Physiological And Molecular Mechanism For Cold Tolerance Of Rubber Tree(Hevea Brasiliensis Muell.Arg.)

Rubber tree(Hevea brasiliensis Muell.Arg.)is a primary source of natural rubber(NR)in the world.The Amazon rainforest originated rubber tree has been commercially planted in large scale in the South or Southeast Asia as well as in the tropical area between the 10 degree latitude near Earth’s Equator where are so-called tranditional rubber tree planting areas.To meet with the demand for NR,rubber tree planting has been continuously expanded and no longer limited to the tranditional areas.This species has been successfully grown in several areas of South China,where are located at the tropical north fringe region and a sudden cold wave frequently occurs.To cope with the cold damage to rubber tree,artificial hybridization and selection have been carried out to develop rubber clones with relatively high potential of cold tolerance.Althouth the process is time-consuming,several clones such as 93-114,Nanhual and Hekou311 have been obtained.In the present study,compatative analysis on the physiological and molecular response of three cold-resistant rubber tree clones and three cold-sensitive rubber tree clones to cold stress was performed.The main results as follows.1.When seedlings of cold-resistant rubber tree clone 93-114 and cold-sensitive rubber tree clone Reken501 were treated at 4℃ for seven days and followed by treatment of 35 ℃for seven days,the difference in cold tolerance between the seedlings of the two clones was obvious.This experimental system is suitable for the early selection of Hevea germplasm with differential cold tolerance in room before identification in the filed.2.Up on cold stress,the level of antioxidant system(SOD,POD,CAT,GST,VC)and the osmotic adjustments such as soluble sugar and prolin were obviously increased in the leaves of rubber tree clone 93-114 than that in the leaves of rubber tree clone Reken501,whereas the level of MDA in the leaves of rubber tree clone93-114 was significantly lower than that in the leves of rubber tree clone Reken501.3.Illumina sequencing generated a total of about 7-8G raw reads from each of the six batches(two batches for control and four batches for cold-treatment)of either rubber tree clone ’Reken501’ or ’93-114’,respectively.By using these reads,167,911 unigenes were obtained by de novo assembly with a series of optimized parameters.Of which,100,455 unigenes(59.8%of the total)were annotated.Analysis of differentially expressed genes in response to cold stress was performed in terms of functional GO and KEGG pathway enrichment assessment and the visible MapMan analysis with the self-constructed annotation file.More down-regulated genes than up-regulated genes was found in the cold-tolerant rubber tree clone ’93-114’ at both early stage(4613↑and 19199↓)and late stage(6847↑and 239111).Of which,the growth related genes were sharply down-regulated while those related to ’heat’ module and ROS-scavenging were activated in the cold-tolerant rubber tree clone ’93-114’.4.The ORFs and 3-UTRs of HbICE1 and HbHsp70-1 were cloned by RACE.Based on the sequences,the specific primers were designed for real-time RT-PCR analysis of the expression pattern of the above unigenes among rubber tree clones with differential potential of cold tolerance.The difference in the expression pattern of HbICE1 and HbHsp70-1 was evident between cold-resistant and cold-sensitive rubber tree clones.And moreover,the level of the unigene transcripts was significantly higher in the cold-tolerant rubber tree clones than that in the cold-sensitive rubber tree clones.5.Upon cold stress,a burst of reactive oxygen species(ROS),HSF and HSP expression was significantly up-regulated and reactive ROS scavenging in the leaves of the cold-resistant rubber tree clone ’93-114’ in response to cold stress by comparison with the cold-sensitive rubber tree clone ’Reken501’.This study concluded:(1)The established cold-stress experimental systerm was useful for selection the Hevea germplasm with differential cold tolerance and the investigation of the molecular mechanism for the cold tolerance of rubber tree;(2)Activation of antioxidant systerm and accumulation of osmotic adjustments may be the physiological bases for rubber tree against cold stress;(3)Down-regulation of growth-related genes and up-regulation of stress-responsive genes may be a strategy for rubber tree to survive from cold stress;(4)Both the transcript abundance and the expression pattern of several stress-responsive genes were significantly different between the cold-tolerant and cold-sensitive rubber tree clones,suggesting the genes may be closely associated with the potential tolerance of rubber tree to cold stress;(5)The results will contribute to further identification of cold tolerance-related genes and development of molecular markers for genetic improvement of Hevea cold-tolerance.