Functional analysis of B and C class floral organ genes in spinach demonstrates their role in sexual dimorphism

Abstract

Background: Evolution of unisexual flowers entails one of the most extreme changes in plant development. Cultivated spinach, Spinacia oleracea L., is uniquely suited for the study of unisexual flower development as it is dioecious and it achieves unisexually by the absence of organ development, rather than by organ abortion or suppression. Male staminate flowers lack fourth whorl primordia and female pistillate flowers lack third whorl primordia. Based on theoretical considerations, early inflorescence or floral organ identity genes would likely be directly involved in sex-determination in those species in which organ initiation rather than organ maturation is regulated. In this study, we tested the hypothesis that sexual dimorphism occurs through the regulation of B class floral organ gene expression by experimentally knocking down gene expression by viral induced gene silencing.

Results: Suppression of B class genes in spinach resulted in the expected homeotic transformation of stamens into carpels but also affected the number of perianth parts and the presence of fourth whorl. Phenotypically normal female flowers developed on SpPI-silenced male plants. Suppression of the spinach C class floral organ identity gene, SpAG, resulted in loss of reproductive organ identity, and indeterminate flowers, but did not result in additional sex-specific characteristics or structures. Analysis of the genomic sequences of both SpAP3 and SpPI did not reveal any allelic differences between males and females.

Conclusion: Sexual dimorphism in spinach is not the result of homeotic transformation of established organs, but rather is the result of differential initiation and development of the third and fourth whorl primordia. SpAG is inferred to have organ identity and meristem termination functions similar to other angiosperm C class genes. In contrast, while SpPI and SpAP3 resemble other angiosperms in their essential functions in establishing stamen identity, they also appear to have an additional function in regulating organ number and identity outside of the third whorl. We present a model for the evolution of dioecy in spinach based on the regulation of B class expression.

Figure 1

SpAP3 silenced flowers. Wild type spinach male flower (a) has four stamen (one designated with arrow) and four sepals (one designated with arrow). Wild type female flowers (b) with two sepals (one marked) that envelop the central carpel (marked) which develops a single ovuled ovary with usually four stigmatic arms. Flowers from pWSRi:SpAP3 treated plants (c through f). c and d. Stigmas from the developing carpels (arrows) are visible in the third whorl, along with stamens (arrows). In flower shown in c, there are an unusual six stigmatic arms. e. A hermaphroditic flower with a carpel developing in the fourth whorl, surrounding by four stamens and four sepals. f. A flower with a central (fourth whorl) carpeloid organ surrounded by four sepals. Abbreviations: st, stamens; se, sepals; c, carpel; s, stigma.

Figure 2

SpPI-silenced flowers. a. Mixed flower with stamen (st) and fourth whorl carpel (c). b. Flower with four sepals (indicated by arrowheads) and four carpels in the third whorl. c. Adjacent male (m) and female (f) flowers.

Figure 3

Plants treated with pWSRI:SpPI were fixed, imbedded and sectioned for in situ hybridization. (a, b) Hybridization with antisense RNA SpPI probe. a. Longitudinal section showing third whorl stamen and third whorl carpel. Strong SpPI expression is present in tapetal cells in stamen, whereas no detectable staining in the carpel. b. Inflorescence cluster with female (f) and early male flower. SpPI is detected in stamen primordium. c. Hybridization with antisense RNA SpAP3 probe. Cross section through a single flower with three stamen and one carpel in third whorl. Strong SpAP3 expression is detected in developing stamen. SpAP3 expression is absent in dehiscing stamen. No discernible SpAP3 staining in carpel. d. Hybridization with antisense SpAG probe. Cross section of flower with stamen and carpel. Strong SpAG expression is detectable in both organs. e. Hybridization with sense RNA SpPI probe. Abbreviations: st, stamen; c, carpel; f, female; m, male.

Figure 4

Schematic of SpAP3 and SpPI gene structures. Dark blue boxes represent amino acid encoding regions. Light blue boxes represent 5' UTR and control regions. Thin red lines represent introns. SpAP3 has seven exons and six introns. The introns are 153 bp, 2522 bp, 96 bp, 295 bp, 103 bp, and 943 bp in length respectively. Due to the lack of reliable 5' RACE data, the first exon start site is uncertain, however, the coding region of exon 1 extends 191 bp starting from the first position of the start codon. The remaining exons 2, 3, 4, 5, and 6 are 67 bp, 62 bp, 100 bp, 42 bp, and 45 bp in length, respectively, while exon 7 starts 138 bp through the stop codon and continues another 238 bp past the stop codon, although only 182 bp of this 3' untranslated region was included in the present survey. SpPI has sevens exons and six introns. The introns are 117 bp, 1504 bp, 388 bp, 127 bp, 912 bp, and 253 bp in length, respectively. Exon 1 extends to position 188 starting from the first position of the start codon. Exons 2 through 6 are 67 bp, 62 bp, 100 bp, 30 bp, and 45 bp long, respectively. Exon 7 starts 120 bp before the end of the stop codon and continues 169 past the stop codon. Positions of potential LEAFY binding elements and CArG boxes are indicated.

Figure 5

SpAG-silenced flowers. a. Flower with complete loss of stamens or carpels in a female silenced plant. b. New inflorescence meristem emerging from center of flower on a male plant. Adjacent flower has four sepals and four opposite sterile organs (arrow). c. Stamens of male flower that failed to mature and produce pollen.

Figure 6

Model for the evolution of dioecy in spinach. In the ancestral hermaphroditic species, upstream elements, including but not limited to GA and LFY, activate both B (PI and AP3) and C (AG) class genes. Both classes of genes retain organ identity functions as described in the ABC model. Mutations in the B class genes, notated by *, result in premature termination of the flower in the third whorl, and thus the loss of the carpel. The resultant flower is male. Inactivation or suppression of expression of the B class genes, modulated by the GA response pathway, results in the expression of AG only. The absence of B class gene products causes a reduction in the number of organs in the first whorl and the formation of a single, terminal carpel. The resultant flower is female.