Functional analysis of Arabidopsis cold shock domain proteins

Abstract

To understand the function of cold shock domain proteins in planta, I analyzed AtCSP3 (At2g17870), which is one of four Abrabidopsis tbhaliana cbold sbhock domain pbroteins (AtCSPs). Taq-Man probe quantitative RT-PCR (qRT-PCR) analysis confirmed that AtCSP3 transcripts were expressed dominantly in reproductive and meristematic tissues. The homozygous loss of function mutant atcsp3 displays a distinct phenotype with an overall reduced sized of seedlings, small sized orbicular rosette leaves, and curled leaf blades. Microscopic visualization of cleared leaves revealed a reduction in size and increased circular shape of palisade mesophyll cells in atcsp3 leaves. Image analysis of palisade cell layers indicated that the reduced size of the circular mesophyll cells is generated by a reduction of cell length and cell number along the leaf-length axis, resulting in an orbicular leaf shape. Also, I determined that leaf cell expansion is impaired for lateral leaf development in the atcsp3 loss of function mutant, but the leaf cell proliferation is not affected. Loss of function of AtCSP3 resulted in a dramatic reduction of LNG1 transcript involved in two-dimensional leaf polarity regulation. Subcellar localization of AtCSP3 in onion epidermal cells revealed nucleocytoplasmic localization. Collectively, these data suggest that AtCSP3 regulates leaf length specific polarity by affecting LNG1 transcript accumulation during leaf blade lateral expansion. I also discuss putative function of AtCSP3 as an RNA binding protein in relation to leaf development.;Abrabidopsis tbhaliana Cbold Sbhock Domain Pbrotein 4 (AtCSP4; At2g21060) contains a well conserved cold shock domain (CSD) and glycine-rich motifs interspersed by two retroviral-like CCHC zinc fingers. GUS staining analysis in pAtCSP4:GUS transgenic Arabidopsis plants confirmed that AtCSP4 was expressed in all tissues but accumulates in reproductive tissues and those undergoing cell divisions. Overexpression of AtCSP4 resulted in a reduced length of siliques and embryo lethality. Interestingly, a T-DNA insertion atcsp4 mutant did not exhibit any phenotypes, implicating that the similar AtCSP2 gene is functionally redundant with AtCSP4. During silique development, overexpression of AtCSP4 induced early browning and shrunken seed formation beginning with the late heart embryo stage. A fifty percent segregation ratio of the defective seed phenotype was consistent with the phenotype of endosperm development gene mutants. Transcripts of FUS3 and LEC1 genes, which regulate early embryo formation, were not altered in the AtCSP4 overexpression lines. On the other hand, transcripts of MEA and FIS2 which are involved in endosperm development were affected by overexpression of AtCSP4 indicating that AtCSP4 may be a regulator of endosperm development via transcriptional or post-transcriptional regulation. Additionally, overexpression of AtCSP4 also affected the mRNA generation of several MADS box…