| Pollination is a key process of flowing plant sexual reproduction.After mature pollen grain falling on the stigma,it undergoes a series of events including adhesion,hydration and germination.Pollen tube grows through the stigma and style to ovary,completing the journey of double fertilization.The interaction between pollen grain and stigma is crucial for plant pollination.Pollen hydration is a critical step for the determination of pollen germination on stigma.Plant Sucrose Nonfermenting Related Protein Kinase 1 complex(Sn RK1 complex),a key factor for sugar signal transduction in plant,is involved in the metabolism regulation and hormone/development signal transduction.Sn RK1 complex is composed of catalytic α subunits and two regulatory noncatalytic subunits: β and γ/βγ.KINβγ is a plant-specific subunit of the Sn RK1 complex that is preferentially expressed in the vegetative cells of Arabidopsis pollen.In pollen of the Arabidopsis kinβγ/+ mutant,the levels of reactive oxygen species(ROS)were decreased and pollen hydration on stigma was compromised compared with the wild type.These findings suggest that KINβγ regulate pollen hydration during Arabidopsis pollination by mediating the level of ROS in pollen.To determine whether ROS is involved in the regulation of pollen hydration on the stigma,the Arabidopsis CATALASE 3(CAT3)was overexpressed in pollen driven by Lat52 promoter.q RT-PCR analysis showed that the expression levels of CAT3 were increased in transgenic lines.The levels of ROS in the pollen of these lines were reduced,and the hydration of the pollen on the stigma was inhibited.Arabidopsis FISSION1A(FIS1A)is a mitochondrion-and peroxisome-targeted protein required for the division of both organelles.The ROS levels in the pollen grains of fis1 A were significantly decreased compared with those of the wild type.And lots of the fis1 A pollen grains on the stigmas could not complete their hydration.These results suggest that ROS signaling functions in pollen hydration on stigma in Arabidopsis pollen.To further dissect the reason of the compromised hydration of the kinβγ pollen grains on the stigma,we performed a RNA-seq analysis for the kinβγ mutant pollen.We identified 213 up-regulated genes and 146 down-regulated genes in kinβγ pollen.To identify critical DNA sequences involved in the KINβγ-regulated gene expression,we analyzed the promoter region(upstream 1000 base pairs from the transcription start site)of the down-regulated and up-regulated genes in kinβγ-2/+.We found that the W-box elements TTGAC(C/T),WRKY transcription factor preferentially binding sequence,were enriched in the promoters of these genes.SPIK is K~+ channel located in plasma membrane and functions in K~+ transmembrane transport in Arabidopsis pollen.We found that the expression level of Shaker Pollen Inward K~+ channel(SPIK)was reduced in the RNA-seq data of kinβγ pollen.Considering K~+ play an important role in osmoregulation,we speculate that SPIK may be involved in KINβγ regulating hydration of pollen.The pollen hydration of spik-1,a mutant of Arabidopsis SPIK,was defective on the wild-type stigma,although spik-1 demonstrated normal pollen hydration on the medium.To test whether the K~+ transport was affected in kinβγ pollen,we determined the K content in pollen grains from the kinβγ-1/+,kinβγ-2/+,and the wild-type.The results revealed that the K content was decreased in kinβγ-1/+ and kinβγ-2/+ pollen,respectively.Our study suggests that in addition to ROS,Arabidopsis Sn RK1 complex regulating SPIK expression functions in determining pollen hydration on the stigma. |
