Functional study of saposin-like proteins in Arabidopsis thaliana

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Sphingolipids and microdomain-associated proteins that are associated with the plasma membrane and endomembrane system are important in plant growth and development. Elucidating functions of these proteins advance understanding of signal transduction from plasma membrane into cytosol and between different intracellular membrane compartments. Saposins and saposin-like proteins (SAPLIP) are among these proteins. In plants, two types of proteins contain saposin B-like domains (SapB-like domains): saposin-domain containing aspartic proteases (ASPAs) and prosaposin-like proteins (PSAPLIPs).

Phenotypic analyses showed that single loss-of-function aspa2 showed delayed seed maturation. Seeds of aspa1-2 aspa2-1 aspa3-3 triple mutant (aspa1 is knock-down, aspa2 and aspa3 are knock-out alleles) showed delayed germination rates and delayed seed storage proteins degradation. Further, protein storage vacuolar fusion was also delayed in the mutant cotyledons. These results suggest that ASPAs process seed storage proteins during seed germination in vivo, and probably also involved in protein storage vacuolar fusion regulation.

ASPAs also have a role in root architecture. Triple mutant showed longer primary root length under low nitrogen conditions. Further analysis suggested that the altered root architecture in the mutants may result from rates of tracheary element (TE) maturation in xylem tissues. Triple mutants were slightly delayed in TE maturation and the ASPA2 overexpression showed slightly early maturation. Together with the expression pattern of ASPA3, this indicates that ASPAs may take part in programmed cell death (PCD) in Arabidopsis. Further studies showed that ASPAs are involved in PCD execution. Results showed that the onset of PCD was not delayed in the triple mutant, but the execution time of PCD was extended. Membrane permeability increased more slowly in the triple mutants and faster in the overexpression plants. This reflects the role of ASPAs in membrane disturbance and permeability regulation during PCD.

The prosaposin-like proteins (PSAPLIPs) have received little study. Sequence alignments identified that prosaposin-like proteins are ubiquitous in plant kingdom. Plant PSAPLIPs show highly conserved in secondary structure of SapB-like domains. This structural similarity was supported by glycosylation analyses of Arabidopsis thaliana AtPSAPLIP1 and AtPSAPLIP2. Both AtPSAPLIP1 and AtPSAPLIP2 traffic to vacuoles. Possible role of PSAPLIPs is facilitating target protein degradation. AtPSAPLIP1 was mainly expressed in inflorescence, especially in sepals, carpels and mature pollen grains, as well as leaves and roots. Young leaves had higher expression level than aged leaves. AtPSAPLIP2 was expressed in inflorescence too, but mainly in young anthers, petals, ovules and developing seeds. This result indicates function differentiation of PSAPLIPs in Arabidopsis. Both genes are important in male gametophyte development.

The significance of this dissertation is that it demonstrates that ASPAs process seed storage proteins during seed germination in vivo for the first time. It also discovered a new role of ASPAs in regulating programmed cell death by promoting memberane permeability, and thus affecting root growth in Arabidopsis. The third is that this is also the first time to characterize the plant prosaposin-like proteins, which are important in male gametophyte development and provide novel sights on how plants regulate reproductive process. These results will broaden our understanding of the protein-lipid interaction in the cell and the biological functions of saposin-like proteins in plant growth and development.