Journal of Experimental Botany, Vol. 66, No. 6 pp. 1627–1640, 2015
doi:10.1093/jxb/erv041 Advance Access publication 19 February 2015
Review Paper
Inside a plant nucleus: discovering the proteins
Beáta Petrovská1,2,*, Marek Šebela2 and Jaroslav Doležel1
1 Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 31, 783 71
Olomouc, Czech Republic
2 Department of Protein Biochemistry and Proteomics, Centre of the Region Haná for Biotechnological and Agricultural Research,
Faculty of Science, Palacký University, Šlechtitelů 11, 783 71 Olomouc, Czech Republic
* To whom correspondence should be addressed. E-mail: [email protected]
Received 25 November 2014; Revised 14 January 2015; Accepted 16 January 2015
Abstract
Nuclear proteins are a vital component of eukaryotic cell nuclei and have a profound effect on the way in which genetic
information is stored, expressed, replicated, repaired, and transmitted to daughter cells and progeny. Because of the
plethora of functions, nuclear proteins represent the most abundant components of cell nuclei in all eukaryotes.
However, while the plant genome is well understood at the DNA level, information on plant nuclear proteins remains
scarce, perhaps with the exception of histones and a few other proteins. This lack of knowledge hampers efforts to
understand how the plant genome is organized in the nucleus and how it functions. This review focuses on the current state of the art of the analysis of the plant nuclear proteome. Previous proteome studies have generally been
designed to search for proteins involved in plant response to various forms of stress or to identify rather a modest
number of proteins. Thus, there is a need for more comprehensive and systematic studies of proteins in the nuclei
obtained at individual phases of the cell cycle, or isolated from various tissue types and stages of cell and tissue differentiation. All this in combination with protein structure, predicted function, and physical localization in 3D nuclear
space could provide much needed progress in our understanding of the plant nuclear proteome and its role in plant
genome organization and function.
Key words: Cell nucleus, chromatin, genome function, nuclear proteins, plants, proteomics.
Introduction
The eukaryotic nucleus has been an object of research attention for a considerable time, primarily because it houses most
of the genetic material of the cell. Early studies revealed a large
variation in nuclear size, shape, and DNA amount, both within
organisms and between species (Tamura and Hara-Nishimura,
2011). It was found that related organisms may have highly
divergent DNA amounts, as demonstrated in angiosperms,
where the C value (DNA amount in the G1 nucleus prior to
replication) ranges >2000-fold. This discrepancy, originally
termed the ‘C-value paradox’ (Thomas, 1971), has been
explained by the presence of repetitive DNA and polyploidy,
and was renamed the ‘C-value enigma’ (Gregory, 2001).
Rapid progress in DNA sequencing technologies made it
possible to establish almost complete genome sequences in a
Abbreviations: AGO, Argonaute protein; CB, Cajal body; CRWN, crowded nuclei; CypRS64, arginine/serine-rich domain-containing cyclophilin 64; 1DE, onedimensional protein electrophoresis; 2DE, two-dimensional protein electrophoresis; ESI, electrospray ionization; GCP, γ-tubulin complex protein; GIP, γ-tubulin
complex protein (GCP)-interacting protein; INM, inner nuclear membrane; KASH, Klarsicht/ANC-1/Syne-1 homology; LC, liquid chromatography; LINC, linker of
nucleoskeleton and cytoskeleton; LINC1–4, little nuclei1–4; MAD, mitotic arrest deficient; MAF1, matrix attachment factor 1 (MFP1-associated factor 1); MALDITOF, matrix-assisted laser desorption/ionization time-of-flight; MAR, matrix attachment region; MFP1, MAR-binding filament-like protein 1; MS, mass spectrometry;
MS/MS, tandem mass spectrometry; NE, nuclear envelope; NMCP1, nuclear matrix constituent protein 1; NPC, nuclear pore complex; NOR, nucleolus organizing
region; NUA, nuclear pore anchor; Nup, nucleoporin protein; ONM, outer nuclear membrane; Plamina, plant lamina; Q-TOF, quadrupole time-of-flight; SINE, SUNinteracting nuclear envelope; siRNA, small interfering RNA; SUMO, small ubiquitin-like modifier; SUN, Sad1/Unc84; TMV, Tobacco mosaic virus; WIP, tryptophan–
proline–proline (WPP) domain-interacting tail-anchored protein; WPP domain, tryptophan–proline–proline domain.
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1628 | Petrovská et al.
number of plant species (for a review, see Vrána et al., 2012).
However, it was becoming increasingly clear that knowledge
of the DNA sequence itself could also explain the function
of the nuclear genome and how its genes and non-coding
sequences regulate plant growth and development. Nuclear
DNA forms complexes with proteins and RNA to form chromatin, and this organization is critical for the arrangement of
DNA in the nucleus and has a direct impact on gene expression (Dunham et al., 2012). The nuclear genome is not static,
and undergoes dynamic changes during mitotic and meiotic
cell cycles, production of gametes, fertilization, and zygote
formation (for a review, see Heslop-Harrison and Schmidt,
2012). These processes require a structural and regulatory
framework, and the cell nucleus must contain all components
necessary to maintain, transcribe, and replicate genetic material, in addition to being capable of synthesizing and assembling components needed for translation. Proteins represent
the most abundant nuclear component (Sutherland et al.,
2001), and thus the characterization of the nuclear proteome,
together with studies on the structure and behaviour of its
individual protein components, is vital to understand the
function of the nuclear genome.
The cell nucleus is composed of two main structural
parts—the nucleoplasm and the nuclear envelope (NE).
Nucleus membrane-less compartmentalization is accepted as
an important feature in the organization of nuclear processes
and plays an essential role in the regulation of gene expression (Taddei et al., 2004). The nucleoplasm (karyoplasm)
comprises chromatin of decondensed chromosomes, which
occupy chromosomal domains, interchromosomal domains,
the nucleolus, and other nuclear bodies such as Cajal bodies (CBs), and nuclear speckles (for a review, see Lanctôt
et al., 2007). The nucleoplasm contains a variety of enzymes
and other molecules and structures participating in packing
DNA, DNA replication, DNA repair, and the transcription
machinery, including products of these processes.
Despite the similarity of cell nuclei in all eukaryotes as far
as their appearance is concerned, it became clear that there
are significant differences between higher plants and mammals (for a review, see Meier, 2009). While it seems that
there is a good knowledge of plant genome organization at
the DNA level, little is known about the organization of the
genome in the plant nucleus or about nuclear compartments.
In line with this, there is almost no information available on
plant nuclear proteins, other than histones and a few other
nuclear proteins. Thus, we are still at the very beginning of
our attempts to understand how the plant genome is organized and how it works. Here, we review the approaches used
to analyse the plant nuclear proteome and current knowledge
of the proteome with the aim of shedding more light on the
nature and function of this vital component of plant cell
nuclei, which has profound effects on the way in which the
genetic information is stored and expressed.
Structural and functional organization of
the cell nucleus
Nuclear envelope
A majority of the hereditary material in the plant cell is contained in the nucleus (Fig. 1), a multifunctional organelle
surrounded by the NE and containing numerous subcompartments (Lorkovic et al., 2004a). The NE is composed of
the inner and outer nuclear membranes (INM and ONM,
respectively), nuclear pore complexes (NPCs), and, in metazoa, the nuclear lamina (Hetzer et al., 2005). The NE controls
the macromolecules trafficking between the nucleoplasm
and cytosol, and anchors the chromatin and cytoskeleton.
Chromatin associated with the NE has been characterized as
silent chromatin (Akhtar and Gasser, 2007; Kalverda et al.,
2008) and interacts with the nuclear lamina. Active chromatin interacts with nuclear pore proteins at inner parts of
the nucleus; proteins of the INM interact with lamina and/
or with chromatin. The NE is a dynamic structure regularly
rebuilt during cell division, and its components participate in
mitosis and cell division (Kutay and Hetzer, 2008). Moreover,
Fig. 1. Schematic representation of a plant cell nucleus. On the left, there is a fluorescent image of 4′,6-diamidino-2-phenylindole (DAPI)-stained barley
nuclei purified by flow sorting; the white bar shows a distance of 10 μm.
Update on plant nuclear proteins | 1629
in plant acentrosomal cells, the NE functions as a microtubule-organizing centre (Stoppin et al., 1994; Murata et al.,
2005; Binarová et al., 2006).
Plant nuclear lamina (plamina)
The nuclear lamina is a major structural component of the
INM in vertebrates and invertebrates (Aebi et al., 1986;
Gerace, 1986). The lamina is made up of a network of lamin
filaments and is associated with residual elements of the NPCs
(Aebi et al., 1986; Krohne and Benavente, 1986). In addition,
it shows continuity with the cytoskeleton of intermediate filaments (Capco et al., 1984; Carmo-Fonseca et al., 1988).
For many years, the existence of a plant nuclear lamin was
controversial. Indeed, to date no lamin homologues have been
identified in plant genomes (Fiserova and Goldberg, 2010)
and it seems that plants have a unique lamina-like structure to
modulate nuclear shape and size. The first ultrastructural and
biochemical characterization of a plant nuclear matrix was
described in 1991 (Moreno Díaz de la Espina et al., 1991),
and the isolated matrix was similar to the metazoan nuclear
lamina (Moreno Díaz de la Espina, 1996). The existence of
plant nuclear lamina was confirmed by Fiserova et al. (2009).
The authors observed a filamentous lattice attached to the
INM of tobacco (Nicotiana tabacum) BY-2 nuclei that was
linked to nucleoplasmic rings of NPCs in a similar way to
those in the oocyte lamina of African clawed frogs (Xenopus
laevis). The authors called this structure ‘plamina’ (i.e. plant
lamina; Fiserova and Goldberg, 2010).
The lack of lamin homologues in the plant kingdom is a
strong motivation to search for plant lamin-like proteins (for
more detail, see Ciska and Moreno Díaz de la Espina, 2014;
Guo and Fang, 2014), and some candidate lamin-like proteins
have now been identified in plants (for reviews, see Kiseleva
et al., 2013; Ciska and Moreno Díaz de la Espina, 2014;
Guo and Fang, 2014). Matrix attachment region (MAR)binding filament-like protein 1 (MFP1), isolated from tomato
(Solanum lycopersicum) in 1996 by Meier et al. (1996) and
from onion (Allium cepa) in 2006 by Samaniego et al. (2006),
was the first plant protein exhibiting specific binding to the
MAR. Based on the sequence similarity of MFP1 and its
predicted structure, the authors suggested that it might form
filaments typical of structural proteins of the nucleoskeleton. Later on, MFP1 was shown to be localized predominantly in plastids and associated with thylakoid membranes
(Jeong et al., 2003; Samaniego et al., 2005). Gindullis et al.
(1999) identified matrix attachment factor 1 MAF1 (MFP1associated factor 1), a protein that specifically interacted with
MFP1. Both proteins localized to the NE and appeared to be
components of the nuclear matrix. Tomato MAF1 was later
shown to be a WPP (tryptophan–proline–proline) domain
protein whose subcellular location was predominantly outside the nucleus (Patel et al., 2004). Thus, the role of MFP1
and MAF1 as lamin-like proteins has not been well defined.
Nuclear matrix constituent protein 1 (NMCP1), first identified in carrot (Daucus carota) (Masuda et al., 1993, 1997),
localized only to the nuclear periphery. Four NMCP1-related
nuclear proteins were described in Arabidopsis thaliana:
LINC1 (little nuclei 1) localized to the nuclear periphery and
LINC2 (Dittmer et al., 2007) localized to the nucleoplasm.
LINC1 and 4 regulate nuclear morphology (Sakamoto and
Takagi, 2013). Because of the name, LINC was often confused with the linker of nucleoskeleton and cytoskeleton (the
same acronym LINC). The little nuclei proteins were therefore renamed as CRWN (crowded nuclei) proteins due to the
reduction in size without a reduction in endoreduplication
levels in mutant lines (Wang et al., 2013). Recently, a novel
nuclear envelope protein KAKU4 interacting with CRWN1
and CRWN4 was identified. This plant-specific protein modulates nuclear morphology and locates at the INM (Goto
et al., 2014). The NMCP family of proteins comprises the
best candidates for plant lamins. They have been characterized in different plant species: a total of 97 NMCP proteins
have been identified from 37 plant genomes (Kimura et al.,
2010; Ciska and Moreno Díaz de la Espina, 2013; Ciska
et al., 2013; Wang et al., 2013).
Linker of the nucleoskeleton and cytoskeleton
Several proteins of the INM, ONM, and nucleoplasm were
identified as components of the LINC (linker of nucleoskeleton and cytoskeleton) complex (Crisp et al., 2006; Tzur et al.,
2006; Worman and Gundersen, 2006). This complex connects
nuclear lamina to the cytoskeleton components and has been
shown to be necessary for nuclear positioning and migration (Tzur et al., 2006; Worman and Gundersen, 2006; Starr,
2009; Tatout et al., 2014). The LINC complex is composed of
Sad1/Unc84 (SUN) and Klarsicht/ANC-1/Syne-1 homology
(KASH) proteins that associate with the INM and ONM,
respectively (Sosa et al., 2012; Zhou et al., 2012).
SUN proteins are the only known INM-specific transmembrane proteins in plants (Graumann et al., 2010a; Oda and
Fukuda, 2011). AtSUN1 and AtSUN2 proteins localize to
the INM and their localization changes during mitosis as
they are tightly coupled with NE dynamics. AtSUN1 and
AtSUN2 interact with three WPP domain-interacting tailanchored (WIP) proteins, AtWIP1, AtWIP2, and AtWIP3
(Meier et al., 2010), and exhibit low similarity to metazoan
KASH proteins (Graumann and Evans, 2010; Graumann
et al., 2010b; Oda and Fukuda, 2011; Zhou et al., 2012; Zhou
and Meier, 2013). The presence of SUN proteins was also
revealed in other plant species, including rice (Oryza sativa),
grape vine (Vitis vinifera), and maize (Zea mays). Five different SUN genes were found in maize: ZmSUN1 and ZmSUN2
are structural homologues of animal SUNs, while ZmSUN3–
ZmSUN5 are novel structural variants of SUN-domain proteins with a predicted specific role at the plant NE (Murphy
et al., 2010). Graumann (2014), using FRET (fluorescence
resonance energy transfer) and FRAP (fluorescence recovery after photobleaching) assays showed that plant SUNdomain proteins interact with LINC1 (CRWN1), a putative
nucleoskeletal protein of the NMCP/LINC (CRWN) family
(Ciska and Moreno Díaz de la Espina, 2013, 2014). These
SUN–LINC interactions are a first indicator of SUN–nucleoskeletal anchorage in plants (Graumann, 2014; Evans et al.,
2014).
1630 | Petrovská et al.
The search for plant KASH-domain proteins took even
longer than that for the SUN-domain proteins. Nevertheless,
some candidates for plant KASH-domain proteins were identified recently. The family of AtWIP proteins (see above)
seems to be a good example (Zhou et al., 2012). Recently,
Zhou et al. (2014) identified a second group of plant KASH
proteins termed SINE (SUN-interacting nuclear envelope)
proteins. SINE1 links with the actin cytoskeleton on the
ONM through its armadillo repeat domain and is required
for proper nuclear anchorage in guard cells. SINE2 contributes to innate immunity towards an oomycete pathogen in
A. thaliana. The authors developed a Java program, called
DORY, to search for putative KASH-domain-containing
proteins. A novel KASH-domain protein, AtTIK, was identified recently by Graumann et al. (2014). AtTIK localized to
the NE and is involved in the control of nuclear morphology
in root cells. TPX2 protein from Arabidopsis is another potential member of the LINC complex (Petrovská et al., 2013).
It is a typical microtubule-associated protein. When TPX2 is
overexpressed together with importin, it reinforces microtubule formation in the vicinity of chromatin and the NE.
Cytoskeletal components associated with the NE
The available data documenting association between the
actin cytoskeleton and the plant NE remain scanty. The
observations of Chytilova et al. (2000) indicate that the actin
cytoskeleton facilitates movement of the nucleus. The movement usually appears in response to different stimuli, such as
blue light (Iwabuchi et al., 2007), and is necessary for migration of nuclei in the emerging root hairs and unequal cell
division (Chytilova et al., 2000). Tamura et al. (2013) showed
that there is a link between plant myosin XI-i and the nuclear
membrane. Myosin XI-i is localized on the ONM and physically interacts with WIT proteins. WIT proteins are required
both for anchoring myosin XI-i to the nuclear membrane and
for nuclear movement.
As early as 2003, Dryková et al. localized γ-tubulin on the
plant NE (Dryková et al., 2003). Subsequently, Seltzer et al.
(2007) demonstrated localization of γ-tubulin complex protein 2 (AtGCP2) and AtGCP3, the γ-tubulin ring complex
proteins, on the plant ONM. More recently, Batzenschlager
et al. (2013) demonstrated a significant role for the γ-tubulin
complex protein 3 (GCP3)-interacting proteins (GIPs) in both
nuclear shaping and NE organization. Small GIPs function
as a component of microtubule nucleation complexes as well
as adaptors and/or modulators of NE-associated proteins
(Janski et al., 2012; Nakamura et al., 2012; Batzenschlager
et al., 2013). Thus, while certain components of the plant
cytoskeleton, such as actin and γ-tubulin, are known to be
associated with the NE, the proteins facilitating this anchorage remain unknown (Graumann and Evans, 2010).
Nuclear pore complexes
NPCs (Fig. 1) serve as mediators for molecular transport
between the nucleus and cytoplasm (Wente and Rout, 2010).
They were identified as a site of transcriptional activation for
inducible genes (Meldi and Brickner, 2011). The NPCs are
the largest cellular multiprotein complex (40–66 MDa), composed of ~30 different nucleoporin proteins (Nups) (Tamura
and Hara-Nishimura, 2013). Although the general morphology of NPCs is conserved from yeast to human and plants, a
complete plant NPC proteome has not yet been established.
The problem is that most nucleoporin homologues cannot be
found in the plant genome using homology-based approaches
(Meier, 2006). To overcome the difficulties, interactive proteomic approaches were used to identify plant NPCs (Tamura
et al., 2010). In Arabidopsis, 30 Nups with a similar domain
organization to their human and yeast counterparts were
identified (Tamura et al., 2010). Plant NPCs seem to lack six
vertebrate components (Nup358, Nup188, Nup97, Nup 45,
Nup37, and pore membrane protein of 121 kDa; Boruc et al.,
2012), and possess Nup136/Nup1 protein. DNA sequence
analysis did not identify any vertebrate homologue of this
protein.
Another protein localized in the nuclear pore of plant cell
nuclei is the NUA (nuclear pore anchor) protein. Arabidopsis
NUA protein functions in SUMOylation and mRNA export
(Xu et al., 2007). Similar to observations in mammals (Lee
et al., 2008), NUA directly interacts with AtMAD1 and
AtMAD2 (Arabidopsis thaliana mitotic arrest deficient
1) (Ding et al., 2012) on the membrane. The AtMAD1–
AtMAD2–NUA complex plays a role in spindle checkpoint
activation during mitosis (Ding et al., 2012). Even though
the function of this complex in the dynamics of the NPC
is interesting, its exact role remains unknown. In the plant
kingdom, Nups play a critical developmental role (Xu and
Meier, 2008; Merkle, 2011; Parry, 2013) and regulate specific
pathways, such as suppression of auxin resistance, plant–
pathogen interaction, and defence signalling (for reviews, see
Matsunaga et al., 2013; Guo and Fang, 2014).
Nuclear bodies
Nuclear bodies are common and dynamic structures of
eukaryotic nuclei. A majority are localized outside the nucleolus, some inside it, and sometimes both options are possible
(Mao et al., 2011). However, information on the mechanisms
by which nuclear bodies are formed and maintained, and how
cells regulate their assembly process, has not yet been provided (Mao et al., 2011).
Plant nuclear bodies are membrane-less subnuclear organelles comprising the nucleolus, CBs, nuclear speckles, cyclophilin-containing speckles, dicing bodies, AKIP1-containing
bodies, and photobodies (Fig. 1).
Nucleoli, the largest bodies in eukaryotic interphase cell
nuclei, are known as producers of ribosomal subunits and
participate in many fundamental cellular activities (for details,
see Stepinski, 2014). The plant nucleolus shows a well-ordered
structure with various components such as fibrillar centres
(both heterogenous and homogenous), a dense fibrillar component, a granular component, nucleolar vacuoles, nucleolar
chromatin, and nucleolonema (Stepinski, 2014). Nucleoli are
mostly composed of proteins (85–90%). RNA represents only
5–10% and the smallest part is rDNA (Gerbi, 1997; Shaw and
Update on plant nuclear proteins | 1631
Brown, 2012). When cells enter mitosis, some nucleolar proteins diffuse into the cytoplasm and some remain attached
to the nucleolus organizing region (NOR) of mitotic chromosomes (Matsunaga and Fukui, 2010). A few nucleolar
proteins have already been characterized in plants, including several nucleolin homologues or nucleolin-like proteins
(Martin et al., 1992; Minguez and Moreno Díaz de la Espina,
1996; Gonzáles-Camacho and Medina, 2004; Sobol et al.,
2006; Medina et al., 2010; Pontvianne et al., 2010), one of the
crucial nucleolar proteins, fibrillarin (Cerdido and Medina,
1995; Pih et al., 2000), and many ribosomal proteins (Brown
et al., 2005; Brown and Shaw, 2008). In addition, using a proteomic approach, 217 nucleolar proteins of Arabidopsis have
been identified (Pendle et al., 2005).
CBs are very dynamic particles. They can move within
the nucleus, into the nucleolus and fuse together (Boudonck
et al., 1999). A major component of CBs is the protein coilin,
which serves as a marker for this structure (Sleeman et al.,
2001; Makarov et al., 2013; Njedojadło et al., 2014). The
plant homologue of coilin, Atcoilin, shows a strong affinity
for snRNA (small nuclear RNA) as well as for non-specific
RNA (Makarov et al., 2013). In addition, a characteristic feature of plant cells is the presence of poly(A) mRNA including
protein-coding RNAs in CBs (Njedojadło et al., 2014). Thus,
the function of plant CBs could be connected with the storage
or retention of poly(A) RNAs. The survival of motor neurons
protein (SMN) is another CB protein involved in the biogenesis of splicing small nuclear ribonucleoproteins (snRNPs)
(Sleeman et al., 2001). As early as 2006, Pontes et al. showed
that ARGONAUTE4 (AGO4) protein co-localized with CBs
in Arabidopsis (Pontes et al., 2006). AGO4 as a member of
the 24 nucleotide siRNA (small interfering RNA) transcriptional silencing pathway could function in the generation of
siRNA–protein complexes that act in RNA-directed DNA
methylation (Li et al., 2006). In Arabidopsis, the number of
CBs depends on the cell cycle stage, cell type, and developmental stage of the root epidermis (Boudonck et al., 1999).
Nuclear speckles or interchromatin granule clusters vary in
size, shape, and number. These dynamics are linked to temperature changes, stress, as well as to transcription or phosphorylation (Reddy et al., 2012). Inhibition of transcription
results in a decrease in the number of nuclear speckles (Ali
et al., 2003; Tillemans et al., 2005, 2006). Nuclear speckles
were characterized as a storage site for splicing factors and
were detected near active transcription sites (Fang et al.,
2004; Spector and Lamond, 2011). Even though the protein
composition of metazoan nuclear speckles has already been
analysed (Mintz et al., 1999; Saitoh et al., 2004), the components of plant nuclear speckles remain elusive (Reddy et al.,
2012).
Plant-specific nuclear bodies
Lorkovic et al. (2004b) showed that arginine/serine-rich (RS)
domain-containing cyclophilin (CypRS64), which interacts
with Ser/Arg-rich proteins as important splicing factors,
localized to a small number of nuclear bodies different from
CBs—cyclophilin-containing speckles.
Dicing bodies are comprised of microRNA processing proteins DCL1 (dicer-like1) and HYL1 (hyponastic leaves1) and
are required for processing of pri-miRNA (primary microRNA) and/or storage/assembly of the miRNA processing
machinery (Fang and Spector, 2007; Song et al., 2007).
AKIP1-containing bodies were observed in 2002 by Li
et al. (2002). AKIP1–green fluorescent protein (GFP) was
localized in the guard cell nuclei and was relocalized to the
speckles after treatment with abscisic acid.
Photobodies are unique to plants and are regulated by
an external light signal (van Buskirk et al., 2012, 2014). The
assembly of nuclear photobodies at the periphery of the
nucleolus was observed recently by Liu et al. (2014).
Nuclear proteomics
Introduction to nuclear proteomics
The lack of information on the majority of plant nuclear proteins has a negative impact on our understanding of plant
genome organization and function. In principle, there are
two possible approaches to characterizing the plant nuclear
proteome. (i) Extrapolating unknown proteins from already
characterized proteins of other species; this approach is commonly used in plant nuclei research, but, given the number
of expected nuclear proteins, progress has been rather slow.
(ii) Direct extraction and identification of unknown or as
yet uncharacterized proteins from purified plant nuclei; this
approach provides the opportunity to identify hundreds or
thousands of nuclear proteins in a relatively short time.
Current proteomics utilizes sophisticated instrumentation, optimized methodologies, and reliable strategies to
study complex biological systems. Consequently, a mosaic
view of biological and biochemical aspects of tissues or cells
is achieved, including information on protein composition,
localization, protein–protein interactions, enzymatic complexes, protein–metabolite complexes, post-translational
modifications, and cellular signalling machinery (Baginsky,
2009; Kersten et al., 2009). Present-day proteomics relies on
a combination of high-resolution separation methods such
as two-dimensional gel electrophoresis and liquid chromatography (LC), and mass spectrometry (MS) as analytical tools
(Thiede et al., 2013). Modern mass spectrometers used for
that purpose are designed to meet the criteria of high sensitivity, resolution, and accuracy, and they employ soft ionization
techniques: electrospray ionization (ESI) and matrix-assisted
laser desorption/ionization (MALDI; Yates, 2004).
As regards the common strategies for protein identification, there are two strategies for the analysis of a protein
sample: bottom-up and top-down proteomics (Chait, 2006).
The former refers to either classical ‘bottom-up’ proteomics
(proteins in a sample are first separated and then enzymatically digested to peptides), or shotgun proteomics (digestion
occurs directly in a complex protein mixture without any
previous separation). Conversely, the top-down proteomics
relies on fragmentation of the isolated protein directly in the
mass spectrometer, thus avoiding the enzymatic digestion.
Obtaining peptide sequences by tandem mass spectrometry
1632 | Petrovská et al.
(MS/MS) leads to highly probable (if not unambiguous)
identifications (Coon et al., 2005). Moreover, many protocols utilizing either chemical labelling with stable isotopes or
label-free approaches have been developed for mass spectrometric quantification of proteins (Bantscheff et al., 2007).
Proteomic experiments usually start with protein extraction, which is always a challenging step due to the presence of
other abundant biomolecules in biological material (such as
lipids, nucleic acids, saccharides, and metabolites) (Isaacson
et al., 2006). Moreover, conducting proteomics on cells and
cellular organelles requires careful protocols for isolation
of these structural units in order to avoid contamination
(Michelsen and von Hagen, 2009). When compared with the
research performed on mammalian species, that on plant
proteomics is still less common. This may be in part due to
the nature of plant cells. They are joined to each other by a
robust extracellular matrix and their rigid cell walls restrict
the release of the cellular contents. Working with plant tissues is thus more demanding with respect to methodology
(Heazlewood, 2011).
From isolation of plant cell nuclei to protein
identification
To date, several methodological approaches that focused
on obtaining pure nuclei have been published (for a review,
see Narula et al., 2013). A majority of the protocols employ
homogenization of plant tissues, filtration of the homogenate
to remove large debris, pelleting, elimination of contaminating organelles (i.e. by solubilization), and finally separation
on a density gradient. This approach was shown to be suitable for identification of nuclear proteins (Fig. 2).
As illustrated in Table 1, proteomic approaches used for
identification of plant nuclear proteins are similar. Generally,
proteins from samples are first separated (using two-dimensional or one-dimensional protein electrophoresis, 2DE and
1DE, respectively) and then digested with suitable proteases
(e.g. trypsin) to obtain peptide mixtures. Finally, protein
identification is achieved either (i) by peptide sequencing
using (nano)LC coupled online to MS and MS/MS with ESI
[(nano)LC-MS and MS/MS]; (ii) by peptide mass fingerprinting on MALDI-TOF (time-of-flight) instruments; or (iii)
by nanoLC-MALDI-MS and MS/MS. Some authors have
exploited combinations of these approaches (Table 1).
Despite the clear opportunities, including the availability
of advanced instrumentation for proteomics, no plant nuclear
proteome has been comprehensively characterized so far.
Early data on the protein composition of plant nuclei were
obtained in model organisms with sequenced genomes, such
as A. thaliana (Bae et al., 2003; Jones et al., 2009), O. sativa
(Tan et al., 2007), and later in other crop species (see Fig. 3 for
a time line overview).
Nuclear proteome of Arabidopsis thaliana
At the early stage of nuclear proteome research, 158 nuclear
proteins were identified in Arabidopsis. The proteins were
associated with various cellular functions (Bae et al., 2003).
Fifty-four of them were up- or down-regulated by cold treatment. Among these, six proteins were selected for further
functional characterization (e.g. gene expression analysis and
localization). This study provided an initial insight into the
Arabidopsis nuclear proteome and its response to cold stress.
The first proteomic study towards the characterization
of the nuclear matrix of higher plants was carried out by
Calikowski et al. (2003). The authors isolated the nuclear
matrix of Arabidopsis and performed its characterization by
confocal and electron microscopy. They identified 36 proteins
by ESI-MS/MS after separation using 1DE. Among the proteins identified, there were known or predicted homologues
of nucleolar proteins, such as IMP4, Nop56, Nop58, fibrillarins, nucleolin, ribosomal components, histone deacetylase,
tubulins, homologues of eEF-1, HSP/HSC70, and DnaJ, as
well as a number of novel proteins with unknown function.
Jones et al. (2009) identified 345 nuclear proteins in this
species, including novel phosphorylation sites and kinases
motifs on proteins involved in nuclear transport, such as Ranassociated proteins, as well as on transcription factors, chromatin-remodelling proteins, RNA silencing components, and the
spliceosome. Surprisingly, the authors found several proteins
involved in Golgi vesicle trafficking that probably contribute
to cell plate formation during cytokinesis (Jones et al., 2009).
Recently, Bigeard et al. (2014) performed proteomic and
phosphoproteomic analysis of chromatin-associated proteins
and identified a total of 879 proteins of which 198 were phosphoproteins participating in chromatin remodelling, transcriptional regulation, and RNA processing. In addition to
the whole nuclear proteome of Arabidopsis, the proteome of
its nucleoli was characterized by Pendle et al. (2005). They
identified 217 plant nucleolar proteins and compared them
with human nucleolar proteins. There were proteins with the
same function in humans, plant-specific proteins, proteins of
unknown function, and proteins that are nucleolar in plants
but non-nucleolar in humans. The comparison of Arabidopsis
and human nucleolar proteomes suggested that plant nucleoli
may have additional functions in mRNA export or control.
Nuclear proteome of rice (Oryza sativa)
Nuclear proteomics of rice, one of the most important crops,
was described by Khan and Komatsu (2004). Using Edman
sequencing and MS, 190 nuclear proteins were identified.
A majority of the identified proteins were involved in signalling and gene regulation, reflecting the role of the plant
nucleus in gene expression and regulation. Subsequently, Tan
et al. (2007) identified 269 unique chromatin-associated proteins by MS. They included nucleosome assembly proteins,
high-mobility group proteins, histone modification proteins,
transcription factors, and a large number of unknown proteins. In addition, using the shotgun approach, the authors
discovered an additional 128 chromatin-associated proteins
and, unexpectedly, they identified 11 variants of histone
H2A. The latter observation points to possible differences
between chromatin in mammals and plants, as only six variants of H2A are known in mammals (Bernstein and Hake,
2006).
Update on plant nuclear proteins | 1633
Fig. 2. Experimental strategy for plant nuclear proteome analysis. Plant nuclei are purified using density gradient centrifugation (or differential
centrifugation, I-A) or flow cytometric sorting (I-B). Nuclear proteins are extracted (II) and separated using 1DE (III-A) or 2DE (III-B) and then digested (IV)
with an appropriate protease (e.g. trypsin) to obtain peptide mixtures. In-solution digestion has also been used as an alternative (see Jones et al., 2009;
Cooper et al., 2011). Proteins are identified (VI) after peptide separation (V), either by peptide sequencing using (nano)liquid chromatography coupled
online to MS and MS/MS with electrospray ionization [(nano)LC-MS and MS/MS], by peptide mass fingerprinting, on MALDI-TOF instruments, or by
nanoLC-MALDI-MS and MS/MS (or by combinations of these approaches).
Li et al. (2008) identified 468 proteins from a nuclei-enriched
fraction of rice endosperm. Among them, transcription factors,
histone modification proteins, kinetochore proteins, centromere/
microtubule-binding proteins, and transposon proteins were
characterized. Of the proteins identified, 39% were hypothetical proteins with nuclear localization signals, indicating that the
endosperm nuclear proteome was still poorly characterized at
that time. Later, Aki and Yanagisawa (2009) identified 657 rice
nuclear and nucleic acid-associated proteins. Their list included
novel nuclear factors implicated in evolutionarily conserved
mechanisms for sugar responses in plants. Simultaneously,
Choudhary et al. (2009) identified 109 rice nuclear proteins
which displayed changes during drought stress. Their function included cellular regulation, protein degradation, cellular
defence, chromatin remodelling, and transcriptional regulation,
supporting the role of the nucleus as the main cellular regulator.
1634 | Petrovská et al.
Table 1. A list of plant nuclear proteomes
Organism
Method for
purification of nuclei
Proteomic approacha
2003
Arabidopsis
Density gradient
158
36
Bae et al. (2003)
Calikowski et al. (2003)b
2004
Rice
Density gradient
2DE, MALDI-TOF-MS
1DE, 2DE, MALDI-TOF-MS
NanoLC-MS, MS/MS
2DE, MALDI-TOF-MS
190
2006
2006
2007
Hot pepper
Chickpea
Rice
Density gradient
Density gradient
Density gradient
6
150
269
2008
Rice
Density gradient
468
Li et al. (2008)b
2009
Barrel clover
Maize
Chickpea
Rice
2DE, MALDI-TOF-MS
2DE, LC-MS, MS/MS
2DE, MALDI-TOF/TOF-MS,
MS/MS
2DE, LC-MS, MS/MS MALDITOF/TOF-MS, MS/MS
1DE, NanoLC-MS, MS/MS
2DE, LC-MS, MS/MS
Khan and Komatsu
(2004)b
Lee et al. (2006)
Pandey et al. (2006)b
Tan et al. (2007)b
Density gradient
143
98
147
109
657
2010
2011
2012
Arabidopsis
Black-stick lily
Soybean
Black-stick lily
Density gradient
Density gradient
Density gradient
2013
Rice
Density gradient
382
78
Repetto et al. (2008)
Casati et al. (2008)
Pandey et al. (2008)
Choudhary et al. (2009)
Aki and Yanagisawa
(2009)
Jones et al. (2009)b
Abdalla et al. (2010)
Cooper et al. (2011)
Abdalla and Rafudeen
(2012)
Mujahid et al. (2013)
Jaiswal et al. (2013)
2014
Chickpea
Arabidopsis
Chickpea
Density gradient
75
879
107
Subba et al. (2013)
Bigeard et al. (2014)b
Kumar et al. (2014)b
Maize
Barley
Flow cytometric sorting
(nuclei at G1, S, and G2
phase)
2DE, LC-MS, MS/MS
1DE, NanoLC-MS, MS/MS
LC-MS, MS/MS
2DE, MALDI-TOF-MS
LC-MS, MS/MS
2DE, LC-MS, MS/MS
1DE, LC-MS, MS/MS
2DE, MALDI-TOF/TOF-MS,
MS/MS
LC-MS, MS/MS
2DE, LC-MS, MS/MS
1DE, NanoLC-MS, MS/MS
1DE, 2DE, MALDI-TOF/
TOF-MS, MS/MS
NanoLC-MS, MS/MS
2DE, MALDI-TOF-MS
1DE
NanoLC-MS, MS/MS
NanoLC-MALDI-MS, MS/MS
No. of nuclear
proteins
identified
345
18
4975
122
163
803 (G1 nuclei)
Reference
Guo et al. (2014)b
Petrovská et al. (2014)b
a
1DE, one-dimensional electrophoretic separation; 2DE, two-dimensional electrophoretic separation; ESI, electrospray ionization; LC, liquid
chromatography; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry.
b
Studies aiming to characterize the complete nuclear proteome.
To better understand the role of nuclear proteins in water
deficit conditions, the nuclear proteome of rice was investigated for possible differences from normal growth conditions
(Jaiswal et al., 2013). 2DE coupled with MS and MS/MS led
to the identification of 78 nuclear dehydration-responsive
proteins. The group of identified proteins interacting in a
concerted manner was mapped into a functional association
network of dehydration-responsive pathways in the nucleus,
possibly acting as an adaptive mechanism against dehydration.
The response to the removal of cell wall in rice suspension
cultured cells was analysed using multiple nuclear proteome
extraction methods by Mujahid et al. (2013). The authors
identified 382 nuclear proteins including histone modification proteins, chromatin structure regulatory proteins, and
transcription factors. Many of the nuclear proteins were not
reported in previous studies on the rice nuclear proteome.
Gene ontology analysis indicated that chromatin and nucleosome assembly proteins, protein–DNA complex assembly,
and DNA packaging proteins were closely associated with
cell wall removal.
Nuclear proteome of hot pepper (Capsicum annuum)
Lee et al. (2006) conducted 2DE in order to determine proteins localized and expressed in the nucleus of hot pepper
as a response to Tobacco mosaic virus (TMV) infection. Six
related protein spots were observed, representing proteins
which might possibly be expressed during the hypersensitive
response against the virus infection. A functional study was
performed on the hot pepper 26S proteasome subunit RPN7
(CaRPN7), showing the possible involvement of CaRPN7 in
TMV-induced programmed cell death.
Update on plant nuclear proteins | 1635
Fig. 3. Diagram representing plant proteome analysis in time. Each colour represents one plant species. Coloured numbers represent the number of
identified nuclear proteins. The label ‘G1’ refers to the G1 phase of the cell cycle at which barley nuclei were analysed.
Nuclear proteome of chickpea (Cicer arietinum)
The first report on the nuclear proteome of chickpea (its genome
has been sequenced only recently by Varshney et al., 2013) was
published by Pandey et al. (2006). A total of 150 nuclear proteins were identified and compared with the proteome data for
Arabidopsis and rice. Only eight identical proteins were found in
all three organisms. Chickpea and rice shared 11 proteins, while
rice and Arabidopsis shared only six. The authors showed that
71% of the chickpea nuclear proteins were novel, emphasizing
the need for further research on the nuclear proteome in plants.
Later on, an insight into the dehydration-responsive nuclear
proteome of chickpea was accomplished by Pandey et al.
(2008). A total of 147 differentially expressed nuclear proteins
were identified during the course of dehydration and included
proteins involved in gene transcription and replication, molecular chaperones, cell signalling, and chromatin remodelling.
To better understand the molecular mechanism of the
dehydration response, Subba et al. (2013) generated data on
the nuclear proteome of a dehydration-sensitive chickpea
cultivar. The LC-MS and MS/MS analyses helped to identify
75 differentially expressed proteins probably associated with
different metabolic and regulatory pathways. A comparative
analysis between dehydration-sensitive and tolerant cultivars
showed unique as well as overlapping proteins, indicating
their contribution to dehydration tolerance.
A recent study by Kumar et al. (2014) identified 107 putative nucleus-specific chickpea phosphoproteins and provided an
insight into the possible function of protein phosphorylation in
plants. The functional categorization of chickpea nuclear phosphoproteins identified various cellular functions, such as protein folding, signalling and gene regulation, DNA replication,
repair, and modification, and metabolism. However, the most
abundant category according to biological and molecular function were stress-responsive and nucleotide-binding proteins.
Nuclear proteome of barrel clover (Medicago
truncatula)
The nuclear proteome of barrel clover, another legume species, has also been analysed. Repetto et al. (2008) published a
list of proteins identified in nuclei isolated from seed tissues.
A number of proteins related to ribosome subunit biogenesis,
chromatin structure/organization, transcription, RNA maturation, silencing, and transport were identified that could regulate gene expression and prepare seed for reserve synthesis
during the filling stage. Altogether, the authors identified 143
proteins and described some novel nuclear proteins involved
in the biogenesis of ribosomal subunits (pescadillo-like) and
nucleocytoplasmic trafficking (dynamin-like GTPase). The
authors speculated that the genome architecture could be
extensively modified during seed development.
Nuclear proteome of maize (Zea mays)
Maize nuclear proteome studies were first performed by
Casati et al. (2008). The authors compared maize nuclear
proteome data prior to and after exposure to UV-B light and
identified 98 proteins that show differences in abundance
between these two conditions. Many identified proteins were
classified as DNA binding and chromatin factors, including
core histones.
A reference map of the maize nuclear proteome in the basal
region of the third seedling leaf was recently constructed
(Guo et al., 2014). In this work, a combination of 2DE and
MALDI-TOF-MS allowed identification of 163 nuclear proteins. As expected, the most abundant representatives from
the identified nuclear proteins were implicated in RNA- and
protein-associated functions. In addition, a comparative proteomic analysis between a highly heterotic hybrid Mo17/B73
and its parental lines was performed. The results showed that
hybridization between two parental lines caused changes in
the expression of different nuclear proteins, which could be
responsible for leaf size heterosis.
Nuclear proteome of black-stick lily (Xerophyta viscosa)
The resurrection plant Xerophyta viscosa is able to survive
long periods without water. Abdalla et al. (2010) analysed its
nuclear proteome and response to dehydration stress. Using
2DE and MALDI-TOF-MS, a total of 438 protein spots
were reproducibly detected and analysed, of which 18 protein
spots were shown to be up-regulated in response to dehydration. Later on, Abdalla and Rafudeen (2012) used LC-MS
1636 | Petrovská et al.
and MS/MS to identify 122 nuclear proteins reproducibly. Many of them were similar to proteins identified in the
nuclear proteomes of Arabidopsis exposed to cold stress (Bae
et al., 2003) and chickpea under dehydration stress (Pandey
et al., 2008). In response to dehydration, 66% of the identified nuclear proteins did not show changes in abundance. The
proteins without changes in abundance during dehydration
were probably structural proteins and proteins associated
with basic metabolic activities without a direct role in desiccation tolerance, but necessary for survival. Of the total proteins identified, 22% were shown to be more abundant and up
to 10% less abundant in response to dehydration stress.
Nuclear proteome of soybean (Glycine max)
Cooper et al. (2011) analysed changes in the soybean nuclear
proteome after rust infection. Approximately 4975 proteins
from preparations of nuclei of soybean leaves were detected
using LC-MS and MS/MS. Statistical analysis revealed sets
of proteins with differential accumulation changes between
isogenic soybeans susceptible and resistant to the soybean
rust fungus. Many of the proteins identified had predicted
nuclear localization signals, homology to transcription factors and other nuclear regulatory proteins, and were phosphorylated. However, the identified proteins also included
those present in cellular compartments other than the
nucleus (e.g. mitochondria, glutamate dehydrogenase and
citrate synthase; cytoplasm, glyceraldehyde-3-phosphate
dehydrogenase, fructose bisphosphate aldolase, and lipoxygenase; chloroplasts, chlorophyll-binding proteins; peroxisomes, catalase) indicating contamination of the protein
extracts by non-nuclear proteins. The authors showed that
numerous plant proteins are post-translationally modified
in the nucleus after infection, and some of these proteomic
changes probably reflect defence responses that confer resistance to soybean rust.
Nuclear proteome of barley (Hordeum vulgare)
In order to characterize the nuclear proteome of barley,
Petrovská et al. (2014) developed a novel protocol to ensure
that the samples were not contaminated by cytoplasmic proteins. This was achieved by flow cytometric sorting of cell
nuclei prior to proteomic analyses. The protocol involved protein extraction and SDS–PAGE separation, in-gel digestion,
and reversed-phase nanoLC of peptides followed by MS and
MS/MS. The results demonstrated efficiency, sensitivity, and
speed without compromising protein integrity. Importantly,
the protocol avoided contamination by non-nuclear proteins.
The analysis of G1 phase nuclei led to the identification of
803 proteins. Among them, 572 were identified by nanoLCMALDI MS and MS/MS. The remainder were unique identifications obtained by nanoLC-ESI-MS and MS/MS on a
Q-TOF instrument (Petrovská et al., 2014). This approach
was found suitable to study the nuclear proteome in different
phases of the cell cycle (G1, G2, and S). In G2 phase nuclei,
~2000 proteins were identified on the same electrospray system (unpublished results).
Conclusions and future directions
A better knowledge of the structure, function, and behaviour
of plant nuclear proteins is urgently needed to understand
the function of the nuclear genome. Unfortunately, most of
the nuclear proteomic studies performed so far in plants did
not aim to characterize the complete protein composition of
cell nuclei. Their primary goal was to identify proteins whose
expression responded to stress conditions (e.g. cold stress,
drought stress, UV-B light treatment, virus infection). Thus, a
complete plant nuclear proteome has not been characterized
to date.
Nevertheless, a broad range of nuclear proteins has been
identified, including histones and histone modification proteins, chromatin-associated protein, kinetochore proteins,
nucleosome assembly proteins, high-mobility group proteins,
several transcription factors, structural proteins, nucleolar proteins, transposon proteins, many ribosomal proteins,
and a large number of new proteins of unknown function.
In order to make sure that the proteins identified are indeed
nuclear, the experimental protocol employed for the separation of nuclei from other cellular components is critical to
avoid contamination, which may compromise the quality of
proteomic data. Flow cytometric sorting of nuclei prior to
protein analysis seems an attractive approach.
Nuclear proteomics alone is not powerful enough to
uncover the functional and structural organization of the
plant nucleus. However, it provides a launch pad to characterize nuclear proteins with unknown function, to identify
proteins not expected to be nuclear, and to verify proteins still
classified as ‘hypothetical’. Moreover, coupling plant nuclear
proteomics with biochemical, molecular biological, immunocytochemical, and morphological approaches, supported by
the analysis of the organization in three-dimensional space
of the cell nucleus, has the potential to provide information
needed to understand the organization and function of the
plant nuclear genome.
A considerable fraction of plant nuclear proteins (10–18%)
identified so far are not similar to known proteins in other
organisms (for a review, see Narula et al., 2013). The biological function of these proteins awaits clarification, and this
may provide clues to understanding the differences between
animal and plant cell nuclei. One way forward is to develop
a comprehensive database of plant nuclear proteins, based
on proteomics data obtained from nuclei at different phases
of the cell cycle (G1, S, and G2), from various types of tissues, and from various stages of cell and tissue differentiation. Combined with protein structure, predicted function,
and physical localization in 3D nuclear space, this may bring
about the much needed progress in our understanding of the
plant nuclear proteome and its role in plant genome organization and function.
Acknowledgements
This work was supported by the Operational Program Education for
Competitiveness–European Social Fund (project CZ.1.07/2.3.00/20.0165
to BP), by grants from the Czech Science Foundation (14-28443S), and
the National Program of Sustainability I (LO1204) from the Ministry of
Update on plant nuclear proteins | 1637
Education, Youth and Sports of the Czech Republic. Professor David Morris
from the University of Southampton is thanked for critical reading of the
manuscript. We sincerely apologize to all colleagues whose relevant works
could not be cited due to space limitation.
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