Analysis of sticky generative cell mutants reveals that suppression of callose deposition in the generative cell is necessary for generative cell internalization and differentiation in Arabidopsis.

Abstract

In flowering plants, double fertilization between male and female gametophytes, which are separated by distance, largely depends on the unique pattern of the male gametophyte (pollen): two non-motile sperm cells suspended within a tube-producing vegetative cell. A morphological screen to elucidate the genetic control governing the strategic patterning of pollen has led to the isolation of a sticky generative cell (sgc) mutant that dehisces abnormal pollen with the generative cell immobilized at the pollen wall. Analyses revealed that the sgc mutation is specifically detrimental to pollen development, causing ectopic callose deposition that impedes the timely internalization and differentiation of the generative cell. We found that the SGC gene encodes the highly conserved domain of unknown function 707 (DUF707) gene that is broadly expressed but is germline specific during pollen development. Additionally, transgenic plants co-expressing fluorescently fused SGC protein and known organelle markers showed that SGC localizes in the endoplasmic reticulum, Golgi apparatus and vacuoles in pollen. A yeast two-hybrid screen with an SGC bait identified a thaumatin-like protein that we named GCTLP1, some homologs of which bind and/or digest β-1,3-glucans, the main constituent of callose. GCTLP1 is expressed in a germline-specific manner and colocalizes with SGC during pollen development, indicating that GCTLP1 is a putative SGC interactor. Collectively, our results show that SGC suppresses callose deposition in the nascent generative cell, thereby allowing the generative cell to fully internalize into the vegetative cell and correctly differentiate as the germline progenitor, with the potential involvement of the GCTLP1 protein, during pollen development in Arabidopsis.