Semester

Summer

Date of Graduation

2009

Document Type

Dissertation

Degree Type

PhD

College

Davis College of Agriculture, Natural Resources and Design

Department

Biochemistry

Committee Chair

Dale T Karlson

Abstract

To understand the function of cold shock domain proteins in planta, I analyzed AtCSP3 (At2g17870), which is one of four A&barbelow;rabidopsis t&barbelow;haliana c&barbelow;old s&barbelow;hock domain p&barbelow;roteins (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.;A&barbelow;rabidopsis t&barbelow;haliana C&barbelow;old S&barbelow;hock Domain P&barbelow;rotein 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 genes in stages of early silique development. Specifically, transcripts of AP, CAL, AG, and SHP2 were up-regulated. Collectively, these results indicate that AtCSP4 plays an important role during the late stages of silique development by affecting the expression of several development related genes.;Cold shock domain proteins (CSPs) have been reported to play an important role in tissue development and cold responses. Eukaryotic CSPs play a crucial role in cell differentiation and cell proliferation which result in regulating the timing of tissue development and cell division. Cold shock domain proteins have been identified in many plants, although functional analyses have been limited to Arabidopsis, wheat and rice and their in vivo functional roles remain unclear. Among the four Arabidopsis thaliana CSPs (AtCSPs) that I have characterized, AtCSP1 is highly similar to AtCSP3 in terms of its predicted amino acid sequence. Transcription of AtCSP1 in increased in the loss of function mutant atcsp3, implicating that AtCSP3 negatively affects AtCSP1 transcription. Using a gene trap line (GT606 defined as atcsp1), which has complete loss of full length AtCSP1 transcript, GUS gene expression was detected preferentially in tissue primordia and highly dividing tissues. The atcsp1 exhibited early germination after stratification but did not exhibit any further atypical phenotype in vegetative tissues. Germination of atcsp1 without stratification also occurred earlier than wild type but the germination time delayed 24 hours. Comparative analysis of GUS expression in seeds with or without stratification confirmed that AtCSP1 expression was affected by cold temperature during radicle emergence. In addition, ABA germination assays revealed a reduced sensitivity to ABA in atcsp1. Taken together, AtCSP1 may function to regulate germination timing which is in turn mediated by cold temperatures to promote embryo expansion. (Abstract shortened by UMI.).

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