In the double-flowered lily cultivar 'Elodie', stamens (floral whorl 3) are converted into petal-like organs to varying degrees, and the degree of this petaloidy correlates with reduced expression of the C-class floral homeotic gene LelAG1 in whorl 3, while whorl-4 (carpel) expression is retained and the LelAG1 coding sequence is essentially wild type. The unresolved problem is the mechanism behind this whorl-3-specific reduction of AGAMOUS-like expression — promoter-level cis-change or DNA methylation versus whorl-3-specific trans-acting regulators — and whether graded reduction of AG-like activity is causally sufficient for the graded stamen-to-petal conversion. Linked secondary questions from the source study concern the in-planta functional impact of the unusually long C-terminal region of Lilium AG-like proteins and the connection between reduced AG activity, jasmonic-acid biosynthesis (via DAD1), and stamen maturation versus identity.

Background

Floral organ identity in flowering plants is explained by the ABC(E) model: combinatorial activity of MADS-box transcription factors (named for the founding MCM1, AGAMOUS, DEFICIENS and SRF genes) specifies organ identity in four concentric whorls — sepals, petals, stamens and carpels. The C-class gene AGAMOUS (AG) specifies stamen and carpel identity in whorls 3 and 4 and terminates the floral meristem; loss of AG function converts stamens into petal-like organs, producing the prized 'double' flowers of many ornamentals. Lily (Lilium) is a petaloid monocot whose two outer whorls both form showy tepals (perianth organs that are not differentiated into sepals and petals) because B-class genes are expressed in whorls 1–3 (the modified ABCE model for monocots). Double-flowered lily cultivars arise mainly through homeotic conversion of whorl-3 stamens into petaloid organs, termed stamen petaloidy. AG-like (class C) cDNAs had been isolated from several Lilium taxa — for example LLAG1 from Lilium longiflorum, whose ectopic expression in Arabidopsis causes floral homeotic modifications (Benedito et al. 2004) — but before the source study no AG-like sequence or quantitative whorl-specific expression analysis existed for a weakly double-flowered lily cultivar showing graded petaloidy. Akita, Nakada and Kanno (2011) cloned the AG-like cDNA LelAG1 (GenBank AB596844; 254 amino acids) from the Asiatic hybrid 'Elodie', in which individual plants reproducibly bear one of three flower types: type I (weak petaloidy; stamen-like, pollenless whorl-3 organs), type II (intermediate; slim petaloid organs) and type III (strong; fully petaloid whorl-3 organs). Northern hybridization and quantitative real-time RT-PCR (reverse transcription followed by quantitative polymerase chain reaction) showed LelAG1 transcript confined to whorls 3 (stamens/petaloid stamens) and 4 (carpels) in all three types, with whorl-4 expression similar across types but whorl-3 expression declining progressively as petaloidy strengthens (to roughly 60% and 20% of the whorl-4 level in type-II and type-III flowers, respectively). The LelAG1 coding sequence, including the conserved C-terminal AG motifs I and II, is nearly identical to that of the wild-type cultivar 'Siberia', so coding variation does not explain the phenotype; notably, Lilium AG-like proteins carry a longer C-terminal region than eudicot AG proteins, a region whose functional importance in Arabidopsis is documented but whose effect in Lilium was never tested. In Arabidopsis, AG also promotes late stamen maturation by directly activating jasmonic-acid (JA; a lipid-derived plant hormone) biosynthesis through genes such as DAD1 (DEFECTIVE IN ANTER DEHISCENCE 1, a phospholipase catalyzing the first JA-biosynthesis step) (Ito et al. 2007), which raises the question whether reduced LelAG1 in 'Elodie' affects stamen maturation, organ identity, or both. Because Agrobacterium-mediated transformation is feasible in some Lilium species, the source study's authors proposed transgenic modification of LelAG1 expression in whorl 3 to test and exploit the correlation — a proposal that has not been executed. The open question is thus the mechanistic chain: what reduces LelAG1 transcript specifically in whorl 3, and is graded AG-like down-regulation the cause, rather than merely a correlate, of graded stamen petaloidy in Lilium.

Problem Statement

What is the molecular mechanism that reduces AGAMOUS-like (LelAG1) transcript accumulation specifically in whorl 3 of the double-flowered lily 'Elodie', and is graded reduction of AG-like activity in whorl 3 causally sufficient for the graded conversion of stamens into petaloid organs? The cause of the whorl-3-specific reduction is open between cis-level explanations (promoter sequence change or DNA methylation of the promoter) and trans-level explanations (whorl-3-specific or reduced-activity trans-acting regulators, identified and tested), and the causal question is whether the direction and degree of AG-like change predict the direction and degree of stamen petaloidy across the graded phenotypes. Two linked secondary questions from the source study's discussion fall within this objective: whether the unusually long C-terminal region of Lilium AG-like proteins alters protein function in planta, and whether reduced AG-like activity affects jasmonic-acid (JA, a lipid-derived plant hormone) biosynthesis and thereby stamen maturation as distinct from stamen identity. The mechanism is expected to be general to graded double-flowered lilies such as 'Red Twin', where it accounts for the same whorl-3-specific, graded AG down-regulation and petaloidy.

References

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