Abstract:

Calcium-dependent protein kinases(CPK)are a class of protein kinases that respond to calcium signals and participate in regulating the polar growth of pollen tubes,which is crucial for plant reproduction and is precisely controlled by calcium signals and the cytoskeleton.To reveal the function of CPK in the polar growth of pollen tubes in Arabidopsis,bioinformatics methods were used to analyze the expression patterns and evolutionary relationships of CPK family members,and constitutively active(CA)and dominant-negative(DN)CPK17 mutants were constructed.Based on gene gun technology,we observed the phenotypic changes of CA-CPK17 and DN-CPK17 overexpressing pollen tubes,compared the effects of high and low calcium stress on the growth of CPK17 and CA-CPK17 overexpressing pollen tubes,and analyzed the co-expression of CPK17 and its homologous protein CPK34.Additionally,the protein interaction network of CPK17 was predicted,and some of the interacting proteins were validated.The results showed that there were seven pollen-specifically expressed CPKs in Arabidopsis thaliana.Among them,CPK17 and CPK34 had a high similarity (93%),and were highly expressed in mature pollen,and low or no expression in other tissues.Overexpression of CPK17 had no significant effect on pollen tube growth,while CA-CPK17 caused depolarized growth and tip swelling of pollen tubes,and DN-CPK17 had no obvious effect.Overexpression of CPK34 also caused similar defects in pollen tube growth,and co-overexpression of CPK17 and CPK34 aggravated the defects.Calcium stress experiments showed that overexpressing CPK17 pollen tubes were insensitive to low calcium conditions,but sensitive to high calcium conditions.While pollen tubes overexpressing CA-CPK17(lacking the regulatory domain)were insensitive to both high and low calcium conditions.The predicted interacting proteins of CPK17 were mainly ROP negative regulators,but yeast two-hybrid experiments failed to confirm the interaction.These results indicated that the C-terminal regulatory domain of CPK17 plays an important role in the response to calcium signals.CPK17 and CPK34 have synergistic effects on pollen tube growth,possibly by regulating calcium influx and affecting intracellular calcium gradient.

Key words: Pollen tube, Polar growth, Gene gun, Calcium-dependent protein kinase, Ca²⁺ signaling pathway