Some viroids—single-stranded, non-coding, circular RNA parasites of plants—are not transmissible through pollen to seeds and to next generation. We analyzed the cause for the elimination of apple fruit crinkle viroid (AFCVd) and citrus bark cracking viroid (CBCVd) from male gametophyte cells of Nicotiana tabacum by RNA deep sequencing and molecular methods using infected and transformed tobacco pollen tissues at different developmental stages. AFCVd was not transferable from pollen to seeds in reciprocal pollinations, due to a complete viroid eradication during the last steps of pollen development and fertilization. In pollen, the viroid replication pathway proceeds with detectable replication intermediates, but is dramatically depressed in comparison to leaves. Specific and unspecific viroid degradation with some preference for (−) chains occurred in pollen, as detected by analysis of viroid-derived small RNAs, by quantification of viroid levels and by detection of viroid degradation products forming “comets” on Northern blots. The decrease of viroid levels during pollen development correlated with mRNA accumulation of several RNA-degrading factors, such as AGO5 nuclease, DICER-like and TUDOR S-like nuclease. In addition, the functional status of pollen, as a tissue with high ribosome content, could play a role during suppression of AFCVd replication involving transcription factors IIIA and ribosomal protein L5.
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.
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