Floral evolution in Croton L. species (Euphorbiaceae): ontogeny and global outline of gene expression

Abstract

Morphological modifications occurred naturally in flowers during evolution of angiosperms, resulting in several forms, whose studies dating from the mid-eighteenth century aim to understand the emergence of the flower. When it comes to floral diversity, Euphorbiaceae receives great prominence, having the genus Croton highlighted, whose staminate flowers present sepals and petals, showing opposite nectaries to the petals with 10-100 stamens, and the female flowers are usually characterized by presenting reduced petals, nectary disc, bifid or multifid styles, 3 - carpel ovary and 3 - locular , with an anatropous ovule per locule. Although the morphological differences in Euphorbiaceae are observed in adult flowers, the evolution of different floral forms is better understood when considering their development. Coupled to the ontogenetic study, the detection of auxin levels performed by several authors in studies with Arabidopsis showed that this hormone affects many stages of development and also in the expression of genes which promote the formation of flower primordia, such as LFY and genes of the molecular model ABC. The objective of this research is to study the floral evolution in three morphologically different species of Croton (Croton fuscescens Spreng., Croton salutaris Casar. and Croton sphaerogynus Baill.). Besides ontogenetic analyzes, immunocytochemistry of the auxin flow and determination of global gene expression outline will be performed. Flower buds and flowers in several stages of development will be collected at different locations in the states of São Paulo and Rio de Janeiro and the species identified by an expert. For ontogenetic analysis, the material will be fixed, dehydrated in alcohol series, embedded in paraffin, sectioned on a rotary microtome and slides will be stained according to conventional techniques. The investigation of the initial stage of floral primordia formation also include SEM analysis, and confocal microscopy. TEM will be performed to try to identify possible changes in subcellular meristematic cells with different development between species and also for the study of the nectaries. The detection levels of auxin during the formation of floral primordia will be conducted through immulocalization. Study of gene expression will make use of sequencing by NGS (Next Generation Sequence) considering 4 zones of cluster in order to identify the expression outline at a given time for the 4 zones demarcated in the first stage of the project. Morphological variations highlighted in Croton, may provide comprehensive understanding as to the origin or suppression of sterile whorls of staminate and pistillate flowers in the family and thus suggest hypotheses about the evolutionary steps that led to the immense floral diversity in Euphorbiaceae.Keywords: floral development, morphoanatomical variations, nectaries, auxin, RNA seq. (AU)