JPROT-02308; No of Pages 10

Journal of Proteomics xxx (2015) xxxxxx

Contents lists available at ScienceDirect

Journal of Proteomics
journal homepage: www.elsevier.com/locate/jprot

Induction and quantitative proteomic analysis of cell dedifferentiation during callus

formation of lotus (Nelumbo nucifera Gaertn.spp. baijianlian)
Yanli Liu a, Palak Chaturvedi b, Jinlei Fu a, Qingqing Cai c, Wolfram Weckwerth b, Pingfang Yang a,
a
b
c

Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, The Chinese Academy of Sciences, Moshan 430074, China
Department of Ecogenomics and Systems Biology, University of Vienna, Althanstr. 14, Vienna 1090, Austria
College of Life science and technology, Huazhong Agricultural University, Wuhan 430070, China

a r t i c l e

i n f o

Article history:
Received 24 June 2015
Received in revised form 29 September 2015
Accepted 8 October 2015
Available online xxxx
Keywords:
Lotus
Cotyledons
Callus induction
Quantitative proteomics

a b s t r a c t
Lotus is an aquatic plant with high nutritional, ornamental and medical values. Its callus formation is crucial for
germplasm innovation by genetic transformation. In this study, embryogenic callus was successfully induced on
appropriate medium using cotyledons at 12 days after pollination as explants. To dissect cellular dedifferentiation
and callus formation processes at the proteome level, cotyledons before and tissues from 10 to 20 days after induction were sampled for shotgun proteomic analysis. By applying multivariate statistics 91 proteins were detected as differentially regulated, and sorted into 6 functional groups according to MapMan ontology analysis.
Most of these proteins were implicated in various metabolisms, demonstrating that plant cells underwent metabolism reprogramming during callus induction. 14.3% proteins were associated with stress and redox, indicating that the detached explants were subjected to a variety of stresses; 13.2% were cell and cell wall-related
proteins, suggesting that these proteins played important roles in rapid cell division and proliferation. Some proteins were further evaluated at the mRNA levels by quantitative reverse transcription PCR analysis. In conclusion,
the results contributed to further deciphering of molecular processes of cellular dedifferentiation and callus formation, and provided a reference data set for the establishment of transgenic transformation in lotus.
2015 Elsevier B.V. All rights reserved.

1. Introduction
Lotus (Nelumbo nucifera Gaertn.) is a perennial aquatic plant of economic, ecological, cultural and religious importance with a wide distribution in East Asia. In addition to its high ornamental value, it is also
widespread cultivated as a food crop for its seeds and rhizomes, and
as a traditional herbal medicine for its seeds, fruit and leaves [13].
According to its agricultural purpose and morphological difference,
N. nucifera has been divided into three types corresponding with three
important organs, separately named as ower lotus, seed lotus and rhizome lotus, which has also been conrmed by random amplication
polymorphic DNA analysis [1,4]. These species can be propagated by
both sexual (seeds) and asexual (rhizomes) methods. Among them, rhizomes reproduction is the predominant reproduction mode used in
horticultural applications. However, it is mother rhizome-consuming,
low reproduction coefcient, difcult storage and transportation, and
can cause the degeneration of good species. In vivo tissue culture acting
as a good alternative method may overcome these obstacles to some degree. Although a remarkable success in lotus regeneration by de novo
Corresponding author at: Key Laboratory of Plant Germplasm Enhancement and
Specialty Agriculture, Wuhan Botanical Garden, The Chinese Academy of Sciences,
Wuhan, China.
E-mail address: yangpf@wbgcas.cn (P. Yang).

shoot organogenesis has been achieved [56], regeneration via callus

phase is still a long unsolved question due to difcult embryogenic
callus induction [79]. In recent years, whole genome of lotus was successfully sequenced [1011], which will accelerate the study of gene
function and then molecular breeding, thereby promoting enhancement of germplasm resources with high quality and high yield by transgenic technology. However, the establishment of highly efcient genetic
transformation systems was restricted to embryogenic callus induction
in lotus.
Callus is a group of unorganized cell masses with totipotency. Under
certain condition, callus obtained by in vitro culture is able to regenerate
the whole plant body through organogenesis or somatic embryogenesis
[12]. Besides the highly frequent usage in the propagation of economically important plants, callus acting as the most suitable acceptor materials of genetic transformation has been extensively used in molecular
genetic research. In general, callus can be induced articially in vitro
by an appropriate ratio of auxin and cytokinin application in various
plants [13]. Additionally, success in culturing embryogenic callus is
also affected by many other factors such as plant genotypes and the concentrations of various supplied substances. However, how these factors
regulated callus formation have long been obscured. Moreover, it has
been demonstrated that differentiated plant tissues or cells can recover
cell division ability and then generate callus on a suitable callus inducing
medium. Which metabolism changed and how metabolism processes

http://dx.doi.org/10.1016/j.jprot.2015.10.010
1874-3919/ 2015 Elsevier B.V. All rights reserved.

Please cite this article as: Y. Liu, et al., Induction and quantitative proteomic analysis of cell dedifferentiation during callus formation of lotus
(Nelumbo nucifera Gaertn.spp. baijianlian), J Prot (2015), http://dx.doi.org/10.1016/j.jprot.2015.10.010

Y. Liu et al. / Journal of Proteomics xxx (2015) xxxxxx

changed during cellular dedifferentiation and callus formation were

also unclear. Although molecular genetic analyses of Arabidopsis mutants impaired in callus formation [14], and proteome or transcriptome
proling of callus formation in nodal segment of Vanilla planifolia
[15], Arabidopsis hypocotyls [16], Arabidopsis multiple organs [17] and
Dimocarpus longan [18] greatly advanced our knowledge of molecular
mechanisms underlying callus formation in recent, the molecular mechanisms that lead to callus formation of lotus cotyledons still remain
unknown.
Proteomics is a high throughput and powerful approach to identify
and characterize a wide range of differentially expressed proteins, and
has been used to dissect the molecular mechanisms underlying callus
formation [16,19]. In the present study, embryogenic callus was successfully induced using developmental cotyledons at 12 days after pollination (DAP) as explants. Subsequently, we employed a label-free
shotgun proteomic approach (GEL-LC-Orbitrap-MS) to compare the
proteome of cotyledons before and tissue from 10 to 20 days after induction. The proteomic results were also further veried at the mRNA
level through qRT-PCR analysis. The ndings may contribute to further
deciphering of molecular mechanisms that lead to callus formation of
lotus and other plants as well.

centrifugation at 12,000 g for 20 min at 4 C. Supernatants were collected, an equal volume of Trisphenol (pH N 8.0) was then added, vortexed
and centrifuged at 12,000 g for 20 min at 4 C. The top phenol phase
was carefully transferred to a new tube. Subsequently, homogenization
buffer was added again, thoroughly mixed and centrifuged at 12,000 g
for 20 min at 4 C. The phenol phase was transferred to another new
tube followed by addition 5 volumes of 0.1 M methanolic ammonium
acetate in 100% methanol and incubation overnight at 20 C. The precipitated proteins were washed one time with 0.1 M methanolic ammonium acetate and two times with acetone. The protein pellets were
vacuum-dried and then stored at 80 C for further use.
2.5. One-dimensional gel electrophoresis (1-DE)
Prior to 1-DE, protein samples were redissolved in lysis buffer (7 M
urea, 2 M thiourea, 4% w/v CHAPS, and 65 mM DTT) and quantied
using the Bradford method [22]. 25 g of total proteins was loaded
onto 12.5% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE)
and run at 50 V for 30 min and 150 V for 1 h. After running, the gels
were stained with Coomassie Brilliant Blue (CBB R-250) solution for
4 h, and then destained until protein bands were clear, scanned with
the scanner (EPSON PERFECTION V700) (Supplementary Fig. 1).

2. Materials and methods

2.6. Quantitative proteome analysis and database searching
2.1. Plant materials and callus induction
Plants of N. nucifera baijianlian were grown in lotus research base
located in Wuhan Botanical Garden, Chinese Academy of Sciences. Developmental seeds of 10, 12 and 15 DAP were harvested, shelled and
surface sterilized in 70% ethanol for 30 s and 2% sodium hypochlorite
for 3 min. After ve times of washing with sterile water, immature
cotyledons were aseptically detached from the developmental seeds
with a scalpel and then cultured in vitro. Callus-inducing medium MS
supplemented with 3% sucrose, 2.5% gelrite, 100 mg/L casein hydrolysate and different hormone combinations was used to induce callus.
The pH of MS medium was adjusted to 5.8 mol/L and then autoclaved
for 15 min. Immature cotyledons were transferred on one 10 cm petri
dish containing callus-inducing medium in ultraclean bench. Subsequently, the explants were cultured at 26 C in dark condition until
the harvest of tissues from 10 to 20 days after induction.
2.2. Harvest of samples for proteomic analysis
Before callus induction, about 0.5 cm width of cotyledons located
near the micropylar region was cut out, pooled and frozen for further use. Additionally, tissues from 10 days to 20 days after callusinduction were also collected, respectively. The samples for proteomic
analysis were harvested during July to September.
2.3. Histological observation
After harvest, the samples were immediately immersed in xative of
formalin-acetic acid-alcohol. Fixed samples were then vacuum-dried for
1 h, successively dehydrated with 30%100% ethanol, transparented, embedded into the parafn, dewaxed and stained with aniline blue. The
embedded samples were cut into 610 m slices using LEICA2150 rotary
microtome (Leica, German). Olympus BX-61 upright metallurgical microscope (Olympus, Japan) was employed to observe histological slices.
2.4. Protein extraction
Proteins were extracted from about 1.0 g cotyledons before and tissues from 10 to 20 days after callus induction using the phenol method
according to previously described protocol [20]. Briey, the samples
were ground into ne power with liquid nitrogen, homogenized in
pre-cooled homogenization buffer used by Yang et al. [21] followed by

Each lane of the gels was divided into four fractions using the sterile
scalpel. Gel pieces were destained, equilibrated, digested with trypsin,
sequentially as described by Valledor and Weckwerth [23]. Afterwards,
tryptic peptides from a lane were dissolved in 4% acetonitrile and 0.1%
formic acid, thoroughly mixed, desalted and then subjected to analysis
using nanoHPLC coupled to LTQ-Orbitrap-MS according to previously
published report [24]. In brief, 10 g of digested peptides was injected
into nano-ow LCMS/MS system equipped with a pre-column
(Eksigent, Germany) following by peptides elution using a Ascentis
Express ES-C18 HPLC column (SUPELCO Analytical, USA) during a
80 min gradient from 5% to 50% acetonitrile and 0.1% formic acid. MS
analysis was performed on an Orbitrap LTQ XL mass spectrometer
(Thermo, Germany) with a controlled ow rate of 500 nL/min. Specic
tune was set to 1.8 kV for spray voltage and 180 C for temperature of
the heated transfer capillary.
MS raw data were searched by SEQUEST algorithm of Proteome
Discoverer version 1.3 (Thermo, Germany) according to previous report
[15]. Briey, the following lter criteria were set: (i) 1% false discovery
rate (FDR); (ii) a variable modication of acetylation of N-terminus
and oxidation of methionine; and (iii) mass tolerance of 10 ppm for parent ion and 0.8 Da for fragment ion.
The newly annotated genome database of lotus (http://lotus-db.
wbgcas.cn) was employed to identify proteins. The assembled genome
of lotus was 804 Mb, which represented 86.5% of the estimated
929 Mb lotus genome. Following repeat-masking and annotation,
26,685 unigenes were inferred, 82% of them have similarity to proteins
in SwissProt [11]. Peptides were matched against lotus database plus
decoys with user-dened searching parameters as follows: (i) medium
or high peptide condence; (ii) minimum Xcorr of 1 for +1, 2 for +2
and 3 for +3 charged peptides, etc. Subsequently, a label-free approach
based on spectral counts (SpCs) was used to quantify the identied proteins followed by a normalized spectral abundance factor (NSAF) normalization strategy [25]. The NSAF was calculated as follows

NSAFi SpCi =Li =

N
X

SpC=Li :

i 1

In this equation, the NSAF for a protein is the number of spectral

counts (SpCs) divided by protein length (L) and then divided by the
sum of SpC/L.

Please cite this article as: Y. Liu, et al., Induction and quantitative proteomic analysis of cell dedifferentiation during callus formation of lotus
(Nelumbo nucifera Gaertn.spp. baijianlian), J Prot (2015), http://dx.doi.org/10.1016/j.jprot.2015.10.010

Y. Liu et al. / Journal of Proteomics xxx (2015) xxxxxx

2.7. RNA extraction and quantitative reverse transcription PCR (qRT-PCR)

Total RNA was extracted from about 0.2 g cotyledons before and tissues from 10 to 20 days after induction using Plant Total RNA Isolation
Kit (Zomanbio, China) according to the operating instruction. Total
RNA was then reverse transcribed into cDNA using M-MLV First Strand
cDNA Synthesis kit (Invitrogen, USA). Transcript levels of each selected
gene were detected by qRT-PCR according to previously published
methods [26]. ACT was used as internal control for normalization. The
relative mRNA level for each gene was calculated as CT values. The
primer pairs were designed according to cDNA sequences downloaded
from lotus genome database and listed in Supplementary Table 1.
2.8. Statistical analysis
One-way ANOVA analysis (Duncan variance analysis) and graph
drawing were performed using SPSS Software Package and Graphpad
Prism 5, respectively. The Venn diagram was produced by online Venn
system (http://bioinfogp.cnb.csic.es/tools/venny/index.html). Principal
components analysis (PCA) was performed using the statistical tool
box COVAIN. For functional categorization of differentially changed
proteins, homology alignment with Arabidopsis thaliana was searched
against UniprotKB (http://www.uniprot.org/uniprot/?query) or NCBI
(http://blast.ncbi.nlm.nih.gov/Blast) database following by online
MapMan (http://mapman.gabipd.org/web/guest/mapcave).
3. Results and discussion
3.1. Induction of embryogenic callus and morphological changes
To determine an optimized developmental stage, the cotyledons of
10, 12 and 15 DAP were used to induce embryogenic callus on MS
media with different hormone combinations. Among them, 12-dayold cotyledons were the most easiest to form callus, and the induction
rate of embryogenic callus was almost up to 100% on MS medium
with the hormone combination of 6.0 mg/L 2,4-dichlorophenoxyacetic
acid, 0.1 mg/L naphthalene acetic acid, 0.5 mg/L 6-benzylaminopurine
and 0.5 mg/L gibberellin, whereas 10-day and 15-day old samples only
generated a few callus. The results revealed that although plant hormones play pivotal roles in callus formation, the developmental status
of cotyledons is also an important inuence factor. Based on the results,
the cotyledons at 12 DAP were collected for further callus induction and
proteomic analysis (Fig. 1B).
Before callus induction, 12-day-old lotus cotyledons exhibited
the characteristics of white, crisp and smooth surface, and rectangle
and regularly arranged monolayer epidermic cells (Fig. 1E, H). After
10 days of callus-induction, cotyledon sections locating at the micropylar region showed a rough surface in which some protuberance cells
generated from cortex parenchyma cells (Fig. 1F, I). After 20 days,
yellowish coloration, loosened, fragile and granular callus appeared
on the explants (Fig. 1G). At the same time, epidermic cells almost
vanished and numerous callus cells occurred through histological observation (Fig. 1J). On the basis of morphological changes, tissues from
three different stages including before callus-induction (NC), 10 days
after callus-induction (CS1) and 20 days after callus-induction (CS2)
were designated as the samples for further proteomic analysis.
3.2. Dynamic proteome proling of cotyledons during callus induction
To further understand the molecular mechanisms underlying
the callus inducing processes, a label-free quantitative shotgun proteomic analysis was performed on the samples from the three stages
mentioned above. Totally, 1149 proteins were successfully identied
(Supplementary Table 2; the mass spectrometry raw data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.
proteomexchange.org) via the PRIDE partner). To be more reliable,

only the proteins identied in all three biological replicates from

samples at least in one stage were subjected to quantitative analysis.
Based on this criterion, 489 proteins were quantied (Supplementary
Table 2). Among them, 415 proteins were in common to all stages,
31 proteins were in common between NC and CS1, 38 proteins were
in common between CS1 and CS2, and only 2 proteins were in common
between NC and CS2. Additionally, 2 and 1 protein was specially
expressed in NC and CS2, respectively, as illustrated by a Venn diagram
(Fig. 2B). Among the quantied proteins, 91 were signicantly differentially changed based on Duncan variance analysis with p 0.05 (Supplementary Table 3, Supplementary Fig. 2).
To evaluate the reliability of samples harvesting, PCA on all samples
from three replicates at three stages was performed using the statistical
tool box COVAIN based on the quantied proteins and their relative
abundance (NSAF scores) [27]. As indicated in Fig. 2A, three stages
were mainly separated by the rst principal component PC1. However,
there is one replicate in each stage that clearly behaves differently,
which may be ascribed to the difference in cotyledons physiological
status before callus induction that caused by the change of natural environment in which lotus lived. Positive loadings of PC1 depicted proteins
with high abundance at CS2 stage followed by CS1 stage, whereas negative loadings represented high level at NC stage. The highest positive loading mainly includes stress and redox-related protein, and a
portion of metabolism-related proteins (N-metabolism, amino acid metabolism, glycolysis, TCA, fermentation and gluconeogenesis-related
proteins), mostly showing a gradual increase from NC to CS2 stage. Negative loading includes a few of metabolism-related proteins and a small
proportion of cell-related proteins (Supplementary Table 3, Supplementary Fig. 2).
3.3. Functional categorization of differentially regulated proteins
To further understand the changes on different pathways during callus induction, functional categorization was performed on the 91 differentially changed proteins through MapMan analysis. According to their
predicted function, 91 proteins were sorted into 6 major functional
groups, including metabolism, stress and redox, signaling and transport,
cell and cell wall, RNA and DNA, and not assigned (Fig. 2C). Among
them, metabolism group is the most abundant one, which can be further
subdivided into 13 subgroups including protein metabolism, hormone
metabolism, N-metabolism, lipid metabolism, amino acid metabolism, carbohydrate metabolism, nucleotide metabolism, co-factor and
vitamin metabolism, secondary metabolism, gluconeogenesis, fermentation, glycolysis and TCA (Fig. 2D); stress and redox group, and cell
and cell wall group are the second and the third largest groups, respectively. The percentage of identied differentially changed proteins is
generally consistent with the studies in Zea mays [33], Vitis vinifera
[55], V. planifolia [19], Cyphomandra betacea [36] and Crocus sativus
[32]. These proteins may be responsible for the changes on physiological
status, cell division and structure.
3.4. Proteins involved in various metabolisms
Plant hormones, acting as inducers imposed on the explants, may
cause plant cells to undergo metabolism reprogramming and energy
consumption in callus formation [28]. In this experiment, over half of
the differentially changed proteins were involved into various metabolism pathways as mentioned above.
Among quantied metabolism-related proteins, proteins associated
with protein metabolism were the most abundant one consisting of
21 proteins. Almost all of them were highly abundant at NC and then
showed a continuous decrease at CS1 and CS2 (Fig. 3G), which was in
contradiction to several published reports [12,24,29]. This may be
ascribed to protein pre-accumulation in developmental cotyledons
(Supplementary Fig. 3) [30]. 7 of 8 amino acid metabolism-related proteins were quantied to be gradually up-regulated at CS1 and CS2, only

Please cite this article as: Y. Liu, et al., Induction and quantitative proteomic analysis of cell dedifferentiation during callus formation of lotus
(Nelumbo nucifera Gaertn.spp. baijianlian), J Prot (2015), http://dx.doi.org/10.1016/j.jprot.2015.10.010

Y. Liu et al. / Journal of Proteomics xxx (2015) xxxxxx

Fig. 1. Morphology of lotus seeds and callus. Embryo (A), cotyledon (B), unshelled seed (C) and developmental seed (D) of 12 DAP. Cotyledon before callus-induction (E). Tissue from
10 days after callus-induction (F). Tissue from 20 days after callus-induction (G). Histological observation of cotyledons before callus-induction (H), tissue from 10 days after callus-induction (I) and tissue from 20 days after callus-induction (J), respectively. Bars = 0.5 cm in AG and 100 m in HJ. Rectangles indicate the samples for histological observation.

one exhibited a reverse tendency (Fig. 3A), revealing that the synthesis
of new proteins may occur in callus. Moreover, two proteins related
to N-metabolism including glutamate dehydrogenase 1 and branchedchain-amino-acid aminotransferase-like protein 3 also showed a continuous increase at CS1 and CS2 (Fig. 3G). The former catalyzes the
transformation of ammonia and -ketoglutaric acid into glutamate [31],
whereas the latter is responsible for the biosynthesis of branchedchain-amino-acid. This indicated that up-regulation of the proteins
would contribute to more substrates for protein biosynthesis. In summary, it is probable that pre-accumulated proteins in developmental
cotyledons concealed the synthesis of new proteins. However, the synthesis of new proteins was required for callus formation [19,32,33].
Carbohydrate and energy metabolism-related proteins play important roles in maintaining carbon skeletons and supplying energy for key
metabolic processes. In this study, 4 glycolysis-related proteins including
pyruvate kinase, fructose-bisphosphate aldolase, pyrophosphatefructose 6-phosphate 1-phosphotransferase subunit beta and NADPdependent glyceraldehyde-3-phosphate dehydrogenase were all upregulated at CS1 and presented at even higher levels at CS2 (Fig. 3B),
demonstrating that they play a crucial regulatory role in carbon ux
and energy supply during callus induction. Recently, several studies

also documented that up-regulation of glycolysis-related proteins

are essential to the formation of embryogenic callus in Z. mays [33],
V. vinifera [34] and V. planifolia [15]. Meanwhile, an increased expression of proteins involved in TCA (1 protein), fermentation (2 proteins),
gluconeogenesis (1 protein) and major CHO metabolism (2 proteins)
was also found (Fig. 3B, 3C), which probably triggered directly by upregulation of glycolytic proteins. Coordinating up-regulation of these
proteins may be prerequisite for the recovery of dedifferentiation ability
and subsequent cell division and proliferation. Taken together, the results enhanced our understanding on the roles of these proteins in
callus formation and the interdependence of different metabolism pathways [35].
Additionally, 8 proteins involved in secondary metabolism (3
proteins), lipid metabolism (2 proteins), nucleotide metabolism (2 proteins), co-factor and vitamine metabolism (1 protein) were also quantied in this study (Supplementary Fig. 2), which may be accountable for
cotyledons-derived callus formation. Collectively, a large number of various metabolisms-related proteins were quantied to be differentially
changed, further indicating that detached cotyledons were subjected
to various metabolisms rearrangement during the recovery of dedifferentiation and callus formation.

Please cite this article as: Y. Liu, et al., Induction and quantitative proteomic analysis of cell dedifferentiation during callus formation of lotus
(Nelumbo nucifera Gaertn.spp. baijianlian), J Prot (2015), http://dx.doi.org/10.1016/j.jprot.2015.10.010

Y. Liu et al. / Journal of Proteomics xxx (2015) xxxxxx

Fig. 2. Multiple analysis of identied proteins. Principal components analysis (A) on all samples from three replicates at three stages based on the quantied proteins and their relative
abundance. Venn diagram analysis (B) of quantied proteins at three biological replicates in one stage at least, the numbers of proteins quantied at NC, CS1 and CS2 are shown in different
segments. Functional categorization of differentially changed proteins (C). Classication of proteins associated with various metabolism pathways (D), the different numbers show identied protein number. NC, before callus-induction; CS1, 10 days after callus-induction; CS2, 20 days after callus-induction.

3.5. Stress and redox-related proteins

Under in-vitro culture conditions, various stresses may be a potent
inducer for embryogenic callus. Stress responsive proteins have been
frequently reported in tissue dedifferentiation and callus development
[3639]. There were six stress-related proteins quantied in this study
(Fig. 3D). Heat shock protein 83 (2 proteins) among them was highly

expressed at NC compared to NC1 and NC2. It is now very clear that

HSPs serve important physiological functions and most of them constitutively presented in normal cells, although their synthesis is often increased under stress conditions [40]. Other 4 proteins including
pathogenesis-related protein STH-21, basic endochitinase (2 proteins)
and major allergen Pru ar 1 were all absent at NC, and accumulated to
a highest level at CS2. An increased expression of these proteins has

Please cite this article as: Y. Liu, et al., Induction and quantitative proteomic analysis of cell dedifferentiation during callus formation of lotus
(Nelumbo nucifera Gaertn.spp. baijianlian), J Prot (2015), http://dx.doi.org/10.1016/j.jprot.2015.10.010

Y. Liu et al. / Journal of Proteomics xxx (2015) xxxxxx

Fig. 3. Expression proles of proteins involved into different functional groups: NSAF scores averaged over three biological replicates.

Please cite this article as: Y. Liu, et al., Induction and quantitative proteomic analysis of cell dedifferentiation during callus formation of lotus
(Nelumbo nucifera Gaertn.spp. baijianlian), J Prot (2015), http://dx.doi.org/10.1016/j.jprot.2015.10.010

Y. Liu et al. / Journal of Proteomics xxx (2015) xxxxxx

recently been reported in wound-healing events of potato (Solanum

tuberosum) tubers [41] and in pro-embryogenic callus of Elaeis
guineensis [35]. Thus, they may be responsible for protective roles in
struggling with pathogen, also be correlated with callus initiation.
ROS, as signal molecules or by-products, can be produced from
various metabolic pathways in all living organisms [4244]. Overproduction of ROS can cause oxidative damage. Therefore, plants have
evolved highly effective antioxidant systems to protect themselves
from oxidative damage. Except for the highest abundance of peroxidase
17 at NC, peroxidase 43, peroxidase 12, 1-Cys peroxiredoxin and probable glutathione S-transferase (3 proteins) were all absent at NC and
gradually increased with the formation of callus in this study (Fig. 3E).
Peroxidase and 1-Cys peroxiredoxin are responsible for detoxifying
hydrogen peroxide. As for glutathione S-transferases, a heterogeneous
superfamily of multifunctional proteins, were possibly involved in scavenging of toxic organic hydroperoxides and protecting against oxidative
damage, also served as stress signaling and binding proteins for phytohormones [45]. Together, this again supported the speculation that
ROS-related proteins were also connected to the increase of cell division
[19,46]. It is very intriguing that three members of peroxidase family
(peroxidase 43, peroxidase 12, peroxidase 17) were highly expressed
at different callus inducing stages, which was also conrmed by qRTPCR at mRNA levels (Fig. 4). However, further study should be required
to explore the different expression of three peroxidases at different callus induction stages.

3.6. Proteins involved in phytohormone metabolism and signaling

transduction
Callus can be generated articially by exogenous application of auxin
and cytokinin in numerous plant species. Other hormones, such as
brassinolides (BRs), may substitute for auxin or cytokinin in certain species [47]. In this study, three phytohormone-related proteins were
quantied (Fig. 3F). Indole-3-acetic acid-amido synthetase (2 proteins),
a primary auxin-response gene, catalyzing the conjugation of IAAamino acid and acting as a feedback regulator of auxin by decreasing
the free IAA concentration [48], showed an up-regulation at CS1 and
CS2. The high abundance of IAA synthetase detected at both proteome
and mRNA levels (Figs. 3F, 4) should be directly induced by the application of high concentration of exogenous auxin in vitro culture, demonstrating it plays a pivotal role in the regulation of auxin homeostasis in
the explants. Cycloartenol-C-24-methyltransferase, catalyzing the

initial step of BR biosynthetic pathway [49], was only accumulated at

NC and absent at CS1 and CS2 stages. Therefore, BR might be of no benet for developmental cotyledons-derived callus formation in lotus. To
obtain more convincing results, cycloartenol-C-24-methyltransferase
(NNU_02744) coding gene was chosen to be checked at mRNA level
through qRT-PCR. However, gene expression was found to be gradually
up-regulated at mRNA level during callus formation, displaying a reverse tendency with its protein level (Fig. 4). Likewise, four genes
below also showed inconsistent trends with their proteins. This may
be ascribed to the regulation at transcriptional, translational and posttranslational levels.
Perception and transmission of signals are the central step of detached explants responded to the high concentration of phytohormone
and wounding. Here, two proteins implicated in signaling transduction
were quantied to be differentially changed. Ras-related protein
Rab11C, a small GTP-binding protein of Rab subfamily, regulating vesicular trafcking pathways and behaving as membrane-associated molecular switches [50], showed a high abundance at CS1 (Fig. 3F); calnexin,
an ER membrane-bound protein [51], acting in Ca2+ homeostasis [52]
and signal transduction by the second messages Ca2 +, displayed a
high expression at NC stage (Fig. 3F). This indicated that Rab11C and
calnexin may be involved in the regulation of cell division and proliferation through signal transduction mediated by vesicular trafcking and
Ca2+. To understand the expression of two proteins, qRT-PCR was carried out. The abundance of two genes (NNU_03699, NNU_22355) was
enhanced at CS1 and CS2 stages (Fig. 4), which is high inconsistent
with their protein expression. Therefore, it is likely that the two genes
were subjected to feed back regulation.

3.7. Cell and cell wall-related proteins

The formation of embryogenic callus requires the coordination of
cell division, expansion and cell structure. 10 cell-related proteins
were quantied to be differentially changed in the present study.
Among them, 6 proteins implicated in cell organization including 5 homologous of -tubulin and villin-2 were down-regulated with callus
formation (Fig. 3G). -tubulin is a subunit of tubulin [53] that constitutes microtubules, whereas villin-2 is an epithelial cell-specic actin
modication protein that makes up microlaments [54]. Both microlaments and microtubules are structural constituents of cytoskeleton.
Therefore, it is probable that the continuous decrease of these proteins
in abundance is ascribed to the status of developmental cotyledons

Fig. 4. Relative mRNA levels of several interested genes. The gene ID in the x-axis stands for differentially changed proteins that have been explained in Supplementary Table 1. The values
are means of three replicates SD. Statistically signicant differences among cotyledons before, tissues from 10 days and from 20 days after callus-induction are indicated with different
lowercase letters. NC, before callus-induction; CS1, 10 days after callus-induction; CS2, 20 days after callus-induction.

Please cite this article as: Y. Liu, et al., Induction and quantitative proteomic analysis of cell dedifferentiation during callus formation of lotus
(Nelumbo nucifera Gaertn.spp. baijianlian), J Prot (2015), http://dx.doi.org/10.1016/j.jprot.2015.10.010

Y. Liu et al. / Journal of Proteomics xxx (2015) xxxxxx

before callus induction. Recently, the changed expression of actin and

tubulin was also reported by several similar studies in Valencia sweet
orange [39], V. vinifera [55,34] and Z. mays [33]. Collectively, this suggested that actin and tubulin may be linked with callus formation.
Cell division cycle protein 48 (3 proteins), a highly abundant type II
AAA-ATPase, may be directly involved in cell division and expansion
[5658]. Here, all of three CDC48 homologs presented higher levels
at NC and gradually decreased at NC1 and NC2 (Fig. 3G). Similarity,
a higher abundance of AtCDC48A was also reported in proliferating
cells of rapidly growing Arabidopsis shoots, roots, and owers [29,59].
Interestingly, through qRT-PCR analysis the mRNA level of CDC48
(NNU_19849) was found to be highly expressed at both NC and CS2
stages (Fig. 4). This suggested that CDC48 is required for cotyledons
development and embryogenic callus formation in lotus. Additionally, peptidylprolyl cis-trans isomerase implicated in cell cycle, a ratelimiting step in protein folding [60], was also quantied. The protein
and its coding gene (NNU_08279) were all detected to be highly
expressed at NC1 (Figs. 3G, 4), suggesting that it is functional in the
early stages of callus formation.
According to histological observation, the degradation of outer cell
wall and the synthesis of new cell call were required for cotyledons
cortex-derived callus formation. Two proteins implicated in cell wall
metabolism including fasciclin-like arabinogalactan protein 10 and
GDP-mannose 4, 6 dehydratase 2 were quantied to be continuously
decreased with callus formation (Fig. 3G) in this study. Fasciclin-like
arabinogalactan protein 10 is correlated with secondary-wall cellulose
synthesis [61], whereas GDP-mannose 4, 6 dehydratase 2 catalyzes
the rst step in de novo synthesis of GDP-L-fucose that acts as a carbohydrate component of glycoprotein and cell wall polysaccharide [62].
We speculated that down-regulation of these proteins maybe contribute to the release of callus cells from the internal layer of tissue. A
qRT-PCR was also performed to evaluate the function of two proteins.
Down-regulated expression was observed in the gene (NNU_24940),
whereas there was no change found in gene NNU_19710 (Fig. 4),
which needs further study.

4. Conclusion
Callus formation and differentiation are crucial for genetic transformation and molecular genetic studies in most plants. However,
the techniques were still undeveloped in lotus. In this study, embryogenic callus was successfully induced from developmental cotyledons. Afterwards, through a shotgun proteomic strategy quantitative
proteomic analysis of callus was performed to dissect the molecular
mechanisms underlying cellular dedifferentiation and callus formation. Totally, 91 proteins were quantied to be signicantly differential changed. Over half of these proteins were metabolism-related
proteins, demonstrating that plant cells underwent metabolism
reprogramming during callus formation. Most of the stress and redoxrelated proteins (14.3%) showed a continuously increased expression
at NC1 and NC2, indicating that the detached explants were subjected
to various kinds of stresses under in vitro culture condition and always
protected themselves from damage by the regulation of protein expression. Furthermore, 13.2% cell and cell wall-related proteins were
also quantied, which may be required for rapid cell division and
proliferation.
Based on our obtained proteomic results, the following model for
molecular mechanisms guiding metabolism progresses of cellular dedifferentiation and callus formation in lotus cotyledons was proposed
(Fig. 5). Based on the model, plant hormones such as auxin (6-BA)
and cytokinin (2, 4-D, IAA) and wounding, acting as inducers, induced
cell proliferation and callus formation in differentiated lotus cotyledons.
Briey, the inducers (stimulus) contributed to a wide range of metabolism reprogramming and imposed various stresses on the explants
following by energy consumption and ROS overproduction. As a result,
the explants activated defense systems to resist all kinds of attacks or
antioxidate systems to scavenge excess ROS, and accelerated carbohydrate and energy metabolism to supply energy and maintain carbon
skeletons. Simultaneously, the addition of exogenous phytohormone
triggered cell division followed by the changes of cell wall. Finally, the
explants generated a mass of irregular cells called callus.

Fig. 5. A work model for callus formation in lotus cotyledons. Numbers located in brace indicate the number of quantied differentially changed proteins in this study.

Please cite this article as: Y. Liu, et al., Induction and quantitative proteomic analysis of cell dedifferentiation during callus formation of lotus
(Nelumbo nucifera Gaertn.spp. baijianlian), J Prot (2015), http://dx.doi.org/10.1016/j.jprot.2015.10.010

Y. Liu et al. / Journal of Proteomics xxx (2015) xxxxxx

Conict of interest
The authors declare that there is no conict of interests.
Acknowledgments
The authors are grateful to Ms. Lei Wang for her help in MS analysis.
This work was supported by The Knowledge Innovation Project (No.
Y455421Z02).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jprot.2015.10.010.
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Please cite this article as: Y. Liu, et al., Induction and quantitative proteomic analysis of cell dedifferentiation during callus formation of lotus
(Nelumbo nucifera Gaertn.spp. baijianlian), J Prot (2015), http://dx.doi.org/10.1016/j.jprot.2015.10.010