The Induced Function And Mechanism Of Nitrite For Rice Embryogenic Callus

Rice?Oryza sativa L.?is one of the world's most important food crops,and it is a model monocotyledon plant when carrying on science research.The Agrobacterium tumefaciens-mediated transformation technique is one of the indispensable tools for the study of rice functional genomics;therefore,establishing a steady and efficient rice tissue culture system is the premise and basis.It has been known that many factors influence the processs of rice tissue culture,such as the sampling time,sampling sites,physiological status and genotype of the explant,the constitution and concentration of the medium salts,the variety of organic components,and the various combination of growth regulators.The nitrogen source is an essential constituent of culture medium,however,whether nitrite can be used as nitrogen source has not been reported.The 'mature embryo-callus tissue-differentiation' procedure represents the typical rice tissue culture system.We systematacially analysed the influence of different nitrogen sources for callus tissue growth and development,especially about nitrite.The main results are as follows.The various combination effects of nitrate,nitrite,ammonium sulfate,and glutamine?Gln?served as nitrogen sources during rice in vitro culture were tested.The induced callus masses,the fold increases of callus masses during subculture,and the regeneration rate were chosen as evaluation indexes.The results indicated that ammonium sulfate and Gln serving as the nitrogen source individually or together failed to induce callus formation during induction,the fold increases of callus masses during subculture were relative lower compared to other nitrogen sources,and the callus could not regenerate into plantlets.The callus during regeneration even did not turn green when the ammonium sulfate served as nitrogen source.An interesting phenomenon is the low concentration nitrite?2 mM and 5 mM?could serve as nitrogen source during rice culture,with no substantial phenotypic differences when nitrate supplemented as nitrogen source.The effect of NO₂^-was credible,rather than the presence of potassium ion or possible contaminant nitrate.The souble protein,nitrate ion,and nitrite ion contents were measured after callus tissue cultivated on different nitrogen sources of media.There were no significant differences in protein content among callus on different media and different culture phases.The nitrite contents of callus cultured on nitrate or nitrite serving as nitrogen source increased during the early stage and then decreased during the late stage.At the same time,the nitrite level did not change significantly in callus inoculated on the ammonium sulfate and Gln serving as nitrogen source individually or together.In a certain concentration range,the nitrite contents of callus were positively correlated with the evaluated indexes.The nitrate levels were uncorrelated with the evaluated indexes.When nitrate reductase?NR?activity is specifically inhibited by increased concentrations of tungstate,a molybdate analogue,the nitrite levels decreased correspondingly,with the growth indexes of callus decreasing on media supplemented with nitrate served as nitrogen source.However,tungstate seemed to have no impact on callus growth on media supplemented with nitrite served as nitrogen source.This was due to the fact that nitrite levels did not change significantly.Four genes were assayed because they were previously reported as nitrate-responsive genes.To test the ability of nitrite to act as a molecular signal,RNA was isolated from callus grown on different culture media,which was then examined by quantitative real-time PCR?qRT-PCR?.The relative expression levels of these genes were highest in callus cultured on media supplemented with nitrate or nitrite as nitrogen source,and were lowest in callus cultured on media supplemented with ammonium sulfate as nitrogen source.It indicated that nitrite probably was the real signal that induced these genes.All these results described above strengthen the conclusion that nitrite may function as a molecular signal for rice in vitro culture,rather than nitrate.The noticeable influence of NO₃^-:NH₄⁺ ratios on the growth and morphogenesis of plant tissue culture have been reported in many plants.We tested the effect of different NO₃^-:NH₄⁺ ratios in the media on callus growth evaluation indexes.When the NO₃^-:NH₄⁺ ratio in culture media was 4:1,the evaluation indexes of Zhonghual1?ZH11,Oryza sativa L.ssp.japonica?were highest,corresponding with the peak nitrite concentration.Nevertheless,the optimal NO₃^-:NH₄⁺ ratio for 9311?Oryza sativa L.ssp.indica?callus was 2:1.Coincidentally,NO₃^-:NH₄⁺ ratios of N6 and MS media are 4:1 and 2:1,respectively.Moreover,N6 medium is widely used for japonica rice in vitro culture.Generally,indica rice in vitro culture are inoculated on MS medium or modified MS medium.However,the mechanism for this general rule is not clear.According to our results,the NO₃-:NH₄⁺ ratio in the culture medium affected the nitrite concentration of callus,that ultimately affected the development and growth of the rice tissue culture.Confirming that the upstream and downstream relationship between nitric oxide?NO?and nitrite.Our results indicated that NO contents of callus were positively associated with nitrite ion contents of callus.If the liquid enzyme supernatant was loaded with increased concentration of nitrite,the NO content increased correspondingly.These results indicated that nitrite-derived NO production is the main pathway of NO generation in rice tissue culture.In addition,in vivo NO levels can be modulated by exogenous treatment with NO donors like SNP or NO scavengers.If NO of callus was eliminated,the callus can not induced during induction and can not regenerated into plantlets during regeneration.This demonstrated that NO probably acts as a signal that associated with callus growth and development.During 9311 callus subculture,SNP increased mass of callus cultured on media supplemented with nitrite,while it had no effect on the mass of callus cultured on media supplemented with ammonium sulfate or Gln.SNP alone did not increase mass,demonstrating that NO cannot function without nitrite.This indicated that nitrite acts as a signal upstream of NO in rice tissue culture.Nitrite maybe act as a reservoir of NO in rice tissue culture,but its role is apparently more than just a reservoir.NO cannot replace nitrite.To test the effects of nitrite on callus transcriptome,we proceeded RNA-seq analyses.The results indicated that nitrite influenced the secondary metabolism pathway of callus during subculture,especially the phenylpropanoid biosynthesis pathway which most genes were down-regulated.This was in accordance with the result that lignin content was lower in callus subcultured with nitrite compared without nitrite.Since lignin content is closely related to the quality of callus,nitrite promotes formation of the embryogenic callus.During regeneration process,nitrite promoted genes relative to stress,disease,alpha-amylase precursor,etc.up-regulated,and had a great influence on genes relative to hormone metabolism and cell wall biosynthesis.Taken together,we have proceeded the pioneering work that nitrite functioned as a signal molecular in rice in vitro culture,and the signal transduction pathway needs more study.We fully expect more research into the effects of nitrite in plants.