Investigating the developmental and genetic basis of nectar spur morphology
Petal spurs are long tubular outgrowths of the petal that fill with nectar to attract pollinators. Across the genus Aquilegia, petals spurs have remarkably diversity of shape and length. Previous studies in the Kramer Lab (Puzey et al., 2011) have found that the spur develops with two distinct phases corresponding to cell division (Phase I) and cell elongation (Phase II). In Phase I cell divisions occur diffusely throughout the petal primordium, then become localized to the incipient nectary. This period of localized, oriented cell divisions completely ceases by the time the petal is ~2mm in length, representing only 3% of its final length in A. coerulea. From this point the spur enters Phase II, in which lengthening of the spur is entirely dependent on highly anisotropic cell elongation. Variation in the duration of elongation accounts for the majority of spur-length diversity across the family.
Having this detailed model now in hand, we can pursue a series of experiments to investigate the genetic basis of spur initiation, elaboration and evolution. For instance, initial transcriptomic studies on the petal spur revealed the upregulation of several hormone response pathways (Yant et al., 2015). Our exploration of the auxin pathway led to the surprising discovery that homologs of the STYLISH gene family play a specific role in nectary development (Min and Kramer, 2019) while analyses of the ARF6/8 homologs indicate that they support cell proliferation (Zhang et al., 2020). Now, we have turned our attention to the Brassinosteroid (BR) response pathway, particularly the Aquilegia homolog of BEH4, a known BR response transcription factor that has been shown to regulate a wide range of developmental processes, including cell elongation. This suggests that BR signaling may play a critical role in the switch from cell division to cell elongation that leads to the formation and length of the petal spur in Aquilegia. We are investigating the role of BEH4 in the Aquilegia petal spur using a combination of gene expression studies, hormone applications and gene silencing. We are also using several inter-specific and inter-organ comparative transcriptomic approaches to identify gene co-expression networks in the Aquilegia petal, which we believe will support a more complete understanding of petal spur morphogenesis in Aquilegia. These datasets are also providing critical reference information for interspecific QTL mapping (Ballerini et al., 2019; Ballerini et al., 2020).
Ballerini, E. S., E. M. Kramer, S. A. Hodges. 2019. Comparative transcriptomics of early petal development across four diverse species of Aquilegia reveal few genes consistently associated with nectar spur development. BMC Genomics, 20: 668. doi: 10.1186/s12864-019-6002-9.
Ballerini, E. S., Y. Min, M. B. Edwards, E. M. Kramer, S. A. Hodges. 2020. POPOVICH, encoding a C2H2-zinc finger transcription factor, plays a central role in the development of a key innovation, floral nectar spurs, in Aquilegia. Proceedings of the National Academy of Science, USA, in press.
Hodges, S.A. and M.L. Arnold. 1995. Spurring plant diversification: Are floral nectar spurs a key innovation? Proceedings of the Royal Society of London Series B-Biological Sciences, 262:343-348.
Min, Y., J.I. Bunn, E. M. Kramer. 2019. Homologs of the STYLISH gene family control nectary development in Aquilegia. New Phytologist, 221: 1090-1100.
Puzey, J. P.*, S. Gerbode*, S. A. Hodges, E. M. Kramer1, and L. Mahadevan1. 2011. Evolution of spur length diversity in Aquilegia petals is achieved solely through cell shape anisotropy. Proceedings of the Royal Academy of Science, London, Series B, 279:1640-1645. *co-first authors, 1joint corresponding authors.
Yant, L., S. Collani, J. R. Puzey, C. Levy, and E. M. Kramer. 2015. Molecular basis for three-dimensional elaboration of the Aquilegia petal spur. Proceedings of the Royal Academy of Science, London, Series B, 282: 20142778.
Zhang, R., Y. Min, L. D. Holappa, C. L. Walcher-Chevillet, X. Dian, E. Donaldson, H.-Z. Kong, E. M. Kramer. 2020. A role for the Auxin Response Factors ARF6 and ARF8 homologs in petal spur elongation and nectary maturation in Aquilegia. New Phytologist, 227:1392-1405.