Tobacco (Nicotiana tabacum) pollen is a well-suited model for studying many fundamental biological processes owing to its well-defined and distinct development stages. It is also one of the major agents involved in the transmission of infectious viroids, which is the primary mechanism of viroid pathogenicity in plants. However, some viroids are non-transmissible and may be possibly degraded or eliminated during the gradual process of pollen development maturation. The molecular details behind the response of developing pollen against the apple fruit crinkle viroid (AFCVd) infection and viroid eradication is largely unknown. In this study, we performed an integrative analysis of the transcriptome and proteome profiles to disentangle the molecular cascade of events governing the three pollen development stages: early bicellular pollen (stage 3, S3), late bicellular pollen (stage 5, S5), and 6 h-pollen tube (PT6). The integrated analysis delivered the molecular portraits of the developing pollen against AFCVd infection, including mechanistic insights into the viroid eradication during the last steps of pollen development. The isobaric tags for label-free relative quantification (iTRAQ) with digital gene expression (DGE) experiments led us to reliably identify subsets of 5321, 5286, and 6923 proteins and 64,033, 60,597, and 46,640 expressed genes in S3, S5, and PT6, respectively. In these subsets, 2234, 2108 proteins and 9207 and 14,065 mRNAs were differentially expressed in pairwise comparisons of three stages S5 vs. S3 and PT6 vs. S5 of control pollen in tobacco. Correlation analysis between the abundance of differentially expressed mRNAs (DEGs) and differentially expressed proteins (DEPs) in pairwise comparisons of three stages of pollen revealed numerous discordant changes in mRNA/protein pairs. Only a modest correlation was observed, indicative of divergent transcription, and its regulation and importance of post-transcriptional events in the determination of the fate of early and late pollen development in tobacco. The functional and enrichment analysis of correlated DEGs/DEPs revealed the activation in pathways involved in carbohydrate metabolism, amino acid metabolism, lipid metabolism, and cofactor as well as vitamin metabolism, which points to the importance of these metabolic pathways in pollen development. Furthermore, the detailed picture of AFCVd-infected correlated DEGs/DEPs was obtained in pairwise comparisons of three stages of infected pollen. The AFCVd infection caused the modulation of several genes involved in protein degradation, nuclear transport, phytohormone signaling, defense response, and phosphorylation. Intriguingly, we also identified several factors including, DNA-dependent RNA-polymerase, ribosomal protein, Argonaute (AGO) proteins, nucleotide binding proteins, and RNA exonucleases, which may plausibly involve in viroid stabilization and eradication during the last steps of pollen development. The present study provides essential insights into the transcriptional and translational dynamics of tobacco pollen, which further strengthens our understanding of plant-viroid interactions and support for future mechanistic studies directed at delineating the functional role of candidate factors involved in viroid elimination.
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.
