Functional Analysis On Genes Related To Cryptochromes In Arabidopsis Thaliana

Cryptochromes are flavoproteins that share similarity in the sequence of amino acids to DNA photolyase but lack the photolyase activity. There are three members of the cryptochromes gene family in Arabidopsis thaliana. Cryptochrome1 (CRY1) and CRY2 mediate primarily blue light inhibition of hypocotyl elongation and photoperiodic promotion of floral initiation, respectively, and they functions redundantly. In order to further study the biological functions of Arabidopsis thaliana cryptochromes, using GUS-CCT2 as a bait, we screened the cDNA library and found that RAT1 (Related to acyltransferase) can interact with GUS-CCT2. The rat1 single mutant flowered at nearly the same time as wild-type under long day conditions (16 h light/8 h dark). We overexpressed RAT1 in the sgs3-11 mutant and found that the RAT1-ox transgenic lines flowered at the similar time as the wild-type. A fusion protein consisting of the green fluorescent protein (GFP) and RAT1 was expressed in Arabidopsis and the localization of the fusion proteins was determined by confocal laser scanning microscopy. The results showed that GFP-RAT1 was localized to not only the cytoplasm and but also the nucleus. Sequences alignment showed that RAT2 shared 89.7% amino acids sequences similarity with RAT1, which were both in the same clade as carbonic anhydrases genes. The rat2 single mutant flowered also at the similar time as wild-type plants under long day conditions. We tried to construct rat1/-rat2/- double mutants and found that the double homozygous rat1/-rat2/- mutants were lethal. Alexander staining showed that the pollen's activity was normal in the heterozygous rat1/+ rat2/- double mutants. About 23.7% of embryos of rat1/+ rat2/- double mutants showed defects. The aborted embryos were cleared and observed under differential interference contrast (DIC) microscopy, showing that they developed slower than those of normal embryos and were incapable of becoming fully developed. This indicated that RAT1 and RAT2 might involve in embryogenesis. We transformed rat2 with the RAT1-RNAi construct. The transgenic r2r1i (rat2/RAT1-RNAi) lines flowered later than that of wild type under long day conditions. Real time Q-PCR results showed that the late flowering phenotype of r2r1i lines might result from the downregulation of Flowering Locus T (FT) and upregulation of Flowering Locus C (FLC). The r2r1i lines were hypersensitive to light and their hypocotyls were shorter than those of wild type under different light conditions, which indicated that RAT1 and RAT2 might act redundantly in photomorphogenesis. We fused RAT2 to yellow fluorescent protein (YFP) and transformed Arabidopsis protoplasts with RAT2-YFP. The localization of RAT2-YFP was determined by confocal laser scanning microscopy, and it showed that RAT2-YFP was localized to the mitochondria. This indicated that RAT2 might play a role in mitochondria.The cry1cry2 double mutant flowered later than wild-type under long day conditions. Through activation tagging, we identified a late flowering mutant ecc2 (enhencer of cry1cry2). TAIL-PCR results showed that a T-DNA insertion located near the open reading frame of At2g45430 on the second chromosome, suggesting that the late flowering phenotype of ecc2 mutant might be caused by overexpression of AHL22 (At2g45430) gene. The electrophoretic mobility shift assay (EMSA) showed that AHL22 can bind specifically to AT-rich DNA sequence, indicating that AHL22 is a bona fide AT-hook protein. The subcellular localization of GFP-AHL22 was observed in the nucleus. The nuclear proteins were purified from seedlings of wild type plants and GFP-AHL22 transgenic lines, and the western blot results showed that AHL22 was expressed in the nucleus. Additionally, GFP-AHL22 was not regulated by light. We also found that AHL protein abundance was higher in roots, indicating that AHL22 might play a role in roots. Then we constructed a GFP-AHL22 fusion gene drived by a root specific promoter TobRB7. The TobRB7:GFP-AHL22 transgenic lines flowered slightly later than that of wild type plants. The GFP-AHL22 fluorescence signal was only observed in the roots of TobRB7:GFP-AHL22 transgenic lines through confocal laser scanning microscopy.Through activation tagging, we identified an early flowering mutant called scc17-D (suppressor of cry1cry2#17-Dominant). TAIL-PCR results showed that a T-DNA insertion located between At1g25440 and At1g25450 on the first chromosome. The scc17-D mutant displayed phenotypes of dwarfism, reduction in leaf size, reduced length of siliques, and the altered shape of cotyledon pavement cells, suggesting that scc17-D had extensive functions on plant development. The results provide a new mutant for genetic studies of cryptochrome functions in Arabidopsis thaliana.