The Response Of Banana AGPs And Its Subfamily FLAs To Lowtemperature Stress

Banana(Musa spp.)is one of the most important fruit and food crops in the tropics and subtropics.The frequent occurrence of low-temperature(LT)stress in later winter and early spring has seriously affected the production of bananas in China and restricted the healthy development of the banana industry.Plant cell walls are the first line of defense against various environmental stresses.Several types of plant cell wall Hydroxyproline-rich glycoproteins(HRGPs)showed resistance to LT stress.Arabinogalactan proteins(AGPs)is the highest glycosylated gene family in HRGPs,while the fasciclin-like AGPs(FLAs)family is a very important subfamily of AGPs.However,studies on the cold resistance function of plant FLAs are not available.In this study,the FLA gene family of the Genome A of Musa acuminata and Genome B of Musa babisiana were identified and characterized based on their reference genome sequences.The chilling-sensitive(CS)(Musa spp.AAA cv.Baxijiao)and chilling-tolerant(Musa spp.ABB cv.Dongguandajiao)banana cultivars were used as materials,and the differences in the spatial and temporal distribution of different types of AGP components in the leaves of CS and CT cultivars,as well as the changes in their expression levels under natural LT stress were analyzed by immunofluorescence(IF)labeling techniques.The differences in expression levels of all 30 Ma FLAs and some other AGP family members in the CS and CT cultivars after exposure to natural LT were analyzed by q PCR.The FLA genes with potential cold resistance were cloned and their cold resistant function was experimentally demonstrated.The results of this study can not only enrich the study of plant cold resistance mechanisms,but also provide cold resistance genes and be benefit for banana cold resistance breeding.The main findings are as follows:1.Thirty and 23 FLA gene were identified from the banana Genome A and B,respectively.The genetic loci were distributed over all the 11 chromosomes of banana except Chr7.Banana FLAs can be divided into four groups A,B,C and D according to their AGP glycosylation regions and fasciclin structural domains.The results of protein structure analysis showed that 24(80%)of 30 Ma FLAs contained N-terminal signal peptides,19(63.3%)contained C-terminal glycosylphosphatidylinositol anchors.It was 18(78.3%)and13(56.5%)for 23 Mb FLAs,respectively.Banana FLAs were conserved well during evolution after sequence comparison,gene/protein structure and phylogenetic analyses.Colinear analysis showed that banana FLAs were amplified as segmental duplication and then the duplicated genes were purified during the long time of evolution.2.IF results showed that the distribution of AGPs in bananas was both tissue-specific and tissue-similar.The levels of AGPs recognized by antibodies JIM4/13/14,LM2/14 and Ma FLA18 was up-regulated in both cultivars and showed significantly higher expression levels in the CT cultivar when compared to the CS one under different natural LT points,suggesting that they may be closely related to the cold resistance of bananas.3.qPCR results showed that natural LT stress resulted in down-regulation of the expression of most Ma FLAs in both cultivars.However,Ma FLA14/18/29 were upregulated by LT in both cultivars with higher expression level recorded in the CT cultivar,while the relative expression of the Ma FLA27 gene was only significantly upregulated in the CT cultivar,but the opposite occurred for the CS cultivar,suggesting that these four FLA genes may be closely related to the cold resistance of bananas.4.The banana MaFLA27 gene was successfully cloned and has been overexpressed in Arabidopsis thaliana.The cold resistance of transgenic plants was investigated.The results showed that the symptom of cold damage in the transgenic plants after LT treatment was not so serious as that of the wild-type Arabidopsis thaliana.The content of malondialdehyde(MDA)in the transformants was significantly lower than that in the wild-type,while the it was on the contrary for Fv/Fm value(PS Ⅱ photochemical efficiency).These results indicated that banana Ma FLA27 gene positively regulated plant cold resistance.