Physiological Mechanism And Micrornas Analysis Of Hyperhydricity Occurrence In Garlic Plantlets In Vitro

Garlic(Allium sativum L.),an economically important vegetable with wide culinary and medicinal applications,is cultivated worldwide.Traditionally,garlic is propagated vegetatively because of sexual sterility.However,a low coefficient of multiplication and viral infection may cause serious losses in yield and quality.Rapid in vitro micropropagation provides an efficient tool for producing virus-free garlic seedlings.However,the 'glassy' morphology of hyperhydricity is one of the main abnormalities observed during tissue culture,which limits the application of in vitro propagation methods and affects the commercial micropropagation industry.The phenomenon of hyperhydricity is a physiological disorder occurring frequently in plant tissue culture.Hyperhydric shoots show a typical morphology of glassy and translucent appearance,which are difficult to muplicate and transplant survival.The physiological malformation and metabolic alteration of shoots seriously affect virus elimination,rapid propagation,gene transformation and commercial micropropogation industry.However,the mechanism of hyperhydricity is still unclear.Studies showed thatreactive oxygen species(ROS)accumulation in plantlets in vitro have been obesrved during hyperhydricity.A large number of researches have proved a close link between ROS metabolic disturbance and hyperhydricity.But their relationship and the mechanism of hyperhydricity are rarely reported so far.To determine the metabolic characteristic of endogenous ROS and investigatethe physiological and molecular mechanism of hyperhydricity in garlic plantlets in vitro,we studied the effects of endougeous ROS on the hyperhyiricity,cell organelles damage,cell membranes abnormality and the expression of relative miRNAs as well as their target genes by tissue observation,physiological and biochemical determination and high-throughput sequencing.The major findings summarized as follows:1.Supplement growth medium with differents concentions of hydrogen peroxide(0,0.5,1.0,1.5,2.0 mM H2O2),hyperhydricity occurrence and physiological characteristic of garlic shoots under different treatments was analyzed.Results showed that 1.5 mM H2O2 enhanced hyperhydric shoots rate significantly.Compared with control shoots,exogenous H2O2 stimulated an obvious increase in content of tissue water and a marked decrease in content of total chlorophyll and soluble protein.SDS-PAGE electrophoresis results showed that the expressions of two protein bands(83 kD and 105 kD-molecular weight)were enhanced in ROS treated-shoots,and exogenous H2O2 treatment induced two new proteins expressions,whose molecular weights were 52kD and 110 kD,respectively.2.The addition of 1.5 mM H2O2 to the growth medium enhanced the superoxide anion(O2·-)generation and H2O2 content in the subcellular compartments.Under exogenous H2O2 stress,ultrastructure of chloroplastic and mitochondrial was disrupted,which was concomitant with aggravated frequency and severity of hyperhydricity.Accumulation of ROS was the highest in apoplasts.Meanwhile,the apoplast was the most sensitive compartment among those investigated.Apoplastic ROS might play a signaling role participating in the coordination of stress adaptation.3.Exogenous H2O2 increased the activities of plasma membrane NADPH oxidases,cell wall peroxidase(POD)and apoplast polyamine oxidase(PAO).Inhibition of each enzyme activity respectively,hyperhydric shoots rate,O2·-generation rate and H2O2 content in H2O2-stressed plantlets decreased significantly.In particular,NADPH oxidases inhibitor could suppress O2·-production at the lower level compared with other enzyme inhibitors.During the entire treatment period,NADPH oxidases activity kept a continuous upward tendency,while the activities of POD and PAO oxidases transient increased and subsequently declined along with H2O2 stress.The results suggested that NADPH oxidases might play the pivotal role on ROS induction during hyperhydricity.4.Hyperhydricity was induced by multiplying plantlets in semi-liquid medium or on medium with high concentration of 6-benzylaminopurine and exogenous H2O2.In contrast to normal shoots,the hyperhydric plantlets presented numerous ultrastructural alterations characterized by larger intercellular spaces,thinner cell walls and incomplete cell membranes.In hyperhydric shoots,the composition of cell wall and plasma membrane decreased significantly.The water-transporting function would be further affected by the structural and compositional abnormality of cell protective layers.5.Hyperhydricity was induced by transplanting normal and uniform shoots to growth medium containing 1.5 mM H2O2.H2O2-treated and normal small RNA libraries were constructed from garlic plantlet leaves to excavate the miRNAs target genes through degradome sequencing and predicted miRNAs.Through filtering,353 conserved miRNAs and 76 novel miRNAs were detected.Among all these miRNAs,133 miRNA members were up-regulated,whereas 76 miRNAs were down-regulated in response to oxidative stress.We also found that 100 sequences of 26 target gene families were cleaved by 21 conserved miRNAs,while 17 sequences of 3 target gene families were cleaved by 17 novel miRNAs.Target gene function description revealed that the target genes were with various functions,while most target genes played a positive role in the response mainly by regulating membrane protein,energy metabolism and ethylene response.