNA insertion in front of the HD coding region (Fig. 1A) and is thought to be a null allele, the frequency of the lack of short stamens was similar to that in the wild type. In the flowers of pdf2–4 hdg1–2 and pdf2–4 hdg2–3, the lack of short stamens was observed with more frequency than in each single mutant (Fig. 2A). They had also defective stamens, although only infrequently (Fig. 2B). We observed a few carpelloid stamens in pdf2–4 hdg1–1 flowers but no homeotic conversion of petals was observed in the above-described pdf2 single and pdf2 hdg double mutants.

Figure 1. Structure of PDF2, HDG1 and HDG2 genes and their effect on floral development. (A) Exon-intron structure and T-DNA insertion sites of PDF2, HDG1 and HDG2. (B) Flowers of each pdf2 single mutant and those of pdf2–1 hdg1–1, pdf2–1 hdg2–3, pdf2–4 hdg1–1 and pdf2–4 hdg2–3 double mutants. Petals, or both petals and stamens are removed from the flowers on the right side. Bars = 1 cm.

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Figure 2. Stamen phenotype in pdf2 hdg double mutants. The early-formed flowers on the primary inflorescence were examined (n ≥ 17). (A) Variation of the number of stamens per flower. (B) Percentage of phenotypes observed in long and short stamens of wild-type and mutant flowers. Fertile stamens had yellow mature anthers with pollen grains, while no pollen grain was observed in infertile stamens.

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These results support that the loss of function of PDF2 affects stamen development in the absence of HDG1 or HDG2. However, it seems more conceivable that, unlike the null pdf2–4 allele, the pdf2–1 allele, which often resulted in homeotic phenotypes in double mutants with some hdg alleles (Fig. 1B),Citation¹¹ does not represent a mere loss-of-function mutation but rather has some additional effect on stamen and petal development. Since the HD-ZLZ domains are sufficient for HD-ZIP IV proteins to form dimers,Citation⁶ truncated Pdf2 proteins in pdf2–1, pdf2–2, and pdf2–3 alleles (Fig. 1A) could form homo- or hetero-dimers by their retained HD-ZLZ domain, if expressed, and interfere with the binding of normal HD-ZIP IV proteins to target L1-box sequences. Considering the position of T-DNA insertion, however, the Pdf2–1 protein would lack most part of the START domain while Pdf2–2 and Pdf2–3 proteins retain some part. It is thus possible that these incomplete START domains in pdf2–2 and pdf2–3 are inhibitory for homo- or hetero-dimer formation. Alternatively, the Pdf2–1 protein might be more stable than Pdf2–2 and Pdf2–3 proteins and have more severe effects. If the latter is the case, the START domain is possibly important for regulating the stability or longevity of HD-ZIP IV proteins. More studies are needed to validate this possibility and clarify the reason responsible for the allele-specificity of pdf2–1.