The Mechanism Investigation Of Arabidopsis Nascent Polypeptide-associated Complex Alpha-like Subunit Protein NACA5 In Pollen Growth

Mature pollen grains are male gametophytes of the plants,and their germination and pollen tube growth are critical for double fertilization.During the processes of pollen germination and ovule-orientated tube-growth,a serial of signal components are involved,including calcium,reactive oxygen species,nitric oxide,phosphatidylinositol and et al.,In addition,the trans-membrane ion is essential for osmotic pressure during pollen germination and tube growth,and cytosolic free Ca2+plays a vital role as a second messenger in these processes.Calmodulin-like proteins(CMLs)are transitory Ca2+sensors whose sequence is highly conserved with calmodulin's,and which could bind the cytosolic Ca2+to generate downstream biochemical and physiological changes.Previous studies have shown that CML25 is highly expressed in pollens and positively involved in pollen germination and pollen tube growth by regulating plasma inward potassium channels.To further reveal the molecular signaling pathway and mechanism of CML25,yeast two-hybrid was employed to screen the CML25 interaction proteins followed with functional analyses of them.The following results were collected from the current study:(1)We screened CML25 interacted protein nascent polypeptide-associated complex subunit alpha-like protein 5(NACA5)by yeast two-hybrid method.And the data collected by using fluorescence bimolecular complementarity technique and the Co-immunoprecipitation also confirmed the interaction between CML25 and NACA5 at the cellular and biochemical levels.(2)The genomic DNA of NACA5 is 1421 bp,and its CDS region is 630 bp and encodes 209 amino acids.The molecular weight of the predicated protein is 22.78 kDa.The NACA5 protein has NAC and UBA domains that are conserved in the NAC family,and with high homology with other known NACs.Which may suggest that NACA5 possess the general function of the NAC proteins.(3)Real-time quantification PCR and promoter+GUS staining results revealed that NACA5 is expressed in pollens;Subcellular localization experiments found that NACA5 is mainly subcellularly localized in the nucleus and cytoplasm.It is suggested that NACA5 plays a physiological regulatory role mainly in the cytoplasm and nucleus.(4)We obtained NACA5 interference strains and CRISPR knockout strains,and found the pollen germination rate and pollen tube length were significantly increased in these mutant pollens than those of wild type.It is speculated that NACA5 may be a negative regulator of pollen germination and pollen tube growth.(5)Through hybridization,double mutants of CML25 and NACA5 were constructed,and the phenotype of double mutants and cml25 tend to be consistent in pollen tube growth;It is speculated that NACA5 is upstream of CML25 and may work together with CML25 in the controlling pollen germination and tube growth.This study revealed an Arabidopsis CML25 interacting protein NACA5,and the NACA5 is negatively involved in pollen germination and pollen tube growth;The genetic analyses indicated that the NACA5 is upstream of CML25 to regulate pollen germination and tube growth.This study will help to further reveal the signaling pathways in which CML25 is located,and it will also help discover the new biological functions of NA CA5.