Molecular cloning and transcriptional activity of a new Petunia calreticulin gene involved in pistil transmitting tract maturation, progamic phase, and double fertilization

Abstract

Calreticulin (CRT) is a highly conserved and ubiquitously expressed Ca2+-binding protein in multicellular eukaryotes. As an endoplasmic reticulum-resident protein, CRT plays a key role in many cellular processes including Ca2+ storage and release, protein synthesis, and molecular chaperoning in both animals and plants. CRT has long been suggested to play a role in plant sexual reproduction. To begin to address this possibility, we cloned and characterized the full-length cDNA of a new CRT gene (PhCRT) from Petunia. The deduced amino acid sequence of PhCRT shares homology with other known plant CRTs, and phylogenetic analysis indicates that the PhCRT cDNA clone belongs to the CRT1/CRT2 subclass. Northern blot analysis and fluorescent in situ hybridization were used to assess PhCRT gene expression in different parts of the pistil before pollination, during subsequent stages of the progamic phase, and at fertilization. The highest level of PhCRT mRNA was detected in the stigma–style part of the unpollinated pistil 1 day before anthesis and during the early stage of the progamic phase, when pollen is germinated and tubes outgrow on the stigma. In the ovary, PhCRT mRNA was most abundant after pollination and reached maximum at the late stage of the progamic phase, when pollen tubes grow into the ovules and fertilization occurs. PhCRT mRNA transcripts were seen to accumulate predominantly in transmitting tract cells of maturing and receptive stigma, in germinated pollen/growing tubes, and at the micropylar region of the ovule, where the female gametophyte is located. From these results, we suggest that PhCRT gene expression is up-regulated during secretory activity of the pistil transmitting tract cells, pollen germination and outgrowth of the tubes, and then during gamete fusion and early embryogenesis.

Calreticulin expression in relation to exchangeable Ca2+ level that changes dynamically during anthesis, progamic phase, and double fertilization in Petunia

“…We previously used aniline blue to characterize the successive growth of the pollen tubes in Petunia pistil (Lenartowski et al 2014 ). Here, using the semi-thin sections of Petunia stigmas, styles and ovules stained with methylene blue, we could detect two main stages before pollination and three main stages after pollination, covering pistil transmitting tract maturation, the progamic phase, and gamete fusion (Fig.…”

“…Given its Ca 2+ -binding/buffering property, CRT could effectively provide transient storage and release of Ca 2+ during these processes. Our previous characterization of PhCRT mRNA expression patterns by Northern blot and in situ hybridization revealed enhanced expression of PhCRT during pistil transmitting tract maturation and at the beginning of the progamic phase, when pollen germinates and pollen tubes outgrow on the stigma (Lenartowski et al 2014 ). Furthermore, we observed the highest level of PhCRT mRNA in the ovary at fertilization and during early embryogenesis.…”

Molecular evidence that rough endoplasmic reticulum is the site of calreticulin translation in Petunia pollen tubes growing in vitro

“…Additionally, high levels of CRT and its transcripts were detected in developing tobacco anthers, especially in pollen tetrads and in the active tapetum, as well as in dry, hydrated, and germinating pollen and growing tubes (Nardi et al 2006). Finally, we observed strong CRT expression in germinated pollen and elongated tubes of Haemanthus (Lenartowska et al 2009) and Petunia (Lenartowska et al 2001, 2002; Lenartowski et al 2014, 2015). On the basis of our findings, we speculated that CRT is translated on ER-associated ribosomes in germinating pollen and growing tubes.…”

Cloning and expression of an APETALA1-like gene from Nelumbo nucifera

“…The downstream primers were B26 (5'-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3') and UPM (5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3'). The middle fragment and the template cDNA of 3'-RACE and 5'-RACE were obtained following protocols for the RevertAid TM First Strand cDNA synthesis and SMARTer TM RACE cDNA amplification kits (Lenartowski et al, 2014).…”

“…RT-PCR and quantitative real-time PCR methods were used to analyze the expression pattern of NnAP1 in various organs and during different developmental stages (Lenartowski et al, 2014). This research provides a basis for control of flowering time and the mechanism of floral formation using genetic engineering technology.…”