Sexual plant reproduction depends on the production and differentiation of functional gametes by the haploid gametophyte generation. Currently, we have a limited understanding of the regulatory mechanisms that have evolved to specify the gametophytic developmental programs. To unravel such mechanisms, it is necessary to identify transcription factors (TF) that are part of such haploid regulatory networks. Here we focus on bZIP TFs that have critical roles in plants, animals and other kingdoms. We report the functional characterization of Arabidopsis thaliana AtbZIP34 that is expressed in both gametophytic and surrounding sporophytic tissues during flower development. T-DNA insertion mutants in AtbZIP34 show pollen morphological defects that result in reduced pollen germination efficiency and slower pollen tube growth both in vitro and in vivo. Light and fluorescence microscopy revealed misshapen and misplaced nuclei with large lipid inclusions in the cytoplasm of atbzip34 pollen. Scanning and transmission electron microscopy revealed defects in exine shape and micropatterning and a reduced endomembrane system. Several lines of evidence, including the AtbZIP34 expression pattern and the phenotypic defects observed, suggest a complex role in male reproductive development that involves a sporophytic role in exine patterning, and a sporophytic and/or gametophytic mode of action of AtbZIP34 in several metabolic pathways, namely regulation of lipid metabolism and/or cellular transport.

KEY MESSAGE : bZIP TF network in pollen. Transcriptional control of gene expression represents an important mechanism guiding organisms through developmental processes and providing plasticity towards environmental stimuli. Because of their sessile nature, plants require effective gene regulation for rapid response to variation in environmental and developmental conditions. Transcription factors (TFs) provide such control ensuring correct gene expression in spatial and temporal manner. Our work reports the interaction network of six bZIP TFs expressed in Arabidopsis thaliana pollen and highlights the potential functional role for AtbZIP18 in pollen. AtbZIP18 was shown to interact with three other pollen-expressed bZIP TFs-AtbZIP34, AtbZIP52, and AtbZIP61 in yeast two-hybrid assays. AtbZIP18 transcripts are highly expressed in pollen, and at the subcellular level, an AtbZIP18-GFP fusion protein was located in the nucleus and cytoplasm/ER. To address the role of AtbZIP18 in the male gametophyte, we performed phenotypic analysis of a T-DNA knockout allele, which showed slightly reduced transmission through the male gametophyte. Some of the phenotype defects in atbzip18 pollen, although observed at low penetrance, were similar to those seen at higher frequency in the T-DNA knockout of the interacting partner, AtbZIP34. To gain deeper insight into the regulatory role of AtbZIP18, we analysed atbzip18/- pollen microarray data. Our results point towards a potential repressive role for AtbZIP18 and its functional redundancy with AtbZIP34 in pollen.

Heat shock transcription factors (Hsfs) are involved in multiple aspects of stress response and plant growth. However, their role during male gametophyte development is largely unknown, although the generative phase is the most sensitive and critical period in the plant life cycle. Based on a wide screen of T-DNA mutant lines, we identified the atren1 mutation (restricted to nucleolus1) in early male gametophytic gene At1g77570, which has the closest homology to HSFA5 gene, the member of a heat shock transcription factor (HSF) gene family. The mutation causes multiple defects in male gametophyte development in both structure and function. Because the mutation disrupts an early acting (AtREN1) gene, these pollen phenotype abnormalities appear from bicellular pollen stage to pollen maturation. Moreover, the consequent progamic phase is compromised as well as documented by pollen germination defects and limited transmission via male gametophyte. In addition, atren1/− plants are defective in heat stress (HS) response and produce notably higher proportion of aberrant pollen grains. AtREN1 protein is targeted specifically to the nucleolus that, together with the increased size of the nucleolus in atren1 pollen, suggests that it is likely to be involved in ribosomal RNA biogenesis or other nucleolar functions.