In Press
Zhang R, Min Y, Holappa LD, Walcher-Chevillet CL, Dian X, Donaldson E, Kong H-Z, Kramer EM. A role for the Auxin Response Factors ARF6 and ARF8 homologs in petal spur elongation and nectary maturation in Aquilegia. New Phytologist. In Press. nph.16633_1.pdf
Filiault DL, Ballerini ES, Mandáková T, Aköz G, Derieg NJ, Schmutz J, Jenkins J, Grimwood J, Shu S, Hayes RD, et al. The Aquilegia genome provides insight into adaptive radiation and reveals an extraordinarilypolymorphic chromosome with a unique history. eLife. 2018;10.7554/eLife.36426. elife-36426-v2.pdf
Min Y, Kramer EM. The Aquilegia JAGGED homolog promotes proliferation of adaxial cell types in both leaves and stems. New Phytologist. 2017;216 :536-548. nph.14282.pdf
Sharma B, Kramer EM. Aquilegia B gene homologs promote petaloidy of the sepals and maintenance of the C domain boundary. EvoDevo. 2017;8 :22. s13227-017-0085-7.pdf
Qin G, Xu C, Ming R, Tang H, Guyot R, Kramer EM, Hu Y, Yi X, Qi Y, Xu X, et al. The pomegranate (Punica granatum L.) genome and the genomics of punicalagin biosynthesis. Plant Journal. 2017;doi:10.1111/tpj.13625.Abstract

Pomegranate (Punica granatum L.) is a perennial fruit crop grown since ancient times that has been planted worldwide and is known for its functional metabolites, particularly punicalagins. We have sequenced and assembled the pomegranate genome with 328 Mb anchored into nine pseudo-chromosomes and annotated 29 229 gene models. A Myrtales lineage-specific whole-genome duplication event was detected that occurred in the common ancestor before the divergence of pomegranate and Eucalyptus. Repetitive sequences accounted for 46.1% of the assembled genome. We found that the integument development gene INNER NO OUTER (INO) was under positive selection and potentially contributed to the development of the fleshy outer layer of the seed coat, an edible part of pomegranate fruit. The genes encoding the enzymes for synthesis and degradation of lignin, hemicelluloses and cellulose were also differentially expressed between soft- and hard-seeded varieties, reflecting differences in their accumulation in cultivars differing in seed hardness. Candidate genes for punicalagin biosynthesis were identified and their expression patterns indicated that gallic acid synthesis in tissues could follow different biochemical pathways. The genome sequence of pomegranate provides a valuable resource for the dissection of many biological and biochemical traits and also provides important insights for the acceleration of breeding. Elucidation of the biochemical pathway(s) involved in punicalagin biosynthesis could assist breeding efforts to increase production of this bioactive compound.

KEYWORDS:  adaption; genome; pomegranate; punicalagin biosynthesis; system evolution

Holappa LD, Ronald PC, Kramer EM. Evolutionary analysis of Snf1-Related Protein Kinase2 (SnRK2) and Calcium Sensor (SCS) gene lineages, and dimerization of rice homologs, suggest deep biochemical conservation across angiosperms. . Frontiers in Plant Science. 2017;8 :396. fpls-08-00395.pdf
Kramer EM. Stranger in a strange land: The utility and interpretation of heterologous expression. Frontiers Plant Science [Internet]. 2015;6 :734. Publisher's Version fpls-06-00734.pdf
Yant L, Collani S, Puzey J, Levy C, Kramer EM. Molecular basis for three-dimensional elaboration of the Aquilegia petal spur. Proc Biol Sci. 2015;282 :20142778.Abstract

By enforcing specific pollinator interactions, Aquilegia petal nectar spurs maintain reproductive isolation between species. Spur development is the result of three-dimensional elaboration from a comparatively two-dimensional primordium. Initiated by localized, oriented cell divisions surrounding the incipient nectary, this process creates a pouch that is extended by anisotropic cell elongation. We hypothesized that the development of this evolutionary novelty could be promoted by non-mutually exclusive factors, including (i) prolonged, KNOX-dependent cell fate indeterminacy, (ii) localized organ sculpting and/or (iii) redeployment of hormone-signalling modules. Using cell division markers to guide transcriptome analysis of microdissected spur tissue, we present candidate mechanisms underlying spur outgrowth. We see dynamic expression of factors controlling cell proliferation and hormone signalling, but no evidence of contribution from indeterminacy factors. Transcriptome dynamics point to a novel recruitment event in which auxin-related factors that normally function at the organ margin were co-opted to this central structure. Functional perturbation of the transition between cell division and expansion reveals an unexpected asymmetric component of spur development. These findings indicate that the production of this three-dimensional form is an example of organ sculpting via localized cell division with novel contributions from hormone signalling, rather than a product of prolonged indeterminacy.

Nikolov LA, Staedler YM, Manickam S, Schonenberger J, Endress PK, Kramer EM, Davis CC. Floral structure and development in Rafflesiaceae with emphasis on their exceptional gynoecia. Am J Bot. 2014;101 :225-43.Abstract

PREMISE OF THE STUDY: The holoparasitic plant family Rafflesiaceae include the world's largest flowers. Despite their iconic status, relatively little is known about the morphology and development of their flowers. A recent study clarified the organization of the outer (sterile) floral organs, surprisingly revealing that their distinctive floral chambers arose via different developmental pathways in the two major genera of the family. Here, we expand that research to investigate the structure and development of the reproductive organs of Rafflesiaceae. METHODS: Serial sectioning, scanning electron microscopy, and x-ray tomography of floral buds were employed to reconstruct the structure and development of all three Rafflesiaceae genera. KEY RESULTS: Unlike most angiosperms, which form their shoot apex from the primary morphological surface, the shoot apex of Rafflesiaceae instead forms secondarily via internal cell separation (schizogeny) along the distal boundary of the host-parasite interface. Similarly, the radially directed ovarial clefts of the gynoecium forms via schizogeny within solid tissue, and no carpels are initiated from the floral apex. CONCLUSIONS: The development of the shoot apex and gynoecium of Rafflesiaceae are highly unusual. Although secondary formation of the morphological surface from the shoot apex has been documented in other plant groups, secondary derivation of the inner gynoecium surface is otherwise unknown. Both features are likely synapomorphies of Rafflesiaceae. The secondary derivation of the shoot apex may protect the developing floral shoot as it emerges from within dense host tissue. The secondary formation of the ovarial clefts may generate the extensive placental area necessary to produce hundreds of thousands of ovules.

Nikolov LA, Tomlinson PB, Manickam S, Endress PK, Kramer EM, Davis CC. Holoparasitic Rafflesiaceae possess the most reduced endophytes and yet give rise to the world's largest flowers. Ann Bot. 2014;114 :233-42.Abstract

BACKGROUND AND AIMS: Species in the holoparasitic plant family Rafflesiaceae exhibit one of the most highly modified vegetative bodies in flowering plants. Apart from the flower shoot and associated bracts, the parasite is a mycelium-like endophyte living inside their grapevine hosts. This study provides a comprehensive treatment of the endophytic vegetative body for all three genera of Rafflesiaceae (Rafflesia, Rhizanthes and Sapria), and reports on the cytology and development of the endophyte, including its structural connection to the host, shedding light on the poorly understood nature of this symbiosis. METHODS: Serial sectioning and staining with non-specific dyes, periodic-Schiff's reagent and aniline blue were employed in order to characterize the structure of the endophyte across a phylogenetically diverse sampling. KEY RESULTS: A previously identified difference in the nuclear size between Rafflesiaceae endophytes and their hosts was used to investigate the morphology and development of the endophytic body. The endophytes generally comprise uniseriate filaments oriented radially within the host root. The emergence of the parasite from the host during floral development is arrested in some cases by an apparent host response, but otherwise vegetative growth does not appear to elicit suppression by the host. CONCLUSIONS: Rafflesiaceae produce greatly reduced and modified vegetative bodies even when compared with the other holoparasitic angiosperms once grouped with Rafflesiaceae, which possess some vegetative differentiation. Based on previous studies of seeds together with these findings, it is concluded that the endophyte probably develops directly from a proembryo, and not from an embryo proper. Similarly, the flowering shoot arises directly from the undifferentiated endophyte. These filaments produce a protocorm in which a shoot apex originates endogenously by formation of a secondary morphological surface. This degree of modification to the vegetative body is exceptional within angiosperms and warrants additional investigation. Furthermore, the study highlights a mechanical isolation mechanism by which the host may defend itself from the parasite.

ann_bot-2014-nikolov-233-42.pdf mcu114supp.pdf
Fei Z, Wang Y, Allen R, Kramer EM, Tadege M. STENOFOLIA recruits TOPLESS to repress ASYMMETRIC LEAVES2 at the leaf margin and promote leaf blade outgrowth in Medicago truncatula. Plant Cell. 2014;26 :650-64.Abstract

The Medicago truncatula WUSCHEL-related homeobox (WOX) gene, STENOFOLIA (STF), plays a key role in leaf blade outgrowth by promoting cell proliferation at the adaxial-abaxial junction. STF functions primarily as a transcriptional repressor, but the underlying molecular mechanism is unknown. Here, we report the identification of a protein interaction partner and a direct target, shedding light on the mechanism of STF function. Two highly conserved motifs in the C-terminal domain of STF, the WUSCHEL (WUS) box and the STF box, cooperatively recruit TOPLESS (Mt-TPL) family corepressors, and this recruitment is required for STF function, as deletion of these two domains (STFdel) impaired blade outgrowth whereas fusing Mt-TPL to STFdel restored function. The homeodomain motif is required for direct repression of ASYMMETRIC LEAVES2 (Mt-AS2), silencing of which partially rescues the stf mutant phenotype. STF and LAMINALESS1 (LAM1) are functional orthologs. A single amino acid (Asn to Ile) substitution in the homeodomain abolished the repression of Mt-AS2 and STF's ability to complement the lam1 mutant of Nicotiana sylvestris. Our data together support a model in which STF recruits corepressors to transcriptionally repress its targets during leaf blade morphogenesis. We propose that recruitment of TPL/TPL-related proteins may be a common mechanism in the repressive function of modern/WUS clade WOX genes.

Sharma B, Yant L, Hodges SA, Kramer EM. Understanding the development and evolution of novel floral form in Aquilegia. Curr Opin Plant Biol. 2014;17 :22-7.Abstract

Flowers of the lower eudicot Aquilegia (columbine) possess morphological innovations, namely elaborate petal spurs and a fifth distinct organ identity, the staminodium, that are well suited to the investigation of key questions in developmental evolution. The recent evolution of these characteristics combined with a growing set of genetic and genomic resources has provided insight into how the traits arose and diversified. The petal spur appears to represent a key innovation that diversified largely via modification of specific aspects of cell expansion. In the case of the staminodium, gene duplication has played a role in allowing a novel organ identity to be carved out of the traditional ABC program.

Pabon-Mora N, Sharma B, Holappa LD, Kramer EM, Litt A. The Aquilegia FRUITFULL-like genes play key roles in leaf morphogenesis and inflorescence development. Plant J. 2013;74 :197-212.Abstract

The APETALA1/FRUITFULL (AP1/FUL) MADS box transcription factors are best known for the role of AP1 in Arabidopsis sepal and petal identity, the canonical A function of the ABC model of flower development. However, this gene lineage underwent multiple duplication events during angiosperm evolution, providing different taxa with unique gene complements. One such duplication correlates with the origin of the core eudicots, and produced the euAP1 and euFUL clades. Together, euAP1 and euFUL genes function in proper floral meristem identity and repression of axillary meristem growth. Independently, euAP1 genes function in floral meristem and sepal identity, whereas euFUL genes control phase transition, cauline leaf growth and fruit development. To investigate the impact of the core eudicot duplication on the functional diversification of this gene lineage, we studied the role of pre-duplication FUL-like genes in columbine (Aquilegia coerulea). Our results show that AqcFL1 genes are broadly expressed in vegetative and reproductive meristems, leaves and flowers. Virus-induced gene silencing of the loci results in plants with increased branching, shorter inflorescences with fewer flowers, and dramatic changes in leaf shape and complexity. However, aqcfl1 plants have normal flowers and fruits. Our results show that, in contrast to characterized AP1/FUL genes, the AqcFL1 loci are either genetically redundant or have been decoupled from the floral genetic program, and play a major role in leaf morphogenesis. We analyze the results in the context of the core eudicot duplication, and discuss the implications of our findings in terms of the genetic regulation of leaf morphogenesis in Aquilegia and other flowering plants.