Responses Of Lignin Synthesis In Haloxylon Ammodendron To Salt And Osmotic Stresses And Functional Identification Of HaLAC15 And HaCOMT

Drought and soil salinization are serious problems and major adversity stresses limiting global agricultural production.Soil pollution is also one of the serious problems facing mankind today.The massive discharge of waste water,liquid and gas has led to the excessive levels of heavy metals such as copper,trichlorophenol and phenolic acid allelochemicals in the soil in some areas,and they pose serious threat to agricultural production and people’s health.Lignin is an important component of plant cell wall and has significant biological functions such as water transport,mechanical support and against to bacterial invasion.The high expression of lignin accumulation and related biosynthetic genes help plants adapt to a wide range of biotic and abiotic stresses.Desert plants have formed their own unique morphological structures and special resistance mechanisms during the long evolutionary process.Due to the long-term exposure to various adversity stresses,desert plants contain rich resistance genetic resources.Haloxylon ammodendron is a desert xerophyte and small tree endemic to desert areas with strong lignification ability,and it equips superb adaptability to abiotic stresses that consist of salinity,drought,high temperature,low temperature and soil infertile.The physiological and molecular mechanisms of adaptation of H.ammodendron to abiotic adversity are not yet clear,especially the relationship between lignin accumulation and resistance to drought,soil salinization and soil pollution has not been reported.In view of this,we conducted the studies to clarify the role of lignin in the resistance to abiotic stresses,and to refine the biological mechanisms of H.ammodendron in adapting to adversity by analyzing the changes of lignin content andrelated physiological indicators under salt and osmotic stresses.On this basis,we searched for important functional genes related to lignin synthesis and stress response in H.ammodendron.We focused on the laccase gene Ha LAC15 and caffeic acid-O-methyltransferase gene Ha COMT,which were significantly up-regulated under salt and osmotic stress.Two genes were cloned,and expression patterns and subcellular localization were analyzed.Finally,we investigated the effects of overexpression of Ha LAC15 in Arabidopsis to salt stress,heavy metal copper,phenolic acid and trichlorophenol（2,4,5-trichlorophenol,TCP）tolerance.And the effect of overexpression of Ha COMT in Arabidopsis to salt tolerance was investigated too.The results of the study provide new genetic resources for improving abiotic stress resistance in crops and new ideas for phytoremediation of contaminated soils.The main research results obtained were as follows.1.Both osmotic stress and salt stress significantly induced the expression of genes related to the lignin synthesis pathway and increased the lignin monomer content in H.ammodendron,and stresses enhanced lignin synthesis by significantly up-regulating the activities of laccase and peroxidase（POD）and the content of hydrogen peroxide（H₂O₂）,which led to a significant increase of lignin content in H.ammodendron.The deposition of lignin in the xylem and thick-walled tissues of H.ammodendron strengthened mechanical strength and enhanced radial stress,thus maintaining the continuity of water transport from the root system to the aboveground.The deposition of lignin in the cell wall reduced the water loss from the assimilated branch cells.2.Ha LAC15 gene with full length of 1855 bp and ORF length of 1695 bp,and Ha COMT gene with full length of 1467 bp and ORF length of 1092 bp were cloned,and two genes were both abundantly expressed under salt and osmotic stress.Ha LAC15was located extracellularly.Ha LAC15 had signal peptide structure and was secretory protein.Ha COMT was localized to the cytoplasmic membrane.The promoter of Ha LAC15 was cloned,and the length was 2259 bp,contained multiple promoter and enhancer region cis-acting elements associated with hormone,signaling,defense and stress responses.Overexpression vectors of Ha LAC15 and Ha COMT were constructed respectively.3.Overexpression of Ha LAC15 reduced the rate of water loss in leaves of Arabidopsis plants and increased the accumulation of proline and betaine in leaves.Overexpression of Ha LAC15 also reduced leaf osmotic potential and plasma membranepermeability,and thus maintained the water status of Arabidopsis.Overexpression of Ha LAC15 reduced Na⁺uptake and translocation by Arabidopsis roots under salt stress,and enhanced antioxidant enzyme activity in leaves.Overexpression of Ha LAC15 in Arabidopsis also reduced the accumulation of H₂O₂,and maintained root activity.Overexpression of Ha LAC15 increased lignin and cellulose accumulation in Arabidopsis plants under Cu stress and increased Cu fixation by the root cell wall.Overexpression of Ha LAC15 in Arabidopsis maintained root vigor and the transport of Fe and Mg elements to the aboveground,and reduced Cu accumulation in the aboveground of the plants.Ha LAC15 was a secreted laccase with certain decomposition ability for sinapic acid,syringic acid and TCP.Ha LAC15 had the potential to remediate contaminated soil.4.Overexpression of Ha COMT promoted melatonin synthesis in Arabidopsis leaves under salt stress,and maintained chlorophyll content and plant growth.Overexpression of Ha COMT in Arabidopsis enhanced antioxidant enzyme activity in leaves,and maintained root vigor.Overexpression of Ha COMT reduced Na⁺uptake and transport by roots,and enhanced Na⁺regionalization in the root system and Na⁺efflux from the root tip.Overexpression of Ha COMT in Arabidopsis also increased leaf proline and betaine accumulation and thus reduced osmotic potential,and maintained leaf water content.Overexpression of Ha COMT improved salt tolerance of Arabidopsis plants.The above results indicate that lignin played an important role in the adaptation of H.ammodendron to salt and osmotic stress.Overexpression of Ha LAC15 significantly improved the tolerance of Arabidopsis to salt,heavy metal copper,phenolic acids and trichlorophenol.Overexpression of Ha COMT also played an important role in the adaptation of Arabidopsis to salt stress.The results of the study provide new research ideas and excellent genetic resources for improving stress tolerance in cultivated crops.