Functional Analysis Of Autophagy Gene CsATG3s In Nitrogen Use Efficiency Of Tea Plant

The content of nitrogen(N)in tea plant accounts for about 4.5% of the dry weight.N is an important component of tea quality such as free amino acids and caffeine.However,high N application has negative effects on the environment.Therefore,breeding tea cultivars with high N use efficiency(NUE)is an important way to improve the NUE of tea plants.Autophagy plays a key role in plant N cycle and has the potential to improve NUE.Many Au Tophagy-related Genes,ATGs,are involved in the autophagy process.In this study,we first identified the expression of autophagy genes and protein in tea leaves with different maturity.Two homologous genes of the ATG3 subfamily,CsATG3 a and CsATG3 b,were identified from the tea plant genome.The role of CsATGs in NUE of tea plants was investigated by overexpression of CsATG3 s in Arabidopsis thaliana.1)The transcriptional level of CsATG3 s in tea leaves increased with maturity.The expression of autophagy-related proteins in mature leaves was higher,indicating that autophagy activity in mature leaves was increased.2)Overexpression of CsATG3 s increased tolerance to LN stress in Arabidopsis.Compared with wild-type(WT)Arabidopsis,CsATG3a-overexpressing(CsATG3a-OE)showed delayed reproductive growth,significantly larger rosette radius and more rosette numbers under LN and HN conditions.On the other hand,the dry weight of APER(Aerial part excluding rosette)in CsATG3b-OE lines under HN condition was significantly higher than that of the WT.Most importantly,the dry weight of rosettes and roots of CsATG3a-OE and CsATG3b-OE lines was significantly higher than that of WT under LN conditions.3)Overexpression of CsATG3 s in Arabidopsis altered the distribution of dry matter and N in roots,rosettes,and APER.Compared with WT,dry matter and N content allocated to rosette were significantly increased in CsATG3a-OE lines under both LN and HN conditions.Similarly,dry matter and N contents allocated to roots by CsATG3a-OE and CsATG3b-OE lines were significantly increased under LN conditions.4)The overexpression of CsATG3 s in Arabidopsis regulated the expression levels of the genes was related with N uptake and transport.Under LN treatment,the expression levels of AtNRT1.1,AtNRT2.1,AtNRT2.2,AtAMT1.1 and AtAMT1.3 in roots of OE plants were significantly higher than those in WT.At the same time,the expression levels of amino acid transport-related genes AtAAP1,AtAAP4,AtAAP5 and AtAAP6 in rosette of OE lines were significantly higher than those of WT under LN and HN condition.5)Overexpression of CsATG3 s in Arabidopsis improved vegetative growth and NUE.The rosette biomass of CsATG3a-OE lines was significantly higher than that of the WT at the seedling stage under soil culture and HN condition.After growth completed,N content and biomass in seeds and stubble of OE lines were significantly increased compared with the WT under HN condition.In addition,the HI,NHI,NUt E and NUE of OE lines were significantly higher than those of the WT under LN condition.All these results indicated that overexpression of CsATG3 s increased seed yield under HN conditions and increased NUE under LN conditions.