Genotypic Differences In Alleviation Effects Of Potassium And Betaine In Tobacco And MiRNA Expression In Response To Cadmium Stress

Cadmium (Cd) pollution affects the yield and quality of tobacco. In this study, we studied the physiological mechanism of alleviation effects of potassium and betaine on Cd toxicity in tobacco, explored the genotypic differences in miRNA expression under Cd stress. The main results are as follows:1. The level depanded alleviation effects of potassium on Cd toxicity in tobaccoHydroponic experiment was conducted using tobacco genotype K326to evaluate the biomass accumulation, photosynthesis, and the activity of antioxidant enzymes in plants under diferent levels of K (K0,0; K1,0.25; K2,0.5; K3,1mM K) against5μM Cd stress. The results showed that there were dose effects of potassium concentration on Cd toxicity alleviation. The physiology treats of K2+Cd were significantly better than other treatments. Cd concentrations in shoots and roots of plants grown in K2+Cd were15.8%and12.9%, respectively, lower than that of the other3Cd treatments. Meanwhile, Zn and Fe contents in tobacco leaf of K2+Cd treatment were much higher than others. Taken together, the protective mechanism for the K2+Cd against Cd toxicity may include:(1) reduced Cd uptake and translacation;(2) improved photosynthesis efficiency and promoted tobacco growth and biomass accumulation;(3) increased the activity of antioxidant enzymes such as SOD, CAT and APX, reduced the membrane lipid peroxidation of MDA content.2. Mechanism of alleviation effects of betaine on Cd toxicity and its genotypic differenceTwo tobacco genotypes Guiyanl (Cd sensitive genotype) and Yunyan2(Cd tolerant) were cultured in hydroponic experiment uder4treatments of0μM Cd (control),500μM betaine (betaine),5μM Cd (Cd) and5μM Cd+500μM betaine (Cd+betaine) to evaluate the alleviation effects of betaine on Cd toxicity and genotype difference. Result showed that foliar spraying500μM betaine (Cd+betaine) significantly alleviated Cd toxicity in tobacco and reduced Cd uptake and accumulation compared with Cd treatment The exogenous betaine reduced Cd concentration of each part of the tobacco plants by11.7%-13.0%(Guiyanl) and11.8%-13.7%(Yunyan2), and reduced MDA content. Meanwhile, Cd significantly reduced tobacco root tip cell vitality and betaine eased this phenomenon. And from the TEM results, we found that betaine reduced the cytoplasm loose, membrane system fuzzy, osmium particles increase and grana lamella decrease caused by Cd stress. Betaine also increased the number of cavitations in the cells of root tips, which fixed free Cd ions. It could be concluded that the mechanism of betaine alleviating Cd toxity as follow:reduced Cd uptake and transportation in tobacco, formatted of a large number of vacuoles in the root to restraint Cd move, reduced Cd toxicity in tobacco from original; improved tobacco photosynthesis, protected PSII under Cd stress, enhanced the vitality of root system, increased the synthesis; protected protein and metabolic enzyme activities, promoted eliminating of the ROS in tobacco leaf and root induced by Cd, reduced Cd damages in cell membrane.3. Genotypic differences of microRNAs expression in response to Cd stressHydroponic experiment was conducted using two tobacco genotypes Guiyanl and Yunyan2under0μM Cd (control) and50μM Cd for root miRNA sequencing. We successfully identifed164conserved miRNAs which belong to53families and29novel tobacco miRNAs, as well as722target genes using Illumina high-throughput sequencing technology and computationally methods from two significantly heavy matal Cd-tolerance tobacco genotypes Guiyanl and Yunyan2, respectively. To get insight into the role of miRNAs in Cd responses, the expression levels of miRNAs were detected; we found dozens of stress related miRNAs which might play an important role in tobacco genotypes Cd-responsive differences. Then we performed qRT-PCR and confirmed in vivo the expression of6identified miRNAs in tobacco roots of both genotypes.