Accumulation Of Glycine Betaine In Transplastomic Potato Plants Expressing Choline Oxidase Confers Improved Drought Tolerance

Potato(Solanum tuberosium L.) is one of staple food crops and plays a vital role in ensuring food supply all over the world.However,due to its sparse and shallow root system,potato varieties are vulnerable to a series of abiotic stresses,including drought,high salinity and severe temperature changes,thus resulting in a reduction of tuber yield and quality.Even short periods of drought stress can result in a severe reduction in tuber production.Therefore,there is an urgent need to develop drought-resistant potato variety to meet the challenge by global warming and desertification.In recent years,the methods to improve the drought resistance of potato include breeding,cultivation techniques optimization,and genetic improvement strategies.The genetic improvement strategies include increasing the concentration of antioxidants to reduce the accumulation of excessive ROS caused by various stresses,over-expressing genes of transcription factors to activate response to drought stress and increase osmolytes accumulation level,such as glycine betaine(GB),which can reduces cell osmotic potential,maintains swell pressure and enhances water absorption of cells,improves the adaptability of plants to stresses,and plays an important role in plant resistance to abiotic stresses.In both higher plants and Escherichia coli,GB is synthesized by two-step oxidation reaction from choline via betaine aldehyde,while one-step synthesis pathway in the soil bacterium Arthrobacter globiformiscan is completed by a single enzyme-choline oxidase,which can catalyze the direct conversion from choline to GB.Moreover,as a low-molecular-weight metabolite,GB is highly soluble in water and non-toxic at high concentration.It has been demonstrated that GB was synthesized in plastid and GB accumulation in plastid could be more effective than in cytosol for protecting transgenic plants against abiotic stresses.Plastid transformation holds couples of unique advantages compared to conventional nuclear transformation,including remarkable high expression levels,absence of epigenetic transgene silencing and the increased biosafety by maternal inheritance.Here we report that the cod A gene from A.globiformis,which encodes choline oxidase to catalyze the conversion of choline to GB,was successfully introduced into potato plastid genome by plastid genetic engineering.Two independent plastid transformed lines were isolated and confirmed as homoplasmy via Southern blot analysis,in which the level of cod A RNA identified by Northern analysis is identical.The phenotypes of two homoplasmic PC lines were phenotypically entirely normal and indistinguishable from WT plants under both mixotrophic growth conditions on synthetic sucrose-containing medium and autotrophic growth conditions in soil.We therefore used one representative transplastomic line for the next physiology experiments.To assess the cod A m RNA transcripts accumulation in leaves and tubers,Northern blot was performed.As can be seen,the cod A m RNA accumulation in leaves was around ten times higher than that in tubers.The GB was analyzed quantitatively by ~1H-NMR,The cod A-expressing plants accumulated GB in both leaves(1.21±0.36μmol g-1 DW)and tubers(1.77±0.21μmol g-1 DW)at the similar level,which were much higher compared to WT plants.Higher levels of relative water content and chlorophyll content were detected in the leaves of PC plants than those of WT plants under drought stress.After re-watering,PC plants returned to normal more quickly,and continued to grow normally.Moreover,PC plants presented significantly higher photosynthetic performance as well as antioxidant enzyme activities during drought stress.For the first time,by using plastid transformation technology,GB synthesis gene cod A from A.globiformis was successfully transformed into potato plastid genome,making GB compartmentation in chloroplast of PC plants.Although high expression of cod A in chloroplast does not lead to a high GB content,PC plants had a significant increased tolerance to drought stress compared to WT plants.The findings suggest that biosynthesis of GB by plastid engineering is an effective method to increase drought tolerance.