Here, we present a protocol for labeling and live visualization of RNA-protein complexes in the form of ribonucleoprotein particles (RNPs) in tobacco pollen tubes. We describe steps for constructing RNA-pp7/MS2 tag and biolistic gene-gun-mediated pollen transformation. We then provide detailed procedures for RNA labeling using PP7 aptamer nascent RNA tagging and a fluorescently labeled Pseudomonas aeruginosa PP7 bacteriophage coat protein (PCP) reporter that binds to PP7 RNA stem loops. This protocol is adaptable to other cell types by employing tissue-specific promoters.

• Keywords
• cell biology, developmental biology, microscopy, plant sciences,
• MeSH
• Staining and Labeling methods   MeSH
• Pseudomonas aeruginosa genetics   metabolism   MeSH
• Pollen Tube * metabolism   genetics   MeSH
• Ribonucleoproteins metabolism   MeSH
• RNA, Plant genetics   metabolism   MeSH
• Nicotiana * genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Ribonucleoproteins MeSH
• RNA, Plant MeSH

• Publication type
• Letter MeSH
• Research Support, Non-U.S. Gov't MeSH

• Keywords
• anther, biotechnology, breeding, crops, pollen,
• Publication type
• Editorial MeSH

Proplastids are essential precursors for multi-fate plastid biogenesis, including chloroplast differentiation, a powerhouse for photosynthesis in plants. Arabidopsis ankyrin repeat protein (AKRP, AT5G66055) is a plastid-localized protein with a putative function in plastid differentiation and morphogenesis. Loss of function of akrp leads to embryo developmental arrest. Whether AKRP is critical pre-fertilization has remained unresolved. Here, using reverse genetics, we report a new allele, akrp-3, that exhibited a reduced frequency of mutant embryos (<13%) compared to previously reported alleles. akrp-3 affected both male and female gametophytes resulting in reduced viability, incompetence in pollen tube attraction, altered gametic cell fate, and embryo arrest that were depleted of chlorophyll. AKRP is widely expressed, and the AKRP-GFP fusion localized to plastids of both gametophytes, in isolated chloroplast and co-localized with a plastid marker in pollen and pollen tubes. Cell-type-specific complementation of akrp-3 hinted at the developmental timing at which AKRP might play an essential role. Our findings provide a plausible insight into the crucial role of AKRP in the differentiation of both gametophytes and coupling embryo development with chlorophyll synthesis.

• Keywords
• embryo development, fertilization, pollen, pollen tube reception, proplastid,
• Publication type
• Journal Article MeSH