Studies On The Physiological Mechanism Of Low Temperature Regulation On Leaf Growth And Development Of Flue-cured Tobacco Seedlings

Tobacco is a kind of thermophilic crop which is native to the subtropical zone.The optimum temperature for growth is 25-28°C,but is sensitive to low temperature.The low temperature of10-13°C causes the growth of tobacco plants retardation.The low temperature of 1-2°C can cause the tobacco plants to die.During the early spring,low temperature has become one of the common problems in the transplanting period of tobacco plants in southern China,such as Fujian province and Jiangxi province.If the extreme low temperature of 4°C occurs in the transplanting period,it will easily lead to growth retardation,early flowering and narrow leaf shape in the early stage,which will affect the yield and quality of tobacco leaves to a large extent.In this study,the low temperature sensitive variety K326 and the low temperature insensitive variety Honghuadajinyuan were used as experimental materials.Firstly,the changes of leaf shape in the recovery stage after low temperature stress were analyzed,and then the leaf tissue structure,chloroplast ultrastructure and related physiological indexes of the varieties with larger leaf shape index response were analyzed.The physiological and molecular mechanisms of auxin involved in leaf shape changes were explored.The specific tubulin gene NtTubB in response to low temperature was locked by iTRAQ technique and the leaf shape after different stress treatments.Through the isolation and cloning of this gene,the function was preliminarily verified by VIGS.The results of this study provided a theoretical reference for further revealing the physiological mechanism of low temperature regulation on leaf growth and development of flue-cured tobacco seedlings.The main results are as follows:1.In order to analyze the change of leaf shape index?leaf length/leaf width?of flue-cured tobacco during the recovery stage after low temperature stress,K326 and Honghuadajinyuan were treated at low temperature and then returned to normal temperature.We found that the response of K326 to low temperature was more obvious than that of Honghuadajinyuan.The low temperature inhibited the leaf growth and development.Among them,the inhibition to leaf width was stronger than the inhibition to leaf length,resulting in an increasd leaf shape index.On the whole,the induction degree of leaf shape index increased with different stress time was 12 h>24 h>6 h>3 h>48 h.And 4°C for 3 h had irreversible effects on leaf growth after recovery.2.To study the effect of low temperature stress on photosynthetic fluorescence characteristics and leaf structure of flue-cured tobacco seedlings,K326 was treated under low temperature stress.The water use efficiency and the stomatal limit value increased continuously with the increase of low temperature stress time.And the net photosynthetic rate and stomatal conductance decreased continuously.In addition,the photoreaction was inhibited and the light energy utilization rate was reduced.At the same time,the leaf tissue structure was damaged and the chloroplast cell layer structure was disintegrated.The low temperature directly affects the chlorophyll synthesis and photosynthetic organ activity of plants,and indirectly affects photosynthesis through water stress and stomatal resistance to CO₂diffusion.3.In order to study the effect of auxin on the growth and development of flue-cured tobacco seedlings under low temperature stress,it was found that exogenous NPA can regulate the leaf shape index of tobacco seedlings,while exogenous auxin NAA can effectively alleviate the trend of narrow leaf shape by HPLC and external application of NAA and NPA.It was preliminarily concluded that the decrease of polar transport of auxin from the shoot tip to the base of stems caused by low temperature stress was one of the physiological mechanisms of the leaf shape of tobacco seedlings in response to low temperature stress.4.To identify the specific proteins and their gene expression changes in response to low temperature stress,the protein expression profile of flue-cured tobacco K326 leaves under low temperature stress was established based on iTRAQ,and 8 tubulins were screened from 2 885 identified proteins.Among them,one NtTubA3 gene?NM₀01326192?and one NtTubB gene?XM₀16581922?were significantly down-regulated only under low temperature stress and had no significant change after normal temperature growth.By comparing the leaf shape index of tobacco seedlings under different stresses,it was speculated that NtTubB may be involved in the establishment of tobacco seedling morphology.5.In order to further clarify the characteristics of NtTubB protein and its biological function,the structure and bioinformatics prediction of NtTubB showed that the full-length cDNA was 2 783 bp,the open reading frame was 2 475 bp,consisting of 3 exons and 2 intron composition.VIGS verification showed that the expression level of NtTubB in TRV-NtTubB-infected tobacco seedlings was significantly down-regulated,and the results preliminarily verified that the NtTubB was involved in the molecular regulation of low-temperature induced leaf-shaped narrow length.