Synchronization Control Of Somatic Embryogenesis And Gene Expression And Protein Phosphorylation Analysis During Somatic Embryogenesis In Cotton

Somatic embryogenesis is the developmental reprogramming of somatic cells toward the embryogenensis pathway and a remarkable illustration of the dictum of plant totipotency.It has been viewed as a potential model system for the study of the basic mechanisms of development reprogramming among the higher eukaryotic organism. Study on cotton somatic embryogenensis could provide a useful way to understand plant development such as embryogenesis and may have merit as a study presenting an application in plant biology due to its biological theory and economical importance.To explore the mechanism of somatic embryogenesis in cotton(Gossypium hirsutum L.), synchronization control technology,histo-cytology,gene expression,protein expression and phosphorylation and casein kinase activity during somatic embryogenesis,and two-dimensional electrophoresis technology were studied by using cotton cultivar Coker 201 as materials.The results are as follows:1.Low efficiency of somatic embryogenesis and asynchronous embryo development results in a lot of difficulties to physiological,biochemical,and molecular biological studies of embryogenesis processes in cotton.A simple and efficient method was developed to improve somatic embryogenesis frequency and synchronous development of mass somatic embryos from cultured cells of the cotton cultivar Coker 201 on the basis of the developed medium which was more suitable for somatic embryo development compared with other tested media.The embryonic calli obtained after several rounds of subculture were scattered in a liquid medium by shaking for 2 d and then resuspended in the same liquid medium after discarding the larger callus aggregates over 30 mesh-size-sieve.The suspensions cultured for 14 d were filtered through 50 mesh-size-sieve and the aggregates over the sieve were incubated for 21 d onto the surface of Whatman filter paper that were placed on the solid medium containing 2.46μmol L^-1 indole-3-butyric acid(IBA) and 0.70μmol L^-1 kinetin.The amount of somatic embryos obtained by this system was 15.5-fold and 3-fold higher than that of suspension culture and solid culture without filter papers,respectively.About 70.2%for globular, 52.3%for torpedo-shaped,and 73.0%for cotyledonary embryos were obtained during the culture.The method combining suspension culture and solid culture(with filter paper) proved to be efficient for synchrony of somatic embryogenesis and mass embryo development.2.Calli were induced from hypocotyl explants of cotton cultivar Coker 201,and individual embryogenic cell clusters with 30-50 mesh sieve,which isolated from embryogenic suspension cultures,were cultured.The development of the calli and single few-celled aggregates was observed through paraffin sections.The results revealed 5 pathways of embryogenic calli origination including origination from both ends of hypocotyls,origination in inner,differentiation from non-embryonic cells,origination from outer cells of calli,and origination in the surrounding of gland.Among these,the origination from both ends of hypocotyls and in inner were the most important pathways that embryogenic calli were differentiated directly from cambium cells of hypocotyls. Cotton somatic embryos originated from individual embryogenic cells,which were in exterior of embryogenic calli in most cases and in inner of embryogenic calli in a few cases.Somatic embryogenesis that the emhryoids were sequentially differentiated through globular,heart-shaped,torpedo embryo and cotyledonary embryo stages was similar to that of the zygotic embryo.The most critical events appeared to be the establishment of apical-basal patterns of symmetry,formation of apical meristems,and differentiation and axial change of procambium during the development of cotton somatic embryos. Differentiation of the protoderm layer marked the beginning of the structural differentiation in globular stage.Incipient procambium formation was the first sign of somatic embryo transition.Axial elongation of inner isodiametric cells of the globular somatic embryo followed by the change in the growth axis of the procambium was an important event in heart-shaped or oblong-stage somatic embryo.Vacuolation in the ground meristem of torpedo-stage embryo began the process of histodifferentiation.Three major embryonic tissue systems,shoot apical meristem,root apical meristem,and the differentiation of procambial strands,were visible in torpedo-stage somatic embryo. Subsequently,partitioning of cotyledon and shoot primordia in the upper layer of torpedo-stage somatic embryos took place.Vascular tissue of the cotyledonary-stage somatic embryos from the two cotyledons to radicle presented V or Y shape.Histological observations of some globular and torpedo-shaped embryos which faired to advance showed that establishment of polarity was the key factor which determined germination of somatic embryos.3.During somatic embryogenesis and zygotic embryos development,the start-up and morphogenesis of embryos are regulated by a variety of factors.In particular,somatic embryogenesis is a temporal and special regulated developmental process.This study attempted to analyze the process of somatic embryogenesis on the expression level of related genes,which involved in the process of transcription,post-transcription and post-translation modification as well as the synthesis,transport,and metabolic biochemical processes of carbohydrate,lipid,amino acids and protein.Among 33 genes in this study,25 were selected from a cotton somatic embryogenesis SSH array, considered differential expressed at different development stages such as the formation of embryogenic callus and somatic embryogenesis,the others including 5 SERK genes and 3 CKⅡgenes were acquired from the Genebank.From the qRT-PCR analysis,some genes were preponderant expressed in special stage of somatic embryogenesis,and these genes could divide into three categories:(1) associating with the start-up and morphogenesis of embryos.Almost all of the genes involving in metabolism and transporter presented a peak expression in this stage.This coincided with the previous studies including remarkable increase of DNA content,evident improving of RNA synthesis rate,mRNA variety,hoist of the levels of amino acids,and stirring synthesis of protein during somatic embryogenesis.Lipid-transporter proteins with the high level of expression were considered as the molecular marker substances of somatic embryogenesis. Post-transcription factors expressed actively at the formative and developmental stage of embryo indicating the complexity of this process.(2) Associating with the development and maturity of somatic embryo.During the development and mature of embryos, somatic and zygotic embryogenesis were different with the genes relating with post-transcription factor,CKⅡ,adenine,amino acid and lipid metabolism-related.Cell wall-related protein genes,adenine and amino acid metabolism-related genes and CKⅡgenes were behaved high expression level during the development and mature of somatic embryos.The high expression level of the post-translation modification genes,such as SERK gene,was considered as a sign that proteins interacted complexly.It was proved that the development and maturity of somatic embryos was complex as the start-up and morphogenesis of somatic embryos.(3) Associating with the development of zygotic embryos.The development and maturity of zygotic embryos required a higher level of acyltransferase.Our results showed that ERT and SCL were unique during somatic embryo development,the rest of transcription factors were not remarkable difference with the expression level between two types of embryo development process.It was related to the difference of the relevant factors identified by the DNA-binding domain of transcription factors.The results of this study first pointed out that HMG transcription factor and GRP protein played a very important role during the development of somatic and zygotic embryo and CKⅡwas very important for somatic embryo development in plant.APRT coding gene was active during the development of somatic embryogenesis, this indicated a way of nucleotide remedial synthesis existed during embryogenesis or embryo development in higher plant.In this study we also found that an oxidoreductase gene exhibited an extremely high expression level in embryogenic callus.It might be a marker gene in this stage.4.Protein kinases and phosphatases are responsible for several cellular events mediated by protein phosphorylation and dephosphorylation.Among these events are cell growth and differentiation and cellular metabolism.CKⅠand CKⅡare involved in the phosphorylation of several substrates.Endogenous protein phosphorylation and casein kinase activity were investigated during cotton somatic embryogenesis.It was observed that a number of different polypeptides are phosphorylated in the five tested development stages including non-embryogenic callus,embryogenic callus,globular embryos, torpedo-shaped embryos,and cotyledonary and mature embryos.Exogenous ATP enhanced protein phosphorylation in these stages.The use of okadaic acid and vanadate in the phosphorylation reactions increased phosphate incorporation in several polypeptides suggesting the presence of serine/threonine as well as tyrosine phosphatases in the five stages of somatic embryogenesis.Also,the results obtained in experiments with CKⅡinhibitor and in-gel kinase assays indicated the presence of CKⅡin somatic embryos. 5.Preparation of high-quality proteins from cotton calli and somatic embryoids is difficult due to the high endogenous contents of polysaccharides,polyphenols,lipin, pigments,and other interfering compounds.To establish a routine procedure of protein extraction for proteomic analysis to cotton embryogenic cultures,a new protocol was developed by optimizing a phenol extraction method combined with methanol/ammonium acetate precipitation.The protein extraction for 2-DE was remarkably improved by the combination of chemically and physically modified processes including polyvinylpolypyrrolidone(PVPP) addition,acetone cleaning and TCA/acetone washes followed by aqueous methanol/ammonium acetate and acetone lavation.The protocol gave a higher protein yield and higher resolution and spot intensity in 2-DE analysis. Furthermore,three lysis buffers for 2-DE were compared on cotton embryogenic calli proteins isolation.Incorporation of chaotrope mixture(7 mol L^-1 urea,2 mol L^-1 thiourea) with 4%CHAPS was found to be the most effective step.In addition,three CBB staining and four silver staining protocols were also compared respectively.A modified Neuhoff's CBB G-250 stain,dubbed "blue silver",exhibited a much higher sensitivity than all other CBB staining recipes and had a good stability.The silver stains were found to increase the sensitivity of protein detection over CBB staining,but had a poor stability due to difficulty in control of developing color.Of all silver stains,one protocol described by Mechin et al.was better on account of its sensitivity and stability.The results in this study laid the foundation for further research.