Molecular cloning and the expression profile of two calnexin genes – CNX1 and CNX2 – during pollen development and pollen tube growth in Petunia

Calnexin (CNX) is a crucial chaperone of the endoplasmic reticulum (ER) that participates in the folding and quality control of glycoproteins. In plants, CNX contributes to multiple physiological processes, including growth, development, and adaptation to abiotic stresses. Nevertheless, its specific function in male gametophyte development and pollen tube growth remains poorly understood. In this work, we report for the first time the molecular cloning of two Petunia hybrida CNX homologs, PhCNX1 and PhCNX2, examine their expression profiles during pollen development, germination, and tube elongation, and discuss their potential functional involvement in these processes. We successfully cloned and characterized full-length cDNAs of PhCNX1 and PhCNX2, which encode proteins containing conserved motifs typical of the CNX/CRT family. Through a combination of qRT-PCR, western blotting, fluorescence in situ hybridization (FISH), and immunocytochemistry, we show that both genes are expressed in Petunia anthers, germinating pollen grains, and elongating tubes. During male gametophyte development, PhCNX1 shows peak expression at the microspore stage, whereas PhCNX2 reaches its highest transcript levels in mature pollen grains. Notably, in dry pollen, both genes exhibit a marked decrease in transcript abundance. FISH analysis indicates that PhCNX mRNAs are detected in both somatic and germline tissues but are predominantly localized to the cytoplasm of tapetal cells from the microsporocyte to microspore stages. Moreover, western blot analysis reveals a progressive increase in CNX protein levels throughout anther development and its accumulation in dry pollen. Immunocytochemical staining confirms CNX localization in all anther cell types, with notable enrichment in tapetal cells and the cytoplasm of pollen grains prior to anther dehiscence; this is further supported by immunogold labeling indicating CNX localization within the ER. In germinating pollen and elongating tubes, PhCNX transcripts accumulate in the cytoplasm near the apertures and along the tube (except the clear zone), whereas CNX protein is concentrated in the ER-dense subapical region. Our findings reveal that PhCNX1 and PhCNX2 are dynamically regulated throughout the course of pollen development within the anther, as well as during pollen germination and tube growth. This spatiotemporal expression pattern supports the notion that CNX functions as a molecular chaperone facilitating the high levels of protein synthesis essential for proper male gametophyte maturation and the polarized growth of the pollen tube.