Effects Of Low Temperature Treatment On Pollen Development And Associated Gene Expression In Chinese Cabbage

The reproduction of plants depends on the viability of pollen, and the abortive pollen leads to severe reduction of crops. There are many factors generating the abortive pollen, which can be summarized as environmental and genetic factors. Studies have shown that flowering stage is the most sensitive period of the temperature stress in the process of plant growth and development. Considering that pollen is more sensitive to temperature than pistil, temperature stress often lead to pollen abortion. In recent years, abnormal change of climate lead to abnormal spring temperature, and the phenomenon of "late spring coldness" intensified, which influence pollen development of the cabbage and other spring flowering plants greatly and hinder the normal development of seed production and plant breeding. Therefore, clarifing the effect of low temperature stress on plant pollen development is important to understand the mechanism of plant response to external stress and the breeding and production practice of vegetables and other crops.In this investigation, we select Chinese cabbage as material. After flowering, plants with uniform growth were exposed at chilling conditions (4℃) for 1,3,5,7 d. In order to understand how cold stress affect the pollen development process, we observed the plant morphology, podding rate and pollen development process changes after low temperature treatment. Then, we analyzed the gene expression in Chinese cabbage before and after the treatment of low temperature using transcriptome sequencing to explore the molecular mechanisms in response to low temperature stress. The results are as follows:(1) The pollen development of Chinese cabbage were divided into five periods according to the characteristics:pollen mother cells, tetrads stage, uninucleate microspores stage, binucleate microspores stage and mature pollen stage. The cytological observation showed that the tetrad stage and uninucleate microspores stage are more sensitive than other stages to cold stress, in which the tetrads stage is most sensitive. Subsequently, we observed each development period of anther using semithin sections and transmission electron microscopy, stained callose in tetrad stage of pollen using aniline blue, detected the mature pollen viability using FDA, pollinated by test cross and statistics podding rate. The results showed that the tetrads stage and uninucleate microspores stage were affected after they were exposed at chilling conditions (4℃) for 3 d and the more sensitive to the extension of treatment time. The tetrads stage is the most sensitive stage to cold stress. The plants were difficult to restore to normal development even after moveing to the normal temperature condition.(2) The transmission electron microscope and aniline blue staining experiments found that advanced degradation of callose in tetrad stage and abnormal degradation of tapetum leaded to abnormalities of pollen wall after low temperature treatment, and eventually leaded to pollen abortion. The abnormal pollen wall was found in tetrad stage after low temperature treatment by transmission electron microscope. The aniline blue staining in tetrad stage showed that callose degraded in advance after low temperature treatment. Compared with the controls, the tapetum degraded abnormally through the observation by semi thin and transmission electron microscope. Therefore, the tetrad stage is the most sensitive stage to cold stress, advanced degradation of callose in tetrad stage. Abnormal degradation of tapetum leaded to abnormalities of pollen wall after low temperature treatment, and eventually leaded to pollen abortion.(3) Based on the transcriptome analysis,1527 genes showed differential expression after cold stress, and 1098 genes were found as new ones beyond the reference. Besides,824 long non coding RNA (lncRNA) were found, among which 81 showed differential expression. We selected the most sensitive (tetrad) stage buds with uniform growth, then exposed them at chilling conditions (4℃) for 7 d for transcriptome sequencing.1527 genes showed differential expression between the treatment group and the control group. In those differential expressed genes, we found two genes (RD29A/COR78/LTI78 andJAI1) were involved in the important regulatory pathways (CBF regulatory approaches).1098 new genes were also found, which haven’t been commented in the genome of Chinese cabbage. In addition,824 lncRNAs were found,81 showed differential expression. By analyzing the target genes of these differentially expressed lncRNA,8 genes were found in the vegetative growth stage in response to low temperature stress. They are:ICE1, JAZ1, WRKY34, EIN3, MYB56, CAMTA1, CAMTA12 and CAMTA13.