Article pubs.acs.org/jpr
iTRAQ Protein Profile Differential Analysis between Somatic Globular and Cotyledonary Embryos Reveals Stress, Hormone, and Respiration Involved in Increasing Plantlet Regeneration of Gossypium hirsutum L. Xiaoyang Ge,† Chaojun Zhang,† Qianhua Wang,† Zuoren Yang,† Ye Wang,† Xueyan Zhang,† Zhixia Wu,† Yuxia Hou,‡ Jiahe Wu,*,§ and Fuguang Li*,† †
State Key Laboratory of Cotton Biology, Institute of Cotton Research, Chinese Academy of Agricultural Sciences, Huanghe Road, Anyang, Henan 455000, China ‡ College of Science, China Agricultural University, No.2 Yuanmingyuan West Road, Beijing 100193, China § Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Beijing 100101, China S Supporting Information *
ABSTRACT: Somatic embryo development (SED) in upland cotton shows low frequencies of embryo maturation and plantlet regeneration. Progress in increasing the regeneration rate has been limited. Here a global analysis of proteome dynamics between globular and cotyledonary embryos was performed using isobaric tags for relative and absolute quantitation to explore mechanisms underlying SED. Of 6318 proteins identified by a mass spectrometric analysis, 102 proteins were significantly up-regulated and 107 were significantly down-regulated in cotyledonary embryos. The differentially expressed proteins were classified into seven functional categories: stress responses, hormone synthesis and signal transduction, carbohydrate and energy metabolism, protein metabolism, cell wall metabolism, cell transport, and lipid metabolism. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis showed that stress response, hormone homeostasis, and respiration and photosynthesis were involved in SED. Quantitative real-time PCR analysis confirmed the authenticity and accuracy of the proteomic analysis. Treatment of exogenous hormones showed that abscisic acid and jasmonic acid facilitate SED, whereas gibberellic acid inhibits SED and increases abnormal embryo frequency. Thus, global analysis of proteome dynamics reveals that stress response, hormone homeostasis, and respiration and photosynthesis determined cotton SED. The findings of this research improve the understanding of molecular processes, especially environmental stress response, involved in cotton SED. KEYWORDS: hormone, iTRAQ, proteomics, respiration, somatic embryo development, stress, upland cotton, Gossypium hirsutum
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INTRODUCTION Cotton (Gossypium hirsutum L.), one of the most important economic crops worldwide, has global socioeconomic impacts worth approximately $56 billion.1 In the past two decades, genetic engineering has been successfully used to improve crop plants, for example, transgenic herbicide-tolerant and pestresistant cotton cultivars2,3 Plant regeneration via somatic embryogenesis is still a limiting step for transgenic cotton development.4,5 In cotton, only a low frequency of somatic embryos are able to mature and regenerate into plantlets. Most embryos develop abnormally, redifferentiate into calli, or become necrotic and die. Techniques to promote somatic embryo development (SED) and maturation and to reduce the frequency of abnormal embryos in cotton have been studied widely, but little progress has been achieved. However, global analyses indicate many genes are involved in (somatic) © XXXX American Chemical Society
embryo development. For instance, LEAFY COTYLEDON1 (LEC1) is required for embryo development of Arabidopsis6,7 and LEC2 induces SED in vegetative cells.8 BABY BOOM (BBM) can promote somatic embryos from transgenic Arabidopsis and Brassica when ectopically overexpressed.9 FUSCA3 (FUS3) is important in embryo development in Arabidopsis.10 MtSERF1 is essential for SED and is induced by ethylene in Medicagotruncatula.11 Accumulation of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1) RNA in carrot cells is a marker for single cell progenitors of somatic embryos.12 Several genes involved in stress signaling pathways, such as xanthoxin Special Issue: Environmental Impact on Health Received: July 1, 2014
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dehydrogenase (ABA2), JASMONATE RESISTANT1(JAR1), JASMONATE ZIM-DOMAIN1 (JAZ1), JAZ2, and JAZ10 are associated with cotton SED.13 Although accumulating evidence indicates that a number of genes and proteins are involved in somatic/zygote embryo development, data on the molecular mechanisms and crosstalk among proteins involved in SED are still missing. Plant SED is a multifactorial event; various factors, especially stress adaptation and response, affect the morphological development and complex physiological processes of zygotic and SED.14 Stress adaptation and response is an important factor that affects plant embryo growth and development in a wide variety of species. During rice embryonic development, the expression of many stress-responsive proteins is actively regulated.15 During cotton embryo development, stress-related genes mainly including these encoding kinase or transcription factors are actively regulated.13 Stress-related genes were differentially expressed in somatic embryogenesis of soybean and Medicago truncatula.11,16 Osmotic stress resulting from exogenous hormones supply also plays a key role in embryo development. For example, appropriate concentration of abscisic acid (ABA) effectively promotes embryo development and maturation and inhibits abnormal embryo proliferation.17 Homeostasis of ABA in metabolism is beneficial for barley seed development.18 Gibberellic acid (GA) affects germination and regeneration of somatic embryos in turf-type Bermuda grass (Cynodon spp.) and alfalfa (Medicago sativa).19,20 On the basis of these above studies, it appears that stress adaptation and response are vital to plant (somatic) embryo development. Similarly, stresses resulting from different environments also have important impacts on cotton development, especially (somatic) embryo development, which affects yield and quality of cotton. Thus, to improve human condition, sustainable cotton productivity is required through controlling and adapting various environmental stresses. Isobaric tags for relative and absolute quantitation (iTRAQ), a powerful tool to detect actively expressed proteins, have been used in proteome studies owing to its advantages over 2D gel electrophoresis.21 Identification of overly large/small, acidic/ basic, and hydrophobic proteins by 2D gel electrophoresis may be difficult. The use of iTRAQ allows simultaneous identification and quantification of proteins from multiple samples. Moreover, some important post-translational modification information, such as proteolytic cleavage, glycosylation, or phosphorylation, may be retained. In addition, the proteome generated by iTRAQ typically consists of millions of peptides, from which substantial information on the proteins can be extracted with the use of bioinformatic tools. In this study, we used iTRAQ to assess proteome changes and identify differentially expressed proteins between somatic globular and cotyledonary embryos of cotton. On the basis of Gene Ontology (GO) annotations, these differentially expressed proteins involved in important SED processes were grouped into seven functional categories. Proteins with functions in respiration and photosynthesis, stress adaptation, and hormones are likely important in somatic embryo maturation and plantlet regeneration. Characterization of the differentially expressed proteins will be helpful to reveal the molecular mechanisms underlying somatic embryo growth, development, and physiological functions. The results of proteomic analysis open new opportunities for genetically engineering cotton, especially in environmental stress control and adaptation, to achieve higher yields and quality, leading to improving human condition.
Article
MATERIALS AND METHODS
Plant Materials
Gossypium hirsutum cv. CCRI24, a cotton genotype that readily produces somatic embryos, was used as the experimental material. Cotton seeds were surface-sterilized by rinsing in 70% ethanol for 1 min and then washing three times with sterile distilled water prior to being treated with 30% H2O2 for 24 h. The seeds were germinated on modified Murashige and Skoog (MS)22 medium (50 mL/L MS + 25 g/L sucrose +5.6 g/L agar). The pH was adjusted to 5.8 to 6.0 prior to autoclaving the medium. Hypocotyls sections (4−6 mm) from 7-day-old seedlings were used as explants for induction of callus. The explants were cultured according to a procedure previously described.23,24 Somatic embryos were formed in the medium following the sequence of callus induction and proliferation, embryogenic callus emergence, and somatic embryo formation. The morphology and vigor of somatic embryos are important determinants of their potential regeneration. Two stages of embryo development were sampled: (1) green globular embryos showing embryonic polarity and (2) green cotyledonary embryos with two normal cotyledons. Globular and cotyledonary embryos were collected with the aid of a stereo microscope (Olympus SZ-DT, Tokyo, Japan), immediately frozen in liquid nitrogen, and stored at −80 °C before protein extraction. For treatment with ABA, GA, paclobutrazol, and JA (Sigma), concentration gradients were generated as follows: ABA (0.01, 0.04, 0.2, 0.4, and 2.0 μM), GA (0.28, 1.4, 2.8, 14, and 28 μM), paclobutrazol (0.01, 0.03, 0.1, and 0.33 μM), and JA (0.1, 0.5, 1, 2, and 10 μM). All treatments were applied to cultures in a greenhouse with a 12 h photoperiod at 28 °C. Each experiment was repeated three times. Protein Preparation
Protein extraction was performed using an a phenol-based procedure described previously.25 Three biological replicates of cotyledonary embryos each with ∼1 g and two biological replicates of globular embryos each with ∼1 g were performed, The protein extracts were stored at −80 °C until further use. Protein concentrations were assayed with the Bradford method.26 Proteome Analysis of Cotton Somatic Embryos by iTRAQ
To reveal the dynamic protein profile of cotton SED, we implemented iTRAQ analysis at the Beijing Genomics Institute, Shenzhen, China. Protein extracted from globular or cotyledonary embryos was digested using trypsin and labeled using iTRAQ 8-plex kits. Globular embryos were labeled with 119 and 121 mass tags, and cotyledonary embryos were labeled with 113, 114, and 115 tags, respectively. The labeled pooled peptides were dried and redissolved for fractionation by strong cation exchange chromatography using a LC-20AB HPLC pump system (Shimadzu, Kyoto, Japan). The procedure used for strong cation exchange chromatography is described in Supplemental Protocol 1 in the Supporting Information. The fractionated samples were analyzed by LC−ESI−MS/MS based on Triple TOF 5600, and the procedure used is outlined in Supplemental Protocol 2 in the Supporting Information. Protein identification and quantification were performed using the Mascot 2.3.02 search engine (Matrix Science, Boston, MA). Search parameters were set as follows: trypsin/P was chosen as the enzyme with one missed cleavage allowed; fragment mass spectrum (MS) tolerance, ± 0.1 Da; peptide MS tolerance, ± 0.05 Da; variable modification, Gln B
dx.doi.org/10.1021/pr500688g | J. Proteome Res. XXXX, XXX, XXX−XXX
Journal of Proteome Research
Article
→ pyro-Glu (N-term Q), oxidation (Met). iTRAQ8plex (Y); fixed modification, carbamidomethyl (C), iTRAQ8plex (Nterm), iTRAQ8plex (K). Complete sequenced cotton AAgenome27 and DD-genome28 sequences were concatenated to organize a protein database, which included 81 147 protein sequences but not any common contaminants (such as trypsin and keratins); each matched sequence with the highest Mascot score was submitted to the cotton protein database for BLASTX analysis to annotate functions for each protein. To reduce the probability of false peptide identification, only peptides with significant scores (≥20) at the 99% confidence interval by a Mascot probability analysis greater than “identity” were counted as identified, and each confident protein identification involved at least one unique peptide; finally we obtained 6318 proteins, of which 3258 proteins with two or more unique peptides (Supplemental Table 5 in the Supporting Information). For protein quantitation, it was required that a protein contained at least two unique peptides. The quantitative protein ratios were weighted and normalized by the median ratio in Mascot. We only used ratios with p values 1.5 were considered as significant. Functional category analysis was performed with Blast2GO software (http://www.geneontology. org) and Clusters of Orthologous Groups of Proteins System (COG) software (http://www.ncbi.nlm.nih.gov/COG/). To take advantage of the current knowledge of biochemical pathways and other types of molecular interactions, we used KEGG databases (http://www.genome.jp/kegg/pathway.html).
Figure 1. Phenotype of normal somatic globular embryo (A) and cotyledonary embryo (B) in cotton. Bar: 1000 μm.
acquired 36 391 unique spectra, 23 922 identified peptides including 16 021 unique peptides, and 6318 identified proteins (Figure 2A). The protein mass distribution above 10 kDa followed a normal distribution (Figure 2B), among which 10−50 kDa covered 43%, 50−100 kDa comprised 41%, and above 100 kDa made up 16% of the proteins. The peptide number distribution is shown in Figure 2C. The proteins with a single peptide, 2−5 peptides, 6−10 peptides, and above 11 peptides comprise 1600, 2651, 1233, and 834, respectively. The distribution of protein sequence coverage is shown in Figure 2D. Protein sequence coverage with 40−100, 30−40, 20−30, 10−20, and under 10% variation accounted for 4.95, 7.50, 14.65, 25.48, and 47.56% coverage, respectively. Functional Categories of Differentially Expressed Proteins
Differentially expressed proteins were defined as those that showed greater than 1.5-fold change in relative abundance and a P value
