ICE1 Regulates Plant Growth And Development By Tuning Gibberellin Synthesis In Arabidopsis

Appropriate low temperature favorably contributes to plant growth and development, for example, cold stratification is widely applied to promote germination both in frequency and synchronization. Phytohormone gibberellins (GAs) play crucial roles in seed germination, stem and hypocotyl elongation, floral development and flowering time. Cold stratification can increase the endogenous GA contents in Arabidopsis, hazel and apple seeds. Therefore, cold and GA signal pathway should be integrated to regulate plant development in a crosspoint which is unclear as yet. ICE1 is a key transcription factor in cold signaling pathway of Arabidopsis. It controls the response of plant to cold signal by initiating CBFs transcription factors and COR (cold-regulated) genes in turn. In this study, the functions of ICE1 in plant growth and development were observed and investigated with emphasis on the regulation of ICE1 to plant growth, flowering and fertility by tuning GA synthesis. The main results are shown as follows:1. ICE1 represses plant growth in ArabidopsisMature WT (Col-0) plant was about 30 cm in height, while ice1-t mutant up to 45 cm. Arabidopsis plants overexpressing ICE1 gene exhibited a dwarf phenotype. The plant height was only 10 cm. So, ICE1 could suppress plant growth in Arabidopsis.2. ICE1 controls male sterility by tuning GAMYB-like genesThe first ten siliques failed to set seed in the base of florescence of ice1-t mutant. The anthers of corresponding flowers dehisced later in mutant than WT. In addition, mutant pollens exhibited abnormal in color relative to WT. Germination in vitro on medium demonstrated that 50% pollens were able to germinate in WT while only 10% in mutant. Furthmore, germination in vivo on pistils artificially pollinated showed that mutant pollens failed to germinate on both WT and mutant pistils, while artificial pollination with WT pollen lead to success in setting seeds in mutant siliques. Those findings indicate that male sterility causes the failure to set seeds in the basal flowers of mutant.Considering the regulation of GAMYB-like genes to pollen development, gene expression patterns were investigated in mutant and WT with RT-PCR and RNA-blot assay. The results showed that the transcript level of four GAMYB-like genes, i.e. AtMYB33, AtMYB65, AtMYB101 and AtMYB120, were up-regulated in ice1-t mutant relative to WT.3. ICE1 controls flowering time by modulating the relative genesice1-t mutant flowered earlier than WT under short day (SD) conditions. Subsequently, the expression patterns of key genes involved in floral initiation were examined with semi-quantitative RT-PCR. The result indicated that the inducing genes LFY, CO and FT for floral initiation were up-regulated in ice1-t mutant, while the suppressor gene FLC was down-regulated.4. ICE1 controls seeds development by tuning LECs genesThe mature seeds of ice1-t were scrawny and shriveled in seed skin. Microsopic observation found that ice1-t produced normal globular, heart and torpedo embryo. However, the embryo development of mutant was inhibited in the following stages and failed to form embryo with curled cotyledon. As a result, the embryos were smaller in mutant than WT, which made the mutant seed looking scrawny.Because LEAFY COTYLEDON (LEC) genes, i.e. LEC1, LEC2 and FUS3, play key roles in controlling embryo development, we detected their expression patterns. Real-time RT-PCR showed that all three genes, especially FUS3, were up-regulated in the siliques of ice1-t mutant relative to WT.5. ICE1 controls endogenous GA level by regulating the expression of genes involved in GA synthesis and metabolism Based on those phenotypes of ice1-t mutant, it was predicted that ICE1 can regulate GA synthesis or metabolism. Real-time RT-PCRs were carried out to verify the hypothesis. It was found that GA synthesis-related genes AtGA3ox1, AtGA3ox2, AtGA3ox3, AtGA3ox4 and AtGA20ox1 were up-regulated in ice1-t mutant relative to WT, while GA degradation-related gene AtGA2ox2 was down-regulated. In fact, it was also found that GA level was much higher in mutant than WT. So, ICE1 repress the synthesis of endogenous GA in Arabidopsis.6. ICE1 protein binds to the promoter of AtGA3ox4Chromatin immunoprecitation (CHIP) showed that ICE1 protein could bind to the promoter of GA biosynthetic gene AtGA3ox4 among 6 candidate genes, i.e. AtGA3ox1, AtGA3ox2, AtGA3ox3, AtGA3ox4, AtGA20ox1 and AtGA2ox2. The binding E-box element is -206 CATTTG -200.