An iTRAQ-based proteomics approach to clarify the molecular physiology of somatic embryo development in Prince Rupprecht's larch (Larix principis-rupprechtii Mayr)

Abstract

Prince Rupprecht's larch (Larix principis-rupprechtii Mayr) is a native high-value forest tree species in North China whose clonal propagation through somatic embryogenesis (SE) has the potential to rapidly capture the benefits of breeding or genetic engineering programs and to improve raw material uniformity and quality. To date, research has focused on clarifying the molecular mechanism of SE, but proteomic studies are still in the early stages. In this study, isobaric tags for relative and absolute quantitation (iTRAQ) analysis was performed on three developmental stages of SE in L. principis-rupprechtii in an attempt to identify a wide range of proteins that are regulated differentially during this process. Proteins were extracted and analyzed from the pro-embryogenic mass (PEM), globular embryo (GE), and cotyledon embryo (CE) stages of embryo development. We detected 503 proteins in total and identified 96 proteins expressed differentially during different developmental stages. The identified proteins were analyzed further to provide information about their expression patterns and functions during SE. Four clusters of proteins based on shared expression profiles were generated. Functional analysis showed that proteins involved in primary metabolism, phosphorylation, and oxidation reduction were upregulated during somatic embryo development. This work provides novel insights into the process of larch embryo development in vitro and a basis for further study of the biological process and opportunities for practical application of this knowledge.

Fig 1. The development of cultured somatic embryos in L. principis-rupprechtii.

Samples were imaged at three developmental stages: PEM (A, bar = 500μm; B, bar = 100μm), GE (C, bar = 200μm) and CE (D, bar = 500μm).

Fig 2. Experimental design and iTRAQ workflow used in this study.

Samples were analyzed by iTRAQ coupling with nanoLC-MS/MS for examining the proteome changes during SE in L. principis-rupprechtii. Proteins were identified using MASCOT software.

Fig 3. Cluster analysis of proteins differentially expressed during SE.

Four clusters were generated to classify proteins during three time points: PEM stage, GE stage and CE stage. The membership values are used to assess how well a given entry fits the consensus profile and allows color coding cluster graph items according to their goodness of fit to the cluster consensus profile.

Fig 4. Functional categorization of differentially expressed proteins during SE.

Ninety-six differentially expressed proteins during three developmental stages (PEM, GE and CE) were categorized based on “Cellular Component,” “Molecular Function,” and “Biological Process” using WEGO.

Fig 5. Water content changes during somatic embryo development.

Percentage of water content during SE was calculated as (FW-DW)/FW. Bars represent standard errors (n = 5). Significantly different groups are indicated by different lowercase letters (P < 0.05).

Fig 6. Comparison and validation of SOD and CAT by iTRAQ profiling and enzyme activity assays.

iTRAQ analysis showed quantitative changes of SOD and CAT during three developmental stages (A). Activity assays for SOD and CAT were conducted to validate the iTRAQ results (B and C, respectively). Bars represent standard errors (n = 3). Significantly different groups are indicated by different lowercase letters (P < 0.05).

Fig 7. Cluster profiles of protein functional clusters during somatic embryo development.

A heat map of the log 2 relative abundance of proteins during SE in relation to the PEM stage was created with the iTRAQ-derived quantitative data. Proteins were grouped according to their known or putative biological functions. For each protein, accession number and protein description are provided. Yellow indicates upregulation, blue denotes downregulation, and black signifies zero difference. The grading represents the ratios of protein expression levels.