Proteomics And Phosphoproteomics Studies On Rhizome Enlargement And Analysis Of CONSTANS-like Gene Family In Nelumbo Nucifera

Lotus is a perennial aquatic plant belonging to the family affiliating to Nelumbonaceae.The Nelumbo genus includes two species,Asian lotus and American lotus.In Asia,especially China,the Asian lotus is widely cultivated,which has the popular name Lotus.Lotus is widely used in ornamentation,traditional medicine and agriculture.Its rhizome is a popular vegetable in Asia.It is rich in nutrients such as vitamins and minerals,and has moderate calories and popular for food processing and people’s daily meals.Asian lotus can be divided into tropical lotus and temperate lotus according to the ecotype,as well with different morphology of lotus underground stem.The underground stems of the tropical lotus are not enlarged,while temperate lotus is known for its underground stems expanded into rhizome.As the temperature decreases and the lotus partly withers,the lotus underground stem,as a substance storage organ for lotus survived from winter,gradually accumulates sugar,starch,and other substances.The rhizome enlargement is completed in about 3 months.The expansion of lotus underground stem determines the material basis of lotus growth and development and the energy source for the next growth cycle,which is a complex physiological process.Besides of this,enlargement of rhizome is also important in agriculture production of lotus.Up-to-date,the molecular mechanism of lotus underground stem swelling is still poorly understood.Studies focusing on this are soly at the early stage.On the purpose of exploring the mechanism underlying this process,we conducted a system study to learn about the dynamic changes of protein levels and their phosphorylation intensities during the expansion of lotus rhizome.Meanwhile,analysis of the CONSTANS-like gene family in lotus genome and functional characterization on its member NnCOL5 were also performed.The main studies and results are as follows:(1)Comparative proteomics analysis was perfomed on the rhizome at three key stages of lotus underground stem development: the stolon stage(S1),middle swelling stage(S2),late swelling stage(S3).After mass spectrometry analysis,a total of 2712 proteins were identified and quantified,including 302 stage-specific proteins(SSPs)and 172 differentially expressed proteins(DEPs).The analysis found that the main functions of proteins in different periods have different emphasis.The S1-specific proteins were mainly enriched in oxidation and metal ion response pathways,the S2-specifc proteins were mainly related to cell growth and division;and the S3-specific proteins were mostly enriched in the biosynthesis of starch.In addition,proteomics research has obtained a large number of proteins related to light signal,auxin signal,and calcium ion signal transduction,which indicated that the light signal,auxin signal and calcium signal pathway might be involved in the expansion process of lotus rhizome.(2)The same set of samples was also subjected to a phosphoproteomics analysis.A number of 5462 phospho-peptides belonging to 2286 proteins were identified.About 57% of proteins could only be identified in the phosphoproteomic analysis,but could not be detected in the proteomics analysis.A total of 835 proteins could be quantified on their phosphorylation.A total of 373 stage-specifically phosphorylated proteins(SSPp),and 230 differentially phosphorylated proteins(DEPp)during the expansion process of lotus rhizome were identified.There are 133 proteins,which are enriched in hormones signaling and starch and sugar metabolism,showed changes at both proteomic and phosphorylation levels.(3)Through the analysis of gene,protein structure and expression data of lotus CONSTANS-like family,we obtained the candidate gene NnCOL5,the expression of which is highly positive related to the development of lotus rhizome.NnCOL5 protein composes of a BBX and a CCT domain,and localized in the nucleus.Overexpression of NnCOL5 in potato resulted in a significant increase in potato microtuber weight and starch under short-day conditions.The transcriptomic analysis of the overexpression potato plant found that overexpression of NnCOL5 affected potato endogenous hormone signaling,especially auxin signaling pathways,followed by starch and sugar metabolism,and affected the expression of key factors in the CO-FT pathway.Regarding the physiological process of lotus rhizome enlargement,NnCOL5 may promote lotus rhizome enlargement by affecting auxin signal transduction and starch synthesis.Combining this study,we have proposed a working model of temperate lotus rhizome enlargement combining proteomics and phosphorylated proteomics and function verification of NnCOL5.Under long-day conditions,lotus senses light signals through PhyA and FYPP to promote the elongation of underground stems.At the same time,FRIGIDA,a flower suppressor under reversible phosphorylation,can inhibit flowering.Auxin signal plays a role in promoting cell division in the early development stage of lotus rhizome.With the advent of short daylight,NnCOL5 negatively affects the auxin signaling pathway and interferes with the expression of key genes in the CO-FT pathway to promote lotus rhizome enlargement.Calcium ion signals are also involved,and proteins such as calmodulin and CPK undergo reversible phosphorylation to regulate protein abundance and promote mass transport during lotus rhizome enlargement.The results might help in not only obtaining new insights into the mechanism of lotus rhizome enlargement,but also in the breeding of rhizome lotus new cultivars as well as its production.