Dynamics of auxin concentration, distribution and transport, and NIT2 expression reveal their involvement in microspore embryogenesis

Abstract

Microspore embryogenesis constitutes an intriguing system in which a cell, the microspore, is reprogrammed from its gametophytic program towards an embryogenic pathway by stress treatments, giving rise to an embryo and a plant, a widely used method to obtain double-haploid plants with many applications for plant breeding. The auxin indole-3-acetic acid (IAA) is a major coordinating signal in the regulation of plant development. Despite the abundant data on the auxin involvement in plant growth and development, no information on the role of endogenous IAA on microspore embryogenesis is available. In this work IAA levels and distribution, expression of BnNIT2, responsible of conversion of indol-3-acetonitrile to IAA, and effects of inhibition of IAA transport and action by N-1-naphthylphthalamic acid (NPA) and α-(p-Chlorophenoxy) isobutyric acid (PCIB) treatments were analyzed during Brassica napus microspore embryogenesis, a system in which the process is induced and embryo developed in vitro from individual isolated cells without addition of plant growth regulators. Experimental approach included immunofluorescence using anti-IAA antibodies and confocal analysis, BnNIT2 expression analysis by qPCR, quantification of IAA levels by LC/ESI-MSMS, and treatments with NPA and BCIP. Results indicated de novo synthesis of IAA at early stages of microspore embryogenesis, progressive IAA increase with microspore-embryo development, and differential distribution pattern in embryo regions at late developmental stages. BnNIT2 gene was up-regulated during microspore embryogenesis. Both, the inhibition of the IAA transport by NPA and the inhibition of IAA action by PCIB negatively affected the microspore-embryo development. NPA also modified the IAA distribution pattern in embryos and at cellular level IAA accumulated in small cytoplasmic compartments suggesting that auxin transport involving the secretory pathway and/or the endoplasmic reticulum could operate during microspore embryogenesis. Taken together, results indicated that endogenous auxin biosynthesis, action and transport are involved in microspore embryogenesis initiation and development.