| Plant root is the major organ for absorbing water and nutrients. As an important part of root system, lateral root(LR) occurs postembryonically and initiates from a few pericycle cells.The anatomy of lateral root development process has already known very well, but the regulatory mechanisms behind many aspects of lateral root formation is unclear. Both intrinsic genetic programs and external signals influence the growth of lateral root. In short, lateral root development is a complicated process affected by a variety of factors. This study used the Low temperature sensitive mutant (Its1) which showed no lateral root at low temperature (below 24 ℃) as a subject to explore the molecular mechanism of regulation of lateral root development at low temperature in rice. The main results were as follows:1. Under the condition of 24 ℃, Its1 displayed a phenotype of no lateral root and the growth of the whole plant was inhibited severely. Methylene blue staining showed that no lateral root primordial (LRP) was observed in Its1 at 24 ℃. Section analysis showed that the length of cortex cell of mutant is only 1/3 that of wild-type. What’s more, no cell division of pericycle cells was observed in Its1.As the temperature increasing, the Its1 phenotype gradually recovered. When the temperature is up to 30 ℃, there was no significantly difference between the mutant and the wild-type.2. To investigate what kinds of signal pathway influence the origination of lateral root in Its1, the rice Affymetrix gene chip was used to examine the differential gene expression between mutant and wild-type at 24 ℃. Expression of 1957 genes were significantly altered in Its1 compared to wild-type (4-fold change), among which 1603 genes were induced and 354 genes were repressed. To investigate the biological process of these genes, we identified significantly enriched Gene Ontology (GO) categories. The most significantly enriched GO category of genes was ’response to stimulus’(p<10-10).Another highly enriched category was ’response to oxidative stress’(p<10-8), for example, the expression of parts of peroxidases had been changed. These results were further validated by qRT-PCR. PIN proteins correlate with estabilishment of auxin gradient in root tip which is a trigger of lateral root initiation. Analysis of expression of PIN family genes in the gene chip showed that among 9 OsPINs,4 were down regulated,2 genes were up regulated and the expression level of the rest of genes were very low in the Its1. The results of qRT PCR were the same as gene chip.3. Under the condition of 24℃ and 30℃, we examined the level of reactive oxygen species (ROS) in the root tips (about 1 cm) of Itsl and wild-type. The result showed that the content of ROS in the mutant was 3 times than that of wild type at 24℃; while there was no significant difference between the mutant and wild-type at 30℃. From the spatial distribution of ROS analysis, low temperature altered the distribution of H2O2 in the root of mutant, which exhibited less H2O2 in the root tip. Yet the tissue distribution of H2O2 is consistent between the mutant and wild type at 30℃. At 24℃, the superoxide(O2·-) concentration was higher in the mature zone of root of mutants, while the root tip between Its1 and wild type had no significant difference. There was no difference between Its1 and wilde-type at 30℃.To sum up, the phenotype of lts1 may be caused by the high level of ROS and the distribution of H2O2 in the root tip at 24℃. The results of this study deepen the understanding of mechanism of plant lateral root development influenced by low temperature. |
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