Expressed sequence-tag analysis of tobacco sperm cells reveals a unique transcriptional profile and selective persistence of paternal transcripts after fertilization

Abstract

Transcript analysis of male gametes of Nicotiana tabacum was conducted to gather gene expression data regarding the specialization of male germ cells and transmission of paternal transcripts during fertilization. We constructed a tobacco sperm cell cDNA library yielding 1,864 expressed sequence tags representing 1,050 clusters; 37.2% of these clusters have no homologs in GenBank, and 42% did not match any functionally classified protein. A comparative analysis of tobacco sperm transcripts with those of Arabidopsis and maize confirms that some genes are conserved in sperm specialization, while some are distinct to tobacco germline cells. Using reverse transcription-PCR (RT-PCR) of selected transcripts, we evaluated expression of sperm-obtained sequences in vegetative tissue, isolated egg cells, zygotes, and two-celled proembryos, identifying sperm cell-specific transcripts as potential markers for fertilization analysis. We further confirmed that two clusters of sperm transcripts were detected in zygotes about 10 h after fertilization, offering new examples of apparently paternally transmitted transcripts that may be involved in egg cell activation and/or early embryogenesis.

Genes of Both Parental Origins Are Differentially Involved in Early Embryogenesis of a Tobacco Interspecies Hybrid

“…In maize, a transgene driven by a 35S promoter in the paternal genome was almost immediately transcribed in the zygote after fertilization [11]. Our previous work also confirmed that paternal transcripts in sperm were also found in fertilized egg cells [19], [20], indicating the possible involvement of sperm-delivered paternal transcripts in zygote development. Meyer and Scholten (2007) reported that paternal transcripts of 24 genes were found in zygote of maize [14].…”

“…8-cell embryos were isolated from SR1 ovaries at 132HAP, Hamayan ovaries at 72 HAP, and hybrid ovaries at 96 HAP, respectively. The method for embryo isolation has been described [17]–[19]. Single cells were transferred to 2×lysis/binding buffer in 0.2 ml tubes and frozen immediately in −80°C refrigerator for later use.…”

The Maternal-to-Zygotic Transition in Higher Plants

“…Such methods, either direct dissection or dissection after brief enzyme treatment, are well established for isolation of sperm cells, egg cells, and early embryos from N. tabacum (Fu, Sun, Zhou, & Yang, 1996;He et al, 2007;Sun, Yang, & Zhou, 1993;Xin et al, 2011), Oryza sativa (Khalequzzaman & Haq, 2005;Ohnishi et al, 2011;Takanashi et al, 2010;Zhang, Dong, Potrykus, & Galli, 1999), and Z. mays (Kranz et al, 1991). Living paired N. tabacum sperm cells can be released after osmotic shock of a pollen tube, and the paired sperm cells washed and collected under an inverted microscope, using a special micropipette, for further analysis .…”

Cis-Regulatory Elements Determine Germline Specificity and Expression Level of an Isopentenyltransferase Gene in Sperm Cells of Arabidopsis

“…5, G-R). Sperm cells produce a complex complement of messenger RNAs contributing to their development, differentiation and fertilization (Gou et al, 2001(Gou et al, , 2009Engel et al, 2003;Borges et al, 2008;Xin et al, 2011). A number of these mRNAs encode key regulators that are required for sperm cell control, identity, and function.…”