Engineering Zinc Defficiency Report Tobacco By Using Promoter Of An Arabidopsis Zinc Transporter Gene

The micronutrient zinc(Zn)is essential for all organisms,and is required as a cofactor in over 300 enzymes and plays critical structural roles in many proteins.However,Zn deficiency is one of the most prevalent nutrient deficiencies in the world,affecting more than 25%of the world's population.Children are the most commonly and severely affected because of the high Zn demands of infant growth.Zinc deficiency is largely related to inadequate intake or absorption of zinc from the diet.In general,lack of Zn in staple foods is the major case for Zn malnutrition.During the past decades,exogenous Zn application is one of the important tools to enhance Zn content in staple foods.However,the physiological range between deficiency and toxicity of Zn is narrow,and unregulated high Zn content in plant can lead to toxicity and damage.Therefore,a technical platform designed for a fine and sensitive detection of Zn fluctuations in plants could be of great significance for proper fertilization and thus for the sustainable development of agriculture.In the past decade,a series of effective biotechnological,chemical and spectroscopic strategies have been introduced for monitoring plant nutrition status.In this study,transgenic tobacco was generated to construct a color-based visual reporter system that facilitates specific,accurate and rapid assessment of plant Zn status either by naked eyes or spectral reflectance.The main results of the present study are listed below:1.By a preliminary screening of the microarray data in public databases,several candidate genes in Arabidopsis(Arabidopsis thaliana)that might be specifically induced by Zn deficiency were selected for further study.Reverse Transcriptase-quantitative Polymerase Chain Reaction(RT-qPCR)analysis was performed to validate the microarray data.The results showed that a gene encoding a ZIP transcription transporter,AtZIP4,was specifically upregulated by Zn deficiency.In addition,bioinformatic analysis of its putative promoter showed that there are two zinc deficiency-responsive cis-element,ZDRE(Zinc Deficiency Responsive Element),residing in the 5' proximal region(within 250 bp).2.To generate a color-based visual Zn deficiency indictor plant,the cauliflower Pr(Brassica oleracea Purple)gene encoding a R2R3-type MYB transcription factor driven by the putative promoter of AtZIP4 was introduced into tobacco(Nicotiana tabacum).As expected,the leaves of the transgenic tobacco turned dark purple due to abundant accumulation of anthocyanin under Zn deficient condition,but not under Zn sufficient condition.Our results demonstrated that the expression of the heterologous Pr gene under Zn starvation condition activated the endogenous genes involved in anthocyanin biosynthesis pathway,resulting in abundant anthocyanin accumulation in tobacco.3.To further evaluate the sensitivity and specificity of the tobacco Zn deficiency reporter system for diagnosis of Zn nutrition status in a temporal manner,transgenic plants were grown under different Zn regimes.With an increase in duration and severity of Zn deficiency,there was a concomitant increase in the dark purple pigmentation in leaves of the transgenic tobacco,and the leaves turned green rapidly upon resupply of Zn.The results demonstrated a high incidence of inverse correlation between Zn content and anthocyanin accumulation in leaves of the transgenic tobacco plants.In contrast to that under zinc starvation condition,no anthocyanin accumulation in the transgenic tobacco plants was detected when the plants were deprived of other major-and micro-nutrients,suggesting the specificity of the designed reporter system.Taken these data together,the AtZIP4pro::Pr system can be successfully applied to specific and accurate assessment of Zn status by both naked eyes and by spectral reflectance.