Biology Reference
In-Depth Information
Chapter 32
Quantitative Proteomic Analysis of Intact Plastids
Takeshi Shiraya , Kentaro Kaneko , and Toshiaki Mitsui
Abstract
Plastids are specialized cell organelles in plant cells that are differentiated into various forms including
chloroplasts, chromoplasts, and amyloplasts, and fulfi ll important functions in maintaining the overall cell
metabolism and sensing environmental factors such as sunlight. It is therefore important to grasp the
mechanisms of differentiation and functional changes of plastids in order to enhance the understanding of
vegetality. In this chapter, details of a method for the extraction of intact plastids that makes analysis pos-
sible while maintaining the plastid functions are provided; in addition, a quantitative shotgun method for
analyzing the composition and changes in the content of proteins in plastids as a result of environmental
impacts is described.
Key words
Chloroplast, iTRAQ, Quantitative shotgun proteomics, Percoll density gradient centrifu-
gation, Protein extraction
1
Introduction
Plastids, of which chloroplasts are a representative type, exhibit a
variety of functions in plant cells. In addition to carbon, nitrogen,
and sulfur assimilation, plastids perform the synthesis of impor-
tant chemical compounds in the plant including storage materials
such as carbohydrates and lipids. The decline and loss of plastid
functions, which can be caused by various environmental stresses
including dehydration, salinity, changes in temperature, and so
on, lead to aging of cells and cell death. Thus, the normal expres-
sion of plastid functions is essential to maintain the living activity
of cells.
The origin of the term
plastid
lies in the Greek word
plastikos
,
which means “molded.” It is important to gain a systematic under-
standing of the “molded” mechanism of these variable organelles.
In former times, plastid research was mainly conducted through
research on photosynthesis in chloroplasts. However, studies have
been actively carried out in recent years on various aspects of the
forms of differentiation, such as control of the differentiation of
