REVIEW

published: 16 June 2015

doi: 10.3389/fpls.2015.00430

Phosphoproteomics technologies
and applications in plant biology
research
Jinna Li 1 , Cecilia Silva-Sanchez 2 , Tong Zhang 3 , Sixue Chen 1, 2, 3 and Haiying Li 1*
1

College of Life Sciences, Heilongjiang University, Harbin, China, 2 Proteomics and Mass Spectrometry, Interdisciplinary
Center for Biotechnology Research, University of Florida, Gainesville, FL, USA, 3 Plant Molecular and Cellular Biology
Program, Department of Biology, UF Genetics Institute, University of Florida, Gainesville, FL, USA

Edited by:
Sabine Lthje,
University of Hamburg, Germany
Reviewed by:
Christof Rampitsch,
Agriculture and Agrifood Canada,
Canada
Mohammad-Zaman Nouri,
Rice Research Institute of Iran in
Mazandaran, Iran
*Correspondence:
Haiying Li,
College of Life Sciences, Heilongjiang
University, 74 Xuefu Rd, Harbin
150080, China
lvzh3000@sina.cn
Specialty section:
This article was submitted to
Plant Proteomics,
a section of the journal
Frontiers in Plant Science
Received: 17 March 2015
Accepted: 27 May 2015
Published: 16 June 2015
Citation:
Li J, Silva-Sanchez C, Zhang T, Chen
S and Li H (2015) Phosphoproteomics
technologies and applications in plant
biology research.
Front. Plant Sci. 6:430.
doi: 10.3389/fpls.2015.00430

Protein phosphorylation has long been recognized as an essential mechanism to

regulate many important processes of plant life. However, studies on phosphorylation
mediated signaling events in plants are challenged with low stoichiometry
and dynamic nature of phosphorylated proteins. Significant advances in mass
spectrometry based phosphoproteomics have taken place in recent decade, including
phosphoprotein/phosphopeptide enrichment, detection and quantification, and
phosphorylation site localization. This review describes a variety of separation and
enrichment methods for phosphoproteins and phosphopeptides, the applications
of technological innovations in plant phosphoproteomics, and highlights significant
achievement of phosphoproteomics in the areas of plant signal transduction, growth
and development.
Keywords: phosphoproteomics, enrichment, quantification, phosphorylation site mapping, plant biology

Introduction
Phosphorylation is one of the most important post-translational modifications (PTMs) of
proteins (Pawson and Scott, 1997). Approximately one-third of the proteins are modified by
phosphorylation (Hubbard and Cohen, 1993). The kinase mediated covalent addition of a
phosphate group to serine, threonine, and tyrosine residues in eukaryotes, and other amino
acids such as histidine, aspartate, glutamate, lysine, arginine, and cysteine in prokaryotes and
the subsequent removal of the phosphate groups by protein phosphatases constitute important
signaling and regulatory mechanisms in living organisms (Batalha et al., 2012). Reversible protein
phosphorylation regulates a wide range of cellular processes such as transmembrane signaling,
intracellular amplification of signals, and cell-cycle control. Protein phosphorylation often leads
to protein structural changes that can directly modulate protein activity, and induce changes in
interaction partners or subcellular localization (Jrgensen and Linding, 2008). The cascade of
protein phosphorylation in a signaling pathway provides the backbone for complex signaling
networks and regulatory processes in plant cells, including hormone sensing (Park et al., 2009),
and environmental stress responses (Mishra et al., 2006). Thus, the analysis of signaling pathways
in plants has often been focused on protein kinases. Traditional studies, however, described
the phosphorylation of a single substrate by a particular kinase. Based on genome annotation,
protein kinases were found to make up about 5.5% of the Arabidopsis genome (The Arabidopsis
Genome Initiative, 2000), which is nearly twice as many as in the human genome (Manning et al.,
2002). This indicates high specificity and complex networks of phosphorylation events in plants

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phosphorylated peptides, and ZrO2 tends to enrich singly

phosphorylated peptides. A serial enrichment procedure with
both TiO2 and ZrO2 can significantly increase the efficiency of
capturing phosphopeptides in biological samples (Kweon and
Hkansson, 2006; Gates et al., 2010). Metal dioxide enrichment
could also be coupled with other peptide fractionation
methods. For instance, a combination of TiO2 enrichment
and hydrophilic interaction liquid chromatography (HILIC)
resulted in identification of 2305 phosphopeptides belonging
to 964 proteins in wheat (Yang et al., 2013). Electrostatic
repulsion hydrophilic interaction chromatography (ERLIC),
a variation of HILIC that uses electrostatic repulsion as an
additional chromatography stationary phase, had also been used
successfully for selectively enrichment of phosphopeptides (Gan
et al., 2008; Loroch et al., 2013).

(Schulze, 2010). Many plant protein kinases have been identified

to play essential roles in response to a variety of stresses including
salt stress, cold stress, and pathogen invasion. Deciphering the
molecular events occurring in stress responses will enhance our
understanding of the biological processes in plants (De la Fuente
van Bentem et al., 2006; Stecker et al., 2014).
The combination of phosphoprotein/phosphopeptide
enrichment techniques, along with technological advancement
in tandem mass spectrometry has been employed as a powerful
tool to study protein phosphorylation and its biological
relevance (Chen and White, 2004). In this review, a variety
of separation and enrichment methods for phosphoproteins
and phosphopeptides, their features as well as applications in
phosphoproteomics research are described.

Phosphoproteomics Technologies

Quantitative Phosphoproteomics
Quantitative phosphoproteomics is aimed to enable a better
understanding of phosphorylation regulated biological events.
Comparative phosphoproteomics of wild-type and mutant plants
or control and treated plants could be conducted in many ways.
In general, the approaches can be grouped into gel-based, gelfree, stable isotope labeling, or label-free. Two-dimensional gel
electrophoresis (2-DE) has been a widely used technology that
resolves thousands of proteins by isoelectric point and molecular
weight. Pro-Q Diamond is a fluorescent stain that provides a
convenient method for selectively staining phosphoproteins in
acrylamide gels. The result shows a global map of the modified
proteins and their relative abundances compared to nonphosphorylated counterparts when a total protein staining is used
after Pro-Q Diamond staining. Differentially phosphorylated
proteins in wild-type and snk2.8 mutant Arabidopsis plants were
analyzed using 2-DE and Pro-Q, and putative substrates of
SnRK2.8 were identified (Shin et al., 2007).
Stable isotope labeling has been applied in plant
phosphoproteomics successfully using a gel-free approach,
for example, stable isotope labeling of amino acids in cell culture
(SILAC).The first SILAC in plants was done by introducing 15 N
in Arabidopsis suspension cells (Benschop et al., 2007) (Table 2).
The methodology has been improved over the years and found
more applications (Schtz et al., 2011; Stecker et al., 2014).
Another labeling approach introduces multiplex isobaric tags to
isolated proteins or digested peptides in vitro. Commonly used
tags include isobaric tags for relative and absolute quantification
(iTRAQ) and tandem mass tags (TMT). The tags are designed
to be isobaric during MS and fragment to reveal differential
low mass ion reporters during MS/MS. Due to its capability of
multiplexing up to 10 samples in a single experiment and the
enrichment effect for low abundance proteins, iTRAQ/TMT
labeling has become popular in plant phosphoproteomics (Jones
et al., 2006; Yang et al., 2013; Fan et al., 2014).
While both in vivo and in vitro label methods are limited by
the number of samples, label free approaches enable quantitative
phosphoproteomics of unlimited number of samples. There
are two main methods in label free quantitation. The first is
based on precursor ion peak intensity/area, and the second
is based on the number of MS/MS spectra acquired for a

Low stoichiometry of phosphorylated proteins and low

ionization efficiency of phosphopeptide are two major challenges
for protein phosphorylation detection. To reduce sample
complexity, it is necessary to enrich the modified proteins and/or
peptides before mass spectrometry (MS) analysis. Commonly
used enrichment techniques were summarized in Table 1, with
enrichment at the peptide level as a popular strategy. A successful
phosphoproteomics study depends not only on the selective
enrichment of phosphopeptides, but also on accurate detection
and quantitation of the peptides, as well as precise mapping of
the phosphorylation sites. Advances in these areas have been
extensively reviewed (Batalha et al., 2012; Fla and Honys, 2012;
Kline and De Luca, 2014; Silva-Sanchez et al., 2015). Most of
the technologies were developed in animal and yeast systems,
and subsequently applied in plants. Here we briefly describe
the advancement of phosphoproteomics technologies in plant
research (Table 2).

Enrichment Strategies
The most widely used enrichment method for phosphopeptides
takes advantage of the affinity binding between negatively
charged phosphate and positively charge metal ions (Fla
and Honys, 2012). Immobilized metal affinity chromatography
(IMAC) is often coupled with strong cation exchange (SCX)
for two-step phosphopeptide enrichment. For example, in a
SCX-IMAC experiment, three times more phosphopeptides were
identified when compared to the use of SCX or IMAC alone
(Trinidad et al., 2006). The first reported SCX-IMAC application
in plants resulted in identification of 283 phosphopeptides
(Nuhse et al., 2004). In addition, Polymer-based Metal-ion
Affinity Capture (PolyMAC) is a variant of IMAC, also showed
high selectivity. For instance, employment of complementary
PolyMAC-Titanium (Ti) and PolyMAC-Zirconium (Zr) ion
affinity chromatography lead to identification of 5386 unique
phosphopeptides (Wang et al., 2013a).
Metal dioxide especially titanium dioxides (TiO2 ) and
zirconium dioxides (ZrO2 ) are gaining popularity for
phosphopeptide enrichment. A comparison of the performance
of TiO2 and ZrO2 performed with -casein and -casein as
standard proteins showed that TiO2 tends to enrich multiply

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TABLE 1 | Phosphopeptide/phosphotprotein enrichment methodologies.

Enrichment method

Description

Advantage

Disadvantage

References

Immunoaffinity enrichment

Use of antibodies directed against pTyr,

pSer, pThr, and more recently against the
surrounding consensus sequences for
pSer/pThr.

Highly specific.

Low efficiency, high cost, use of

different antibodies for different
phosphorylation motifs.

Stokes et al., 2012

Immobilized metal affinity

chromatography (IMAC)

Negatively charged phosphate groups on

the phosphorylated amino acids interact
with positively charged metal ions such as
Ni2+ , Fe3+ , Ga3+ , Zr4+ , and Ti4+ that
are chelated with silica or agarose through
nitriloacetic acid or iminodiacetic acid.

Good for both phosphoproteins and

phosphopeptides. When used with
peptides, it can enrich mono- and
multiple phosphorylated peptides.

Tends to bind strongly to

monophosphorylated peptides,
which makes it difficult for
elution. Non-specific binding of
acidic peptides can occur.

Fla and Honys,

2012

Metal oxide affinity

chromatography (MOAC)

Similar to IMAC, the phosphate groups on

the amino acids interact with positively
charged metal oxides, e.g., titanium or
zirconium that acts as anchoring
molecules to trap phosphopeptides
through the formation of multi-dentate
bonds.

Good for both phosphoproteins and

phosphopeptides. When used with
peptides, it can enrich mono- and
multiple phosphorylated peptides.

Tends to binds strongly to

multiple phosphorylated
peptides, which makes it difficult
for elution. Nonspecific binding
of acidic peptides can occur.

Gates et al., 2010

Phos-Tag
chromatography,

Uses 1,3-bis[bis(pyridine-2ylmethyl)amino]propan-2-olato dizinc(II)

complex as a selective phosphate binding
tag in aqueous solution at neutral pH.

Increased sensitivity due to

complete deprotonation of
phosphoproteins/ phosphopeptides
at neutral pH. Elution at the
physiological pH allow for protein
activity and functional analysis.

Mainly used to confirm the

phosphorylation state in relatively
pure proteins, but not with
complex mixtures.

Kinoshita et al.,
2006

Prefractionation by strong
cation exchange (SCX)
and strong anion
exchange (SAX)

In SCX, tryptic peptides often carry a

charge of +2, except for phosphopetides
with a net charge of +1, making them
elute early in the chromatography. SAX
retains phosphor-peptides, allowing
separation based on the number of
phosphorylated residues.

Used for fractionation of highly

complex mixtures, it can be
performed on-line with mass
spectrometry.

Similar degree of unspecific

binding as IMAC and MOAC.

Leitner et al., 2011

Hydrophilic interaction
liquid chromatography
(HILIC)

Phosphopeptides with polar phosphate

groups are strongly retained on the HILIC
stationary phase resulting in separation
from non-phosphorylated species.

Good for both phosphoproteins and

phosphopeptides. When used with
peptides, it can enrich mono- and
multiple phosphorylated peptides.

Similar degree of unspecific

binding as IMAC and MOAC.

(Yang et al., 2013)

Electrostatic repulsion
hydrophilic interaction
chromatography (ERLIC)

ERLIC is a variation of HILIC using

electrostatic repulsion as an additional
phase to adjust selectivity by varying pH or
organic solvents.

Good for both phosphoproteins and

phosphopeptides. When used with
peptides, it can enrich mono- and
multiple phosphorylated peptides.

Similar degree of unspecific

binding as IMAC and MOAC.

Gan et al., 2008

Hydroxyapatite
chromatography

It takes advantage of the strong interaction

between positively charged hydroxyapatite
and phosphate ions.

Good for fractionating mono-, di-,

tri-, and multi-phosphorylated
peptides when using gradient of a
phosphate buffer.

Developed with phosphoprotein

standards, not tested with
complex samples.

Mamone et al.,
2010

given peptide (known as spectral counting). Both methods

were used in plant phosphoproteomics (Reiland et al., 2011;
Engelsberger and Schulze, 2012; Wang et al., 2013a). For instance,
Reiland et al. (2011) characterized the function of a thylakoidassociated kinase STN8 in the fine-tuning of cyclic electron flow,
which is regulated by the phosphorylation/dephosphorylation
event.
In addition to these large scale discovery phosphoproteomics
approaches, multiple reaction monitoring (MRM) has been
used for quantification of targeted phosphopeptides (Glinski

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and Weckwerth, 2006; Schulze et al., 2012; Minkoff et al.,

2015). A triple quadrupole is typically used for the MRM
measurement, in which the first quadrupole (Q1) is set as
a filter for the precursor ion with predetermined mass and
Q3 is set to measure a specific fragment ion. The specific
combination between a precursor ion and a fragment ion
is called a transition and multiple transitions can be used
to determine the relative and absolute (with synthesized
peptide standards) levels of phosphopeptides (Schulze et al.,
2012).

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TABLE 2 | Representative plant phosphoproteomics work in the past decade.

Plant

Phosphopeptides/

Enrichment

Quantitation

Phosphorylationsite MS instrument

materials

phosphoproteins

method

method

mapping

References

Arabidopsis plasma
membrane

283 phosphopeptides

IMAC

None

Mascot

QTOF Ultima (Waters)

Nuhse et al.,
2004

Arabidopsis leaves

317 phosphopeptides

Phospho- protein kit

iTRAQ

Mascot

QTRAP (AB Sciex)

Jones et al.,
2006

Arabidopsis leaves

16 phosphopeptides

None

MRM

MS3 de novo

TSQ Quantum (Thermo)

Glinski and
Weckwerth,
2005

Arabidopsis
suspension cells

1168 phosphopeptides

TiO2

SILAC

MSQuant

LTQ FT-ICR (Thermo)

Benschop et al.,
2007

Arabidopsis leaves

111 phosphoproteins

Pro-Q Diamond

2-DE

Mascot

QSTAR XL (AB Sciex)

Shin et al., 2007

Arabidopsis plasma
membrane

67 phosphopeptides

IMAC

Precursor ion intensity

MSQuant

LTQ (Thermo)

Niittyl et al.,
2007

Tomato leaves

48 proteins

TiO2

Precursor ion intensity

VEMS

QTOF (Micromass)

Stulemeijer
et al., 2009

Arabidopsis leaves

3589 phosphopeptides

TiO2 and FeCl3

Spectral counting

Mascot

Orbitrap (Thermo)

Reiland et al.,
2011

Arabidopsis leaves

3 phosphopeptides

None

MRM

Previously
determined

TSQ Quantum (Thermo)

Schulze et al.,
2012

Arabidopsis leaves

5386 phosphopeptides

PolyMAC

Precursor ion intensity

PhosphoRS

Orbitrap Velos (Thermo)

Wang et al.,
2013b

Arabidopsis leaves

1 phosphopeptide

None

MRM

Previously
determined

4000 QTRAP (AB Sciex)

Prado et al.,
2013

Wheat leaves

2305 phosphopeptides

TiO2 and HILIC

TMT

Mascot

Orbitrap Velos (Thermo)

(Yang et al.,
2013)

Arabidopsis
microsome

1229 phosphopeptides

TiO2

SILAC

Mascot

Orbitrap XL (Thermo)

Stecker et al.,
2014

Cotton leaves

1315 phosphopeptides

TiO2

iTRAQ

PhosphoRS

Q Exactive (Thermo)

Fan et al., 2014

Arabidopsis leaves

14 phosphopeptides

TiO2

MRM

Mascot

QTRAP 5500 (AB Sciex)

Minkoff et al.,
2015

LTQ, linear ion trap; VEMS, Virtual Expert Mass Spectrometrist; MRM, multiple reaction monitoring; TOF, time of flight; FT-ICR, Fourier transform ion cyclotron resonance. Please refer
to the text for other abbreviations.

Applications of Phosphoproteomics in
Plant Biology Research

(MAP2K) and a MAPK, which activate in a sequential manner

via phosphorylation (Figure 1). An activated MAPKKK firstly
phosphorylates two serine and/or threonine residues (S/T-X35-S/T) located within the activation loop of the MAPKK.
Activated MAPKKs in turn trigger MAPK activation through
dual phosphorylation of a highly conserved T-X-Y motif in
the activation loop of MAPKs (Hamel et al., 2012). In a
proteomic analysis of plasma membrane isolated from maize
roots, four isoforms of Pto-interacting-like kinase 1 (PTI1)
showed increased levels in response to low and high iron
conditions (Hopff et al., 2013). Interestingly, a previous oxidative
stress study in Arabidopsis demonstrated that interaction of a
PTI1-like kinase (PTI1-4) with another serine/threonine protein
kinase, oxidative signal-inducible 1 (OX1), mediates oxidative
stress signaling. In addition, PTI1-4 was found to interact with
MPK6 in the same protein complex (Forzani et al., 2011). These
results imply that the PTI signals may function through the
OXI1 and MPK6 signaling cascades. Recently, Hoehenwarter
et al. (2013) developed a two-step chromatography combining
phosphoprotein enrichment using Al(OH)3 -based metal oxide
affinity chromatography with phosphopeptide enrichment using
TiO2 -based metal oxide affinity chromatography to enrich
phosphopeptides from complex A. thaliana protein samples. The

Phosphoproteomics of Signal Transduction

Protein phosphorylation in signal transduction is an important
area of current plant biology research. Many key proteins
such as kinases, transcription factors, and ubiquitin ligases
function through reversible protein phosphorylation in the signal
transduction cascade (Hunter, 2000). In recent years, it has
become apparent that analysis of signaling networks is required
for the understanding of the dynamic and complex mechanisms
underlying cellular functions and outputs. Most of the studies
in plants have often been focused on protein kinases and
identification of the phosphorylated substrates.
The mitogen-activated protein kinases (MAPKs) constitute
one of the most important signaling mechanisms in plants,
and they play essential roles in linking the perception of
different stimuli with cellular adaptive responses. The MAPK
signal transduction pathways are evolutionarily conserved in
all eukaryotic organisms such as plants, yeast, fungi, insects,
nematodes, and mammals (Mishra et al., 2006). A MAPK
cascade is minimally composed of distinct combinations of at
least three protein kinases: a MAPKKK (MAP3K), a MAPKK

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FIGURE 1 | A typical mitogen-activated proteins (MAP) kinase cascade. The MAPK cascades are generally organized as modular pathways, in which the
activation of upstream MAPKKKs leads to the sequential phosphorylation and subsequent activation of downstream MAPKKs and MAPKs.

particular, this study provided insights into the ABA signaling

pathway by identifying potential substrate proteins of SnRK2s
(Umezawa et al., 2013).

method was successfully applied to identify MAPK substrates.

A large number of novel phosphorylation sites and 141 MAPK
substrate candidates (mostly novel) have been identified. For
example, time for coffee (TIC) and non-phototropic hypocotyl 3
(NPH3), which are involved in circadian clock and phototropism,
were found to be MPK3/6 substrates. The result suggests that
plant circadian rhythm and phototropism may be regulated by
the MAPK signaling network.
Abscisic acid (ABA) is a phytohormone that plays an
important role in many aspects of plant life. For example,
ABA is essential for regulating seed maturation and stomatal
closure under abiotic and biotic stresses (Hubbard et al.,
2010). Protein phosphorylation and dephosphorylation play a
central role in ABA signaling. Multiple signaling components
have been found to undergo phosphorylation/dephosphorylation
regulation to control stomatal movement in response to ABA
(Zhang et al., 2014). A simplified ABA signaling model consists of
the soluble ABA receptors (members of the pyrabactin resistance
1 (PYR1) and PYR1-like (PYL) proteins, also known as regulatory
component of ABA receptor (RCAR) family, and collectively
referred to as PYR/PYL/RCAR), a subgroup of type 2C protein
phosphatases (PP2Cs), and the SNF1-related protein kinase 2
(SnRK2) family (Umezawa et al., 2010). Umezawa et al. (2013)
studied protein phosphorylation networks in ABA signaling
using phosphoproteomics of Arabidopsis treated with ABA
and dehydration stress, as well as snrk2 mutants to identify
SnRK2-dependent protein components. Comparative analysis
between ABA treatment and dehydration stress revealed that
dehydration stress induced multiple protein phosphorylation
pathways in addition to the ABA-dependent pathway, supporting
that multiple protein kinases are involved in dehydration stress
signaling, including SnRK2s, MAPKs, and calcium-dependent
protein kinases (CDPKs) (Umezawa et al., 2013). Further studies
will be required for understanding how multiple kinases mediate
dehydration stress signaling. It appeared that subclass III SnRK2s
may be uniquely employed during ABA responses, and subclass
II SnRK2s are the main subclass that regulates dehydration
stress responses, although they are also activated by ABA. By
integration of genetics with phosphoproteomics, it is possible to
connect protein kinases with their in vivo signaling pathways. In

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Phosphoproteomics of Subcellular
Compartments
Phosphoproteomics studies were often performed in a shotgun
fashion, with the identification of hundreds and thousands of
proteins that lead to a very complicated set of phosphoproteins
across subcellular compartments and organelles (Table 2),
leading to a poor understanding of the networks that regulate
the cellular activities (Jung et al., 2000). Compartmentalization
in eukaryotes offers a practical approach to study subcellular
phosphoproteomics networks, with a reduced population of
identified proteins. There are about 3000 proteins in the
chloroplasts of Arabidopsis, but only four kinases were previously
identified. It may be feasible to find specialized kinases
or families of kinases that can potentially show differential
activities in the chloroplasts (Millar and Taylor, 2014; van
Wijk et al., 2014). A meta-analysis of 27 publications of
phosphoproteomics data sets in Arabidopsis comprises 60,366
phosphopeptides matched to 8141 non-redundant proteins.
The phosphoproteins showed predicted subcellular distribution
in the following categories: nucleus, secretory (containing
endoplasmic reticulum, Golgi, plasma membrane, cell wall, and
vacuolar), cytosol, other/unknown, intra-plastid, mitochondria,
and peroxisome (van Wijk et al., 2014). The study of
phosphoprotein compartmentalization supports the hypothesis
that a fine mechanism helps to maintain and regulate
protein translation, post-translational metabolism, signaling, and
trafficking through the cells (Millar and Taylor, 2014). Some
studies have already started to focus on PTMs in subcellular
compartments and here we describe a few examples.
Jones et al. (2009) performed a phosphoproteomic analysis
of the nuclei-enriched fractions prepared from suspension
cell cultures and seedlings of A. thaliana. The work led to the
identification of 416 phosphopeptides from 345 proteins. Two
thirds of the proteins are known or predicted to be nuclear
localized, and one half of the nuclear localized proteins have
novel phosphorylation sites. Many phosphorylation sites and

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regulators, respectively (Seo et al., 2006). Many protein kinases

and phosphatases participate in ABA signaling to regulate
seed germination. Recently, Han et al. (2014) used PolyMAC
phosphopeptide enrichment and gel-free proteomics identified
a total of 933 phosphorylated peptides corresponding to 413
proteins in rice embryos during early stages of germination.
By quantitative normalization of phosphoprotein abundance
and One-Way ANOVA testing, 149 phosphorylated proteins
were found to be significantly changed in abundance during
germination. Among the phosphoproteins, seven brassinosteroid
(BR) signaling pathway-related proteins were identified and three
(BR signaling kinase 1, BR-insensitive 2, and BR-insensitive 1
suppressor 1) showed significant increases in phosphoprotein
abundance during the early stages of germination. In addition,
treatment with brassinolide promoted the rice seed germination.
These results suggest that brassinosteroid signal transduction
plays an important role in triggering seed germination.
Plant vegetative growth is important for biomass
accumulation and potential biofuel applications. A recent
phosphoproteomic study of Brachypodium distachyon as a
model biofuel plant using TiO2 enrichment and LC-MS/MS
has identified a total of 1470 phosphorylation sites in 950
phosphoproteins (Lv et al., 2014). Among the phosphoproteins,
there were 58 transcription factors, 84 protein kinases, 8 protein
phosphatases, and 6 cellulose synthases. Through bioinformatic
analysis, a protein kinase and phosphatase centered network
related to rapid vegetative growth was deciphered. For example,
a MAPK signaling cascade might play an important role in leaf
growth and development (Lv et al., 2014). This finding is very
interesting, considering MAPK cascade is generally involved in
plant stress responses (Mishra et al., 2006).

kinase motifs were identified on proteins involved in nuclear

transport (e.g., Ran-associated proteins), and on transcription
factors, chromatin remodeling proteins, and spliceosome
components. Surprisingly, many novel phosphopeptides from
proteins involved in vesicle trafficking such as components
of the exocyst complex (SEC10, SEC51, and SEC5a-like) were
identified. How phosphorylation of these SEC proteins plays a
role in vesicle trafficking is intriguing. Recently, phosphorylation
of Sec31 by a casein kinase 2 was found to control the duration
of COPII vesicle formation, decrease its association with ER and
promote ER-to-Golgi trafficking (Koreishi et al., 2013).
Subcellular proteomics can address conserved mechanisms
underlying plant responses to stresses. By analyzing the
phosphorylation changes in proteins of microsomal fractions
from A. thaliana and Oryza sativa, Chang et al. (2012)
found similar phosphoproteins between the species including
photosystem II reaction center protein H PsbH. Both Arabidopsis
and rice showed an increased ratio for a diphosphorylated
peptide (ApTQpTVEDSSRSGPR) of PsbH as a response to salt
stress. Interestingly, the two phosphorylation sites (Thr2 and
Thr4) are found to be evolutionarily conserved in many plants
using sequence alignment.
Light plays a crucial role in the regulation of protein
phosphorylation in photosynthetic thylakoid membranes. In
Arabidopsis, the thylakoid Ser/Thr protein kinase 7 (STN7)
and STN8 kinases are light regulated and participate in
phosphorylation of thylakoid membrane proteins and stroma
proteins. Ingelsson and Vener (2012) performed a thylakoid
phosphoproteomics study using Arabidopsis wild-type and the
STN mutants stn7, stn8, and stn7stn8. The results showed that
STN7 is required for the phosphorylation of pTAC16 at the
Thr451, and pTAC16 was found to be distributed between
thylakoids and nucleoid. In addition, the results suggest that
pTAC16 could anchor DNA to the thylakoid membrane, and it
was proposed that STN7-dependent phosphorylation of pTAC16
may regulate membrane-anchoring functions of the nucleoid.

Identification and Functional Analysis of

Novel Phosphorylation Sites
The identification of protein phosphorylation sites has been
difficult in the past. Nowadays, high throughput modern
technologies such as tandem MS have promoted large-scale
discoveries of new phosphorylation sites and phosphoproteins
in recent years. Rao and Mller (2012) initiated a large-scale
study of phosphorylation site occupancy in eukaryotic proteins.
They analyzed the occurrence and occupancy of phosphorylation
sites in a large number of eukaryotic proteins, and provided
insights into protein phosphorylation and related processes.
Phosphorylation probability was found to be much higher in both
termini of protein sequences (much more in the C-terminus)
than middle parts of the sequences. A large proportion (51.3%)
of the occupied sites had a nearby phosphorylation within a
distance of 10 amino acid residues. This proportion is very high
compared to the expected value of 16.9%. More than half of the
phosphorylated sites fall within a small number of motifs.
A large phosphoprotein, the RNA surveillance protein UPframeshift 1 (Upf1) in Saccharomyces cerevisiae, has only
been partially characterized for phosphorylation sites, but the
functional relevance of the phosphorylation has not been
studied. Lasalde et al. (2014) used tandem MS and in vitro
phosphorylation assays to identify novel phosphorylation sites

Phosphoproteomics of Plant Growth and

Development
Sucrose non-fermenting 1 related kinase (SnRK1) acts as a
sensor of energy levels in plant development, and regulates
plant growth by maintaining energy homeostasis during stress
conditions (Tsai and Gazzarrini, 2014). It is activated by sugar
depletion, energy depletion in the dark and hypoxia (BaenaGonzlez and Sheen, 2008). Trehalose 6-phosphate (T6P) is
a signaling molecule involved in the regulation of embryonic
and vegetative development, flowering time, and meristem
determinacy. An increase in the levels of T6P led to metabolic
changes that promote plant growth. However, T6P regulates
SnRK1 by inhibiting its activity. SnRK1 and T6P are global
regulatory molecules that also interact with plant hormones,
and along with ABA modulate several crucial cellular activities
such as seed maturation and germination, ABA sensitivity
and signaling, vegetable growth, and flowering regulation (Tsai
and Gazzarrini, 2014). Seed germination is known to be
controlled by phytohormones, including gibberellins (GAs) and
ABA, which play antagonistic roles as positive and negative

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Li et al.

Phosphoproteomics and applications

greatly improved over the years. For instance, combining the

titanium (Ti4+ )-based IMAC and the reverse phase (RP)strong cation exchange (RP-SCX) biphasic trap column-based
online RPLC is a great example of the advancements (Bian
et al., 2012; Wang et al., 2013b). The recent development of
specific labeling techniques has greatly aided the quantification
of phosphorylation profiles and their stress-induced changes.
Especially, iTRAQ and TMT in vitro labeling and SILAC in vivo
labeling have shown to be successful in combination with IMAC
and MS (Isner et al., 2012; Yang et al., 2013; Zhang et al., 2013;
Stecker et al., 2014). These studies have revealed novel nodes
and edges in signaling pathways and regulatory processes that
are dependent on phosphorylation. Despite many new insights
gained from quantitative phosphoproteomics, improvements are
required to enable a comprehensive description of total and PTM
proteomes. Currently, LC-MS/MS based phosphoproteomic
technologies have established as an indispensable tool in
identification of novel phosphorylation sites and signaling
pathways. As large data sets accumulate, informatics tools will
be indispensable, e.g., informatics has revealed phosphorylation
probability to be frequent at the termini of protein sequences.
Taken together, researchers have provided not only new insights
into the complex phosphorylation regulatory networks in plants,
but also important resources for future functional studies of
protein phosphorylation in plant growth and development.

in UPF1. A total of 11 phosphorylated residues of UPF1 were

identified. Sequence alignment of UFP1 from lower and higher
eukaryotes showed complete conservation of the phosphorylated
residue Y-754. Residues corresponding to S. cerevisiae UPF1
T-194, S-492, Y-738, and S-748 were similar to those in
the homologs of Homo sapiens, Mus musculus, Drosophila
melanogaster, and A. thaliana. Since the phosphorylated residues
in UPF1 were clustered in four small regions, each one was tested
to determine its importance by independently deleting the four
individual regions. The deletion mutant lacking phospho-motif4 was not able to complement the Nonsense-Mediated mRNA
Decay (NMD) defect as revealed by Northern blot analysis.
To test the role of phospho-motif-4 in translation termination
efficiency, a well-established dual luciferase assay was used. The
deletion-mutant lacking phospho-motif-4 was not able to rescue
this defect, indicating that this motif has a role in translation
termination efficiency. To dissect the sequences within phosphomotif-4 required for NMD activity, PCR-mediated mutagenesis
was used to generate three additional deletion mutants (736745,
746750, 751751). The results revealed that deletion of residues
736745 reduced NMD activity as measured by Northern blot
analysis. To test the functional role of Y738 and Y742, sitedirected mutagenesis was used to create phosphorylation mimic
mutants. Interestingly, the Y738F and Y742F fully rescued
NMD activity of a chromosomal UPF1 deletion-mutant strain,
indicating that they are not compromised in their ability to
function in NMD. These results provided strong evidence that
UPF1s ability to promote translational termination fidelity is
depended on the conserved C-terminal phosphorylation motif,
which is important for its NMD activity.

Acknowledgments
Research in the HL lab was supported by the National Science
Foundation of China (Project 31471552: The response of
antioxidant enzymes to salt stress in sugar beet M14, and Project
31401441: Identification of root variation related proteins in
sugar beet (Beta vulgaris L.) monosomic addition line M14
using iTRAQ analysis), the National Science Foundation
of Heilongjiang Province (Project C201202: Comparative
proteomics analysis of sugar beet M14 under salt stress), and the
Common College Science and Technology Innovation Team of
Heilongjiang Province. The paper represents serial 016 from our
innovation team at the Heilongjiang University (Hdtd2010-05).

Concluding Remarks
Phosphopeptide enrichment and MS have been essential tools
for studying protein phosphorylation. It is challenging to
directly detect phosphoproteins in biological samples due to
the low abundance and low stoichiometry of phosphorylation
in different biological processes. The enrichment methods of
phosphoproteins/phosphopeptides from complex mixtures have

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Conflict of Interest Statement: The authors declare that the research was
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