Tropical Plant Biol.

DOI 10.1007/s12042-016-9175-2

Molecular Genetic Dissection of Sugarcane

Flowering under Equatorial Field Conditions
Amanda L. Medeiros 1 & Cristiane M. Furtado 1 & Francinaldo S. Leite 1 &
Valeska S. Souto 1 & Nathalia de Setta 2,3 & Marie-Anne Van Sluys 2 & Joo Paulo Kitajima 4 &
Ana Paula P. Costa 2,5 & Vagner A. Benedito 6 & Katia C. Scortecci 1

Received: 21 November 2015 / Accepted: 9 June 2016

# Springer Science+Business Media New York 2016

Abstract Sugarcane is a tropical crop used for sugar and

biofuel production in tropical and equatorial regions of the
globe. Sugarcane flowering is intrinsically induced in equatorial regions due to long-day conditions. Flower development
is problematic for this crop because it halts vegetative growth,
leading to a reduction of the sugar accumulated in the stalks,
and significant yield loss. Notwithstanding, the identification
of genes differentially expressed in contrasting cultivars can
potentially reveal markers and tools to generate genotypes
more suitable for expanding the geographical limits of this
crop. Thus, dissecting the flowering gene expression network
under field conditions is highly relevant for breeding. We
report the analysis of subtractive cDNA libraries produced
from shoot apical meristem of cultivars contrasting for

Communicated by: Paul Moore

Electronic supplementary material The online version of this article
(doi:10.1007/s12042-016-9175-2) contains supplementary material,
which is available to authorized users.
* Katia C. Scortecci
kcscortecci@ufrnet.br
1

Departamento de Biologia Celular e Gentica, Centro de Biocincias,

Universidade Federal do Rio Grande do Norte,
Natal, RN 59072-970, Brazil

Departamento de Botnica, Instituto de Biocincias, Universidade de

So Paulo, So Paulo, Brazil

Universidade Federal do ABC, Santo Andr, Brazil

Mendelics Anlise Genmica, So Paulo, Brazil

Centro de Cincias Biolgicas e da Sade, Universidade

Presbiteriana Mackenzie, So Paulo, Brazil

Laboratory of Plant Functional Genetics, Division of Plant & Soil

Sciences, West Virginia University, Morgantown, USA

flowering time growing in production fields under equatorial

conditions. Transcripts with homology to POLYPHENOL
OXIDASE (PPO), CALMODULIN (CAM),
PHOSPHATIDYLCHOLINE/PHOSPHATIDYLINOSITOLTRANSFER PROTEIN (SEC14), OBTUSIFOLIOL-14-DEMETHYLASE (CYP51), 143-3, and the
phosphotransferases SHAGGY KINASE (GSK), PROTEIN
KINASE C INHIBITOR (PKCI), and SERINE/THREONINE
PHOSPHATASE (PP1) were identified as differentially
expressed in the subtractive libraries and further chosen for
RT-qPCR validation and in silico interactome analyses. Our
results suggest that ScPPO, ScSEC14 and Sc143-3 may act
as flowering inhibitors. RT-qPCR data also revealed two 14
3-3 isoforms as potential flowering markers. Sc143-3 was
structurally and phylogenetically characterized and its genomic architecture was analysed in two BAC clones, showing
that they probably correspond to two different loci with confirmed synteny to other grass genomes. This work reveals
potential novel mechanisms of flowering in grasses with implications to crop breeding.
Keywords Early flowering . Plant development . Field
production . Subtractive cDNA libraries . RT-qPCR .
Interactome

Introduction
Sugarcane (Saccharum spp.) is the leading first-generation
biofuel crop in the world. Flowering is a highly undesirable
trait in sugarcane because the energy accumulated in the stem
as sucrose is used to develop reproductive structures (Araldi
et al. 2010; Scortecci et al. 2012). Sugarcane flowering is
induced in the field by several factors, including high temperatures, short photoperiod, plant maturity, and poor mineral

Tropical Plant Biol.

nutrition (Araldi et al. 2010). Photoperiods of 1212.5 h light

are usually sufficient to induce flowering in most cultivars
(Moore and Nuss 1987; Moore and Berding 2014). The varieties currently available were generated from many intricate
crossings, which themselves had been originally derived from
crosses between S. spontaneum and S. officinarum (Daniels
and Roach 1987; Scortecci et al. 2012). Derived hybrids therefore have highly complex polyploid (generally decaploid and
aneuploid) genomes (Waclwoysky et al. 2010). Consequently,
in sugarcane the genotype plays a central role in determining
the plants promptness to respond to the environmental cues
that trigger flowering.
Sugarcane cultivars are classified according to their responsiveness to flowering in a given environment. In Brazil, two
main regions are responsible for sugarcane production: the
southeast (with subtropical climate) and the northeast (tropical
and equatorial climates). These regions have contrasting environmental conditions, in particular soil type, temperature, water availability, photoperiod, light quality, and UV irradiance
level. Consequently, early flowering is a major problem in the
northeast of Brazil, where temperatures are consistently high
and the photoperiod is close to twelve hours all year round
(Araldi et al. 2010). Furthermore, with the prospect of global
climate changes, the southeast might also be affected by early
flowering in sugarcane crops. Thus, a major goal of sugarcane
breeding programs is to produce varieties that are late or recalcitrant to flowering in order to allow more sucrose accumulation and an extended harvest season. Important unanswered questions in this regard involve the identity of the
exogenous and endogenous factors that trigger the switch
from vegetative to reproductive development in this crop.
Flowering transition is an important trait in plants and it has
been studied using physiological, genetic and molecular approaches. It is well know that plants respond to different environmental and endogenous signals that trigger the transition
from vegetative to reproductive state at the shoot apical meristem (SAM). Furthermore, this transition involves an intricate
signal network of flowering repressors and promoters
(Matsoukas et al. 2012; Khan et al. 2014). In a simple way,
flowering induction depends on five pathways: autonomous,
temperature (vernalization), gibberellin, photoperiod, and aging. Moreover, light quality (blue, red and far red), high temperatures, and temperature fluctuations also play important roles
in flowering (Yuan-li and Wei-jiang 2012; Song et al. 2013;
Pos et al. 2013; Lee et al. 2013; Khan et al. 2014). The balanced output of these pathways ultimately induces expression
of FLOWERING LOCUS T (FT). The FT protein translocates
from leaf to SAM where it interacts with the FD protein and this
FT/FD complex plus 143-3 proteins promotes ontological
changes from vegetative to reproductive state in SAM
(Matsoukas et al. 2012; Khan et al. 2014; Song et al. 2015).
This work aimed to compare sugarcane cultivars with contrasting flowering induction that were growing in commercial

fields under equatorial conditions, and to identify the genetic

agents (inducers and inhibitors) that are differently expressed
in the SAM. Eight flowering-related genes with clear differential expression identified from the subtractive libraries were further studied by RT-qPCR over a seven-month
time-course analysis. From this study, a putative 143-3
protein was identified as having the greatest potential to
act as an important regulator of flowering in sugarcane.
We further examined its genomic architecture, evolutionary features, and expression profile. Furthermore, the
genes identified in this work may be important transducers of flowering signaling in sugarcane, warranting further
functional assessment of the integrative genetic network
leading to flowering in this crop.

Results
Functional Categorization of Differentially Expressed
Genes in the Shoot Apical Meristem
Our approach aimed at identifying differentially expressed
genes in sugarcane that may be coordinating flowering
transition time a reduction of inhibitors or increase of
promoter signals For this, four subtractive cDNA libraries
were designed to identify differentially expressed genes
from contrasting, early- and late-flowering cultivars at
different SAM development (L1 and L2 libraries), as well
as transcripts related to signals that trigger flowering transition (L3 and L4 libraries used only meristems of SP81
3250, an early-flowering cultivar, at vegetative and reproductive development stages). We obtained 321 sequences
for L1, 322 for L2, 245 for L3, and 335 for L4, ranging
from 350 to 550 nucleotides (Table 1).
Table 2 shows functional categorization of genes differentially expressed in each subtractive cDNA library.
Metabolism-related gene expression was higher in libraries
denoting transcripts from vegetative SAM (i.e., L2 and L4).
On the other hand, energy-related transcripts were remarkably
more abundant in libraries from early-flowering cultivar during reproductive development (i.e., L1 and L3), with respective values of 71 % and 45 % of the transcript pool when
compared to the contrasting L2 and L4 libraries, with 11.2
and 20.5 %, respectively. Whereas sequences related to cell
cycle represented less than 2 % in L1, it constituted 10 % in
L2; signal transduction transcription was more represented in
L2 (25 %) than any other library. Transposable elements were
in the range of 310 % across all libraries. Lower representation of transcription factors was observed in L1, with
about half (4 %) of that found in other libraries (7.5
9 %). It was noteworthy that sequences of unknown function were 14 % in L1 to up to 30 % in L4, a category that

Tropical Plant Biol.

Table 1

Subtractive cDNA libraries

Library

Subtraction

Number of clones
sequenced

L1 - SAMER

Early-flowering induced* (SP813250) subtracted against late-flowering (RB75126)

321

L2 - SAMLV

Late-flowering* (RB75126) subtracted against early-flowering induced (SP813250)

322

L3 - SAMEVR
L4 - SAMERV

Early-flowering induced* (SP813250) subtracted against early-flowering not-induced

Early-flowering not-induced* (SP813250) subtracted against early-flowering induced

241
335

*Sequences cloned correspond to transcripts from in this library

can potentially include interesting novel candidate

flowering genes for functional analyses.
Table 3 shows sequences represented at least four times in
each contrasting cDNA subtractive library. No differentially
expressed transcripts were identified in L1. On the other hand,
several transcripts were identified specifically in the L2 library, which contains transcripts from late-flowering cultivar
at vegetative SAM development after the subtraction. Among
these, we identified POLYPHENOL OXIDASE (PPO, 4
times), CALMODULIN (CAM, 7 times), and three different
kinases: PROTEIN KINASE C INHIBITOR (PKCI, 10 times);
143-3 (4 times); and SERINE/THREONINE
P H O S P H ATA S E ( P P 1 , 5 t i m e s ) . F u r t h e r m o r e ,
PHOSPHATIDYLINOSITOL-PHOSPHATIDYLCHOLINE
TRANSFER PROTEIN (SEC14) showed remarkable representation in L2 (32 times, constituting 10 % of the sequences).
The subtractive cDNA libraries L3 and L4 were synthesized using only the early-flowering cultivar (SP813250).
They reflect differential expression of reproductive and vegetative SAM, respectively. L3 library is enriched in transcripts
associated with flowering inducers, or alternatively lacking
switch inhibitors from the vegetative to reproductive development. The main sequence identified in the L3 library was 14
3-3 (6 times). In contrast, L4 is enriched in transcripts related
to maintaining SAM in the vegetative development state. We
found sequences coding for CAM (once), SHAGGY-like kinase (4 times), PKCI (5 times), CYP51 (4 times), and

Table 2 Identified sequences organized into functional classes using

Gene Ontology
Class

L1 (%)

L2 (%)

L3 (%)

L4 (%)

Metabolism
Energy
Transposable elements
Signal transduction
Cell-cycle
Transcription factors
Plant defense
Unknown sequences

1.6
71.0
4.5
2.2
1.9
4.2
0.3
14.3

9.6
11.2
5.8
25.5
10.0
9
12.2
24.7

4.8
45.0
2.7
8.5
8.6
8.2
0.0
22.2

9.0
20.5
9.7
6.3
5.7
7.5
10.9
30.4

SEC14 (3 times). PP1 was represented by a single sequence

found in each of the libraries, whereas 143-3 was represented
by only one sequence in L4, compared with six sequences
present in L3.
In order to better understand the expression dynamics of
these genes, we performed a time-course experiment during a
seven-month period for all cDNAs identified: from August,
when sugarcane is in a juvenile vegetative phase in both cultivars, up to February, when the plant is mature and competent
to receive signals and, genotype and environment allowing,
proceed to reproductive development. Gene expression of
transcripts with homology to PPO, SEC14, CAM, CYP51,
kinases SHAGGY, PP1, PKCI (Fig. 2), and 143-3 (Fig. 1)
was analyzed via RT-qPCR.
ScPPO expression remained constant during the time
course for the late-flowering cultivar, with levels kept above
those of the early-flowering cultivar, except for the first harvest (in August), which showed identical expression level in
both cultivars (Fig. 1a). ScSEC14 is a member of a gene superfamily that is not very well characterized. Over time, we
observed a similar gene expression pattern in both the cultivars, with essentially higher levels in the late-flowering cultivar, especially in December and February (Fig. 1b). ScCAM is
associated to Ca+2 transport and cell signaling. The RT-qPCR
data showed higher transcription levels in the late-flowering
cultivar relative to the early cultivar, although this difference
was not significant (Fig. 1c). ScCYP51 showed a slight but
significantly lower expression in the late-flowering cultivar
than the early cultivar by December (Fig. 1d).
For the kinases studied, the SHAGGY-like gene showed
distinct and inverted expression pattern between the cultivars.
There was a significant decrease in ScSHAGGY expression for
the early-flowering cultivar, at the time when SAM is probably changing from the vegetable to reproductive phase (OctDec) (Fig. 1e). On the other hand, for the late-flowering cultivar, there was an increase in ScSHAGGY expression during
the same period (Fig. 1e). PP1 is a serine/threonine phosphatase involved in diverse biological pathways. Its expression
pattern remained rather constant in both cultivars, despite the
small fluctuation in February (Fig. 1f). Finally, PKCI (protein
kinase C inhibitor) the RT-qPCR data did not show any significant difference (Fig. 1g).

Tropical Plant Biol.

Table 3 Identification of the
transcripts differentially
expressed in the subtractive
cDNA libraries

L1a

L2a

L3a

L4a

Homology

143-3 protein

TC75568

1e-23

0
0

4
7

0
0

0
1

Polyphenol oxidase (PPO)

Calmodulin (CAM)

TC104861
TC82574

3e-10
9e-41

0
0

2
10

0
1

4
5

Shaggy-like kinase (SHAGGY)

Protein Kinase C Inhibitor (PKCI)

TC103395
TC77161

7e-33
2e-46

Serine/Threonine Phosphatase 1 (PP1)

TC78689

4e-29

TC73046

0.0

32

Cytochrome P450 (obtusifoliol 14-alpha

demethylase-CYP51)
Sec14

TC78676

7e-68

DFCIb

e-valuec

Number of transcripts identified in each cDNA subtractive library

Accession number from DFCI sequences that had homology to the sugarcane sequences identified in the
subtractive cDNA library (ftp://occams.dfci.harvard.edu/pub/bio/tgi/data/Saccharum_officinarum/SOGI.
release_3.zip)
c

E-value obtained for the subtractive cDNA sequences identified against BLASTx (non-redundant protein sequences - nr) at DFCI database

Expression of the FAC Complex at the Shoot Apical

Meristem
Flowering is the result of a balance between different pathways, which induces the FLOWERING LOCUS T (FT). We
therefore analyzed the expression pattern of FT and the bZIP
transcription factor FD at SAM (Fig. 2). Considering that the
FT mRNA is associated to the FT protein transport to SAM
and there the FT protein interacts to FD forming the FAC
complex (Li et al. 2011; Jackson and Hong 2012; Liu et al.
2013), the mRNA levels for both genes were analyzed
(Fig. 2). FT expression was quite similar in both cultivars,
except in January, when it increased in the early-flowering
cultivar while decreased in the late-flowering cultivar
(Fig. 2a). FD expression pattern was similar in both cultivars,
except in October, when flowering in the late cultivar was
more induced than the other cultivar; however, this difference
was not sustained throughout the season and both cultivars
showed similar expression levels in a plateau reached in
January and February (Fig. 2b).
In Silico Interactome Analysis
Except for 143-3, the sequences identified in the subtractive
cDNA libraries are not recognized as canonical players in
flowering transition. These sequences correspond to signal
transduction pathways and their relation to flowering is currently unknown. Therefore, these results lead to ask about
their potential roles in flowering. In order to answer this question, we used an in silico approach using the Stringer program
to identify the potential interactome with Arabidopsis homologs of the differentially expressed genes. This approach
should reveal possible interacting proteins connected with
flowering transition pathways. From this analysis, we found
that SEC14 potentially interacts to phospholipase D, which

are related to phospholipid hydrolysis (Fig. 3a). The sugarcane CAM has highest sequence similarity to Arabidopsis
CAM7. CAM7 protein interacts with an auxin responsive
protein (AT5G20810), as well as gene products involved in
cytoskeleton, post-transcription regulation, and jasmonate metabolism (Fig. 3b). Although in animals, CYP51 is a
demethylase associated with steroid metabolism, and some
of its related proteins are involved with cell division and embryo formation (e.g., FACKEL FK; Fig. 3c). Furthermore,
PP1 potentially interacts with WD-40-motif transducins, calcineurin like-proteins and FY (Fig. 3d). Interestingly, FY is
associated with flowering as it decreases FLC mRNA in
leaves and regulates the autonomous pathway via FCA.
SHAGGY kinase interacts with others members of the
SHAGGY superfamily, such as BRASSINAZOLERESISTANT1 (BZR1), which is associated to plant development and protein phosphatase 2C (PP2C) (Fig. 3e). PKCI
interacts with F-box protein and many CDK kinases and
cyclin-dependent kinase, which are related to cell division
and potentially important for meristem morphogenesis during
flowering transition (Fig. 3f). Interestingly, we did not find
any close PPO homologs in the Arabidopsis Stringer database, revealing a potential monocot-specific flowering regulator. This analysis shows that the cDNAs identified may be
related to flowering by direct or indirect genetic networks and
may trigger signals under equatorial field conditions.
Characterization of Sc143-3
It is well established that 143-3 interacts with FT protein in
plants. It is also clear that FT interacts with FD in the SAM to
form the complex that induces flowering transition
(Matsoukas et al. 2012). Based on the data presented in
Table 3, Sc143-3 transcripts (TC75568) were present in libraries L2, L3 and L4. Given this relation to flowering, this

Tropical Plant Biol.

Fig. 1 Expression of flowering-related genes in sugarcane plants growing in equatorial conditions via RT-qPCR. The Y-axis corresponds to gene
expression levels. Ct is the cycle which SYBR fluorescence reaches the
established threshold. 40-Ct determines the expression level relative to
a reference gene, protein elongation factor 1 alpha (EF1)
The X-axis corresponds to the analysis period. Flowering induction occurred in October for the early-flowering cultivar, while the late cultivar is

only induced around February. CT was normalized for E = 2 [CT = CT x

(log2 E/log2 2)], the errors bars corresponds to standard error using the all
different biological and technical replicates. The data obtained were then
analyzed using One-way ANOVA and Tukeys test with p 0.05.
Asterisks represent significant differences within each harvest time between early to late-flowering cultivar. a PPO; b SEC14; c CAM; D)
CYP51; e SHAGGY; f PPI and g PKCI.

sequence was chosen for further characterization. In

Arabidopsis, there are thirteen 143-3 isoforms (Paul et al.
2012), with variations occurring largely in the 3-UTR (Paul
et al. 2009). Therefore, isoform-specific primers were designed within unique regions for each of the three sugarcane

EST isoforms identified at DFCI database (TC106943,

TC143247 and TC143749). The expression patterns were analyzed by RT-qPCR in a time course for both sugarcane cultivars (Fig. 4). Based on the data presented in Figs. 1 and 2, we
considered that plants were in the vegetative phase from July

Tropical Plant Biol.

Fig. 2 Expression analysis via RT-qPCR of genes identified in the subtractive sugarcane cDNA libraries. The Y-axis corresponds to gene expression levels. Ct is the cycle which SYBR fluorescence reaches the
established threshold. 40-Ct determines the expression level relative
to a reference gene, protein elongation factor 1 alpha (EF1). The Xaxis corresponds to the analysis period. Flowering induction occurred in
October for the early-flowering cultivar, while the late cultivar is only

induced around February. CT was normalized for E = 2 [CT = CT x (log2

E/log2 2)], the errors bars corresponds to standard error using the all
different biological and technical replicates. The data obtained were then
analyzed using One-way ANOVA and Tukeys test with p 0.05.
Asterisks represent significant differences within each harvest time between early to late-flowering cultivar. a FT and b FD

until October. In December, SAM was presumed competent to

receive signals for flowering transition, since it was may expected to have already undergone transition and converted to
reproductive state in JanuaryFebruary, at least in the inductive SP813250 (early-flowering) cultivar. The gene expression pattern revealed that TC106943 and TC143247 isoforms
peaked in September for the late-flowering cultivar, whereas
in the early-flowering cultivar their expression levels decreased over this period. TC143749 isoform peaked in
October for late-flowering cultivar. Furthermore, expression
in the early-flowering cultivar remained consistently lower
during the October for the three isoforms (Fig. 4). The most
noticeable expression differences between the cultivars occurred between SeptemberOctober for the early-flowering
cultivar, exactly during the reproductive transition period.
The genomic architecture of the Sc143-3 gene was further
explored by sequencing two sugarcane BAC clones containing this gene. Figure 5 shows the structure of the BACs,
SHCRBA_121_P14 and SHCRBA_268_K21 (P14 and
K21, respectively). The results suggest that P14 is syntenic
to chromosome 6 of sorghum and chromosome 4 of rice and
that K21 is syntenic to sorghum chromosome 5 and rice chromosome 11 (for rice microsynteny, see Fig. 5a). This suggests
that these BACs contain two paralogous copies of the gene,
rather than being allelic versions of the same locus in sugarcane. Furthermore, while the P14 BAC contains 13 predicted
genes present in the syntenic region of sorghum
(Sb06g019020 to Sb06g019120), in rice the syntenic region
encompasses 18 genes (Os04g38740 to Os04g38910). As the
rice region encompasses 18 genes, it was represented in
Fig. 5a only the syntenic region for rice to sugarcane.
The K21 BAC region contains three predicted genes in
sugarcane. The syntenic region of sorghum revealed four

genes (Sb05g021000 to Sb05g021030) while the syntenic region of rice contains three genes (Os11g34440 to
Os11g34460) (Fig 5a). Our analysis shows that, like rice, three
genes are present in K21, including the Sc143-3 isoform:
phospholipase A2, 143-3 protein, and OsFBO10, which is
an F-box protein that may play a role in blue light-dependent
circadian cycle.
By analyzing the genomic region of both 143-3 paralogs
in sugarcane, we observed that these regions were composed
of five exons, with predicted proteins of different sizes: 218
and 254 amino acids for P14 and K21, respectively (Fig. 5b).
These sequences are 64 % identical at the amino acid level
(Fig. 5c). A 1.1-kb region upstream of the start codon was
analyzed using the program PLANTCARE
(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).
The results revealed differences and similarities in conserved
DNA-binding motifs between the homologous sequences
(Table 4). The potential promoter regions have similar cisacting regulatory elements related to response to light, meristem activation, drought, and MeJA response. On the other
hand, whereas just P14 has conserved motifs related to gibberellin response, only K21 has response elements to ABA,
heat stress and zein metabolism regulation. Altogether, the
microcollinearity (microsynteny) and gene structure analyses
suggest that the two sugarcane BACs analyzed have
paralogous copies of the Sc143-3 gene, rather than just being
allelic versions.
Fig. 3 In silico analysis of protein interactome. The figure representation
of protein interaction using STRING database (http://string-db.org).
Closest homologs in Arabidopsis were chosen via BlastP to retrieve its
associated network in the heterologous model. a SEC14; b CAM; c
CYP51; d PPI; e SHAGGY; and f PKCI

Tropical Plant Biol.

Tropical Plant Biol.

Fig. 4 Gene expression analysis via RT-qPCR of Sc143-3 isoforms

from sugarcane growing in equatorial field conditions. The Y-axis corresponds to gene expression levels. Ct is the cycle which SYBR fluorescence reaches the established threshold. 40-Ct determines the expression level relative to a reference gene, protein elongation factor 1 alpha
(EF1). The X-axis corresponds to the analysis period. Flowering induction occurred in October for the early-flowering cultivar, while the late

cultivar is only induced around February. CT was normalized for E = 2

[CT = CT x (log2 E/log2 2)], the errors bars corresponds to standard error
using the all different biological and technical replicates. The data obtained were then analyzed using One-way ANOVA and Tukeys test with
p 0.05. Asterisks represent significant differences within each harvest
time between early to late-flowering cultivar

In order to better understand the functional and evolutionary relationships between the three transcript isoforms and the
two Sc143-3 paralogous genes identified in sugarcane, we
performed a phylogenetic reconstruction using 143-3 sequences from Arabidopsis and model grass species. The protein identity from these sequences was from 61 % to 96 %
(Table 5). The phylogenetic tree allowed us to distinguish two
clades with high bootstrap support (>80 %; Fig. 6).
Arabidopsis and grass sequences fell into the two major
clades. Sugarcane TC143749 is closely related to that encoded
by the P14 locus, being possibly an allele within the same
locus (Fig. 6). The other two sugarcane sequences fell closest
to s org hu m seq uen ces : TC1 069 43 grou pe d w ith
Sb07g025680 while TC143247 is more closely related to
Sb07g020990. The K21 143-3 grouped only with sequences
from sorghum and rice (Fig. 6, Table 5). These results indicate
that there are at least four different paralogous Sc143-3 genes
in sugarcane. Importantly, this analysis reveals a powerful
method to distinguish gene paralogy and potential allelisms
in a highly complex genome, such as sugarcane.

by various external cues (derived from the environment) as

well as endogenous signals (genetics) (Matsoukas et al. 2012;
Khan et al. 2014; Song et al. 2015). Flowering pathways have
been well characterized especially in the long-day model,
Arabidopsis. In monocots, there are additional pathways,
genes and environmental and endogenous signals involved
in the reproductive development program (Colasanti and
Coneva 2009; Pautler et al. 2013).
Sugarcane has a highly complex genome structure due to
its domestication and breeding history of interspecific hybridization especially between S. officinarum and S. spontaneum
(Scortecci et al. 2012). A large number of duplicated genes are
the result of polyploidization events, which played important
roles during the evolution of this crop. This phenomenon resulted in a high level of heterozygosity that provides a vast
reservoir of new alleles for selection, mutation, and gene evolution (Adams 2007).
In this work, we also compared gene expression between
cultivars contrasting for flowering induction in order to identify differentially expressed transcripts from early- versus lateflowering cultivars growing on commercial field under equatorial climate conditions. Through this approach, we identified
differentially expressed genes potentially as a result of distinctive endogenous signals transduced from environmental cues
to which plants from commercial field are exposed daily.
Diatchenko et al. (1996) have shown that subtractive cDNA

Discussion
The switch from vegetative to reproductive development is
extremely important for crop production and it is triggered

Tropical Plant Biol.

Fig. 5 Analysis of Sc143-3
genomic structure using BAC
sequences. a Representation of
the genomic structure of two
BACs from the R570 cultivar.
Gene homology is represented by
lines connecting syntenic
chromosomal regions between
sugarcane (BAC) and rice. The
black box represents the 143-3
gene and gray boxes, the adjacent
genes. For P14, numbers above
the boxes represent the following
genes in both species, sugarcane
and rice: #1, MYB transcription
factor (TF); #2, plastidial 30S ribosomal protein 3; #3, MADSbox TF; #4, a transcription factor;
#5, a putative protein; #6, formin;
#7, LTPL81 (Protease inhibitor/
seed storage/LTP family protein
precursor, expressed, Oryza
sativa Japonica); #89, putative
proteins; #10, 143-3 protein;
#11, dynein light chain type 1
domain containing protein; and
#1213, prohibitin. For K21: #1,
phospholipase A2; #2, 143-3
protein; #3, OsFBO10 (F-box). b
Representation of gene structure
of the putative Sc143-3 genes
(identified as #10 above) in BACs
SHCRBA_121_P14 and
SHCRBA_268_K21. Lines and
boxes represent non-coding regions and exons, respectively. c
Alignment of conceptuallytranslated 143-3 protein sequences encoded in each BAC

library allowed to clone even low abundance transcripts due to

the normalization and suppression PCR methods. From the
subtractive libraries produced, we identified eight transcripts
involved in signal transduction pathways with putative roles
during transition to flowering in sugarcane observed by RTqPCR and/or in silico interactome. Tran et al. (2012) identified
that in plants PPO genes underwent duplication and diversification in cellular localization and function. PPO also
showed differential expression during development of flower
primordia and floral organs (Shahar et al. 1992; Goldman et al.
1998; Tran et al. 2012). In our study, sugarcane PPO was

found in the late-flowering cultivar and RT-qPCR data supported a possible role as an important signal to keep plant
development at vegetative stage. We will further pursue the
functional analysis of this gene product and test its participation in inhibiting flowering in sugarcane.
SEC14 proteins belong to a superfamily first identified in
yeast that functionally related with a multitude of cellular activities. It has been suggested SEC14 plays important roles in
signal transduction pathways and lipid metabolism regulation,
such as transfer of phospholipids in cell membranes (Bankaitis
et al. 2010). Our in silico interactome data suggested that

Tropical Plant Biol.

Table 4 Motifs found at
promoter regions 1.100 bp
upstream from start codon of the
Sc143-3 gene

Promoter Motifs
(PLANTCARE)

SHCRBa_121_P14

SHCRBa_268_K21

Potential Function

A-box

AAGAA-motif

Box I
CCGTCC-box

X
X

X
X

Light response
Meristem specific activation

CE3

ABA response

CGTCA-motif
G-Box

X
X

MeJA response
Light response

GC-motif

GCN4_motif

I-box

MBS

P-box

Skn-1_motif
Sp1

X
X

TC-rich repeats
TGA-element
ABRE

X
X

Unknown
Unknown

Enhancer-like element involved in

anoxy response
Endosperm expression
Light response
X

MYB binding site involved in

drought response
GA response

X
X

Endosperm expression
Light response

Defense and stress response

Auxin response
ABA response

ARE
Box 4

X
X

Anoxy response
Light response

HSE
O2-site
TGACG-motif
CAAT-box
TATA-box

X
X
X
X
X

Heat stress response

Zein metabolism regulation
MeJA response
promoter and enhancer regions
Core promoter element of transcription start

X
X
X

SEC14 may interact with phospholipases D (PDL), which are

heterologous enzymes playing many functions in plant cells
and that are often stimulated by Ca+2 levels (Wang and Wang
2001). In sugarcane, 32 clones with homology to SEC14 were
identified in the L2 library. Expression analysis of this gene
showed similar trends in both cultivars differing only in expression levels, and being consistently higher in the lateflowering cultivar.
SHAGGY kinases encompass a protein family divided into
four groups and related to diverse biological processes (Saidi
et al. 2012). It was initially associated with the brassinosteroid
pathway. However, it is currently know that SHAGGY kinases
regulate growth, root, vascular, flower development in response
to environmental cues, such as light, and biotic and abiotic
stress (Patade et al. 2011; Youn and Kim 2015). Moreover,
AtSK11 and AtSK12 were demonstrated in Arabidopsis to regulate flowering patterning (Dornelas et al. 2000). Thus,
SHAGGY kinases might be linking stress responses and
flowering mechanisms in sugarcane. Our RT-qPCR data suggest a potential role as an inhibitor factor since its expression in
December was higher in the late-flowering cultivar. Our in

silico interactome analysis with Arabidopsis homologs showed

potential interaction with BIN2 and other SHAGGY kinases.
The genes such as CAM, CYP51, PPI and PKCI were
identified at subtractive cDNA libraries, however the RTqPCR did not show any significant different. Additionally,
the in silico interactome data showed the possible protein patterns and connections to flower transition. Despite the fact that
these genes were not differentially expressed, their roles in
flowering transition may still be related to others modifications like mRNA stability, translational regulation as well as
protein modification, although this goes beyond our analyses.
For example, for CAM, it has been shown that in Arabidopsis
that CAM7 interacts with the HY5 promoter (Abbas et al.
2014). The HY5 levels increase during flowering transition
(Hardtke et al. 2000). Tsai et al. (2007) showed that the
CAM CLM23 induces flowering transition by affecting CO
and FLC expression and NO accumulation. The Arabidopsis
cml23/clm24 mutant was late-flowering due to accumulation
of FLC mRNA. Additionally, the interactome showed that
PP1 may interact with the flowering repressor, FY (Feng
et al. 2011; Feng and Michaels 2011).

Tropical Plant Biol.

Table 5 Protein identify from 14
to 3-3 proteins in plants

Sugarcane GF1412
(SCCCRZ1001D02.g)
Sugarcane GRF7

Sugarcane GF1412
(SCCCRZ1001D02.g)

Sugarcane GRF7

Sugarcane GF1422

(TC106943)

(SCCCLR1022D05.g)

Sac002

Sac003

Sac001

______________

96 %

83 %

96 %

________________

82 %

(TC106943)
Sugarcane GF1412

83 %

82 %

_______________

(SCCCLR1022D05.g)
shcrba_121_p14 (sac005)

81 %

80 %

78 %

shcrba_268_k21 (sac004)
Arabidopsis episilon

80 %
70 %

79 %
71 %

75 %
71 %

Arabidopsis omicron

70 %

98 %

71 %

Arabidopsis phi
Arabidopsis omega

80 %
81 %

80 %
79 %

82 %
81 %

Arabidopsis kappa
Arabidopsis iota

80 %
72 %

79 %
68 %

79 %
72 %

Arabidopsis Pi

49 %

48 %

48 %

Arabidopsis Vu
Arabidopsis lambda

85 %
79 %

85 %
78 %

86 %
78 %

Rice 13,101.m01198
Rice 13,102.m04043
Rice 13,103.m05467

62 %
89 %
86 %

61 %
87 %
87 %

63 %
88 %
86 %

Rice 13,104.m03742
Rice 13,108.m03474
Rice 13,108.m03952
Rice 13,111.m03345

87 %
96 %
77 %
83 %

87 %
93 %
76 %
82 %

87 %
95 %
76 %
82 %

The other sequence identified, 143-3, belongs to a conserved gene family comprising of thirteen members in
Arabidopsis and seven in rice. These proteins are involved
in different signal pathways as red light signaling, abiotic
stress response, flowering and others (Schoonheim et al.
2007; Paul et al. 2008; Paul et al. 2012). FT-FD complex
interacts with 143-3 protein to form the FAC complex that
switches on the flowering program (Pautler et al. 2013). Then,
the 143-3 protein family is considered an important key regulator, although the precise function of this protein has not
been revealed (Paul et al. 2012; Liu et al. 2013; Ho and
Weigel 2014). We identified three homologous transcripts in
the EST database and two genes in sugarcane BACs, corresponding to at least four paralogs. 143-3 proteins not only
bind to a diverse set of phosphoproteins, but they also seem to
coordinate their targets via diverse mechanisms (Scheeff and
Bourne 2005; Schoonheim et al. 2007). Sun et al. (2011) identified 31 143-3transcripts in cotton. They observed that
some isoforms played a role in salinity and drought
conditions. On the other hand, Mayfield et al. (2007)
showed that some 143-3 isoforms are involved in
connecting a light sensor to the central hub that regulates
flowering transition. Purwestri et al. (2009) proposed the

143-3 isoform, GF14c in rice is an inhibitor of flowering

by interacting with Hd3a. Their expression data showed
some isoforms had differences in its expression level according to tissue. Here, we show four sugarcane paralog
isoforms. The BAC sequence for two isoforms showed a
similar structural gene organization, but the putative promoter region has different motifs. Our gene expression data
captured differences between early and late-flowering sugarcane cultivars and isoforms (especially for the TC143749
and TC143247). Together, the expression data support that
the isoform TC143749 may have a potential role as a
flowering inhibitor and a marker for flowering transition
in commercial field or in plant breeding.
Altogether, our molecular data support field observations
that early-flowering induction may occurs between October
February in equatorial conditions, and that this switch to
flowering does not happen before February in the lateflowering cultivar. Moreover, it has been shown that the FT
mRNA may be promoting the movement of FT protein into
the SAM (Li et al. 2011; Jackson and Hong 2012; Liu et al.
2013). Then, the increase of FT mRNA may represent an
increase of FT protein at SAM, while this process is delayed
in the late-flowering cultivar happen. Then, the FT RT-qPCR

Tropical Plant Biol.

Fig. 6 Phylogenetic analysis of
plant 143-3 isoforms. Unrooted
phylogenetic tree inferred with
MEGA 6.0 software using the
maximum-likelihood method.
The scale bar shows estimated
amino acid substitution per site.
At, Arabidopsis thaliana; Os,
Oryza sativa; Sc, Saccharum sp.;
Sb, Sorghum bicolor

data is in agreement to this for the early flowering variety.

Coelho et al. (2014) performed phylogenetic analysis using
five sugarcane FT homologue sequences. They found that
ScFT4 and ScFT5 were closely related to the grass flowering
inductor, Hd3a. In the same work, ScFT1 was functionally
analyzed in Arabidopsis. Surprisingly, ScFT1 showed a role
as floral repressor in the heterologous model. Collectively, this
information reinforces the idea that FT proteins play important
roles in flowering not only as an activator but potentially also
as a repressor (Matsoukas et al. 2012; Coelho et al. 2014).
In conclusion, our results represent the first steps towards
the molecular identification of key genes expressed in SAM
that are related to flowering in sugarcane. Except for 143-3,
the genes identified in this study have not been classically
associated with flowering. Essentially, our data support the
importance of stress-response signal transduction pathways
interacting with classic flowering genes. We propose that a
crosstalk occurs between flowering induction and stress
response pathways that involve the genes identified here,
which are induced especially by high temperatures, high
UV levels and drought, which are common conditions in
equatorial regions. Furthermore, the central messengers of
these pathways are possibly FT and 143-3 protein (Li
et al. 2011; Tsuji et al. 2011; Jackson and Hong 2012;
Matsoukas et al. 2012; Paul et al. 2012; Liu et al. 2013;
Khan et al. 2014; Nakamura et al. 2014).

Material and Methods

Plant Material
Two sugarcane cultivars with contrasting flowering time (early-flowering, SP813250 and late-flowering, RB75126)
were planted as ratoon-cane in February 2005 in an equatorial
climate at the commercial field of Usina Estivas S.A. (Arez
town, 61140 S 350937 W). Importantly, climatologic
data recorded at Arez town were similar for the 20052006
a n d 2 0 0 7 2 0 0 9 s e a s o n s ( h t t p : / / w w w. i n m e t . g o v.
br/portal/index.php?r=tempo2/mapasPrecipitacao). In the
equatorial zone, the rain season is from March to August,
when the sugarcane plants are the vegetative state whereas
during the drought season, which corresponds to September
to February, is when the plants are triggered to flower.
SAM tissues were collected in the field for the subtractive
cDNA libraries in July 2005 (vegetative SAM) and February
2006 (vegetative SAM for RB75126 and reproductive SAM
for SP813250). For RT-qPCR experiments, SAM tissues
were harvested from the same commercial field every four
weeks from August 2007 to February 2008, and again during
the same period the following year. Twelve stalks from each
cultivar were collected per sampling and dissected in the lab.
The plant material (apical meristem) was snap frozen in liquid
nitrogen, and kept at -80 C until processing.

Tropical Plant Biol.

Subtractive cDNA Library Synthesis

Phylogenetic Relationships

Four subtractive cDNA libraries were synthesized, in order to identify mostly which are the signal that were
changed at the early-flowering cultivar (vegetative and
reproductive development). SAM development was preliminarily determined visually in the field (inflorescence
development is determined in the field by the distance
from stalks, and by section by the presence of Bcandle
flame^ at the tip of the meristem, which is actually the
appearance of a flame shape through tissue oxidation
when the apical bud is longitudinally cut in half Suppl.
Fig. 1), and then in the laboratory via stereomicroscopy
analysis. In July 2005, both cultivars presented vegetative
SAM in the field. In February 2006, the early-flowering
cultivar (SP813250) showed SAM transitioning to
flowering but the late-flowering cultivar (RB75126)
remained in the vegetative phase under the equatorial field
conditions (Suppl. Fig. 1). The four contrasts studied in
the subtractive libraries are defined in Table 1. L1 and L2
libraries were produced using contrasting cultivars; the
early-flowering cultivar collected in February (reproductive SAM), and the late-flowering cultivar collected in
July (vegetative SAM). The L1 library was designed to
identify differentially expressed genes in the earlyflowering reproductive SAM (SAMER). The L2 library
is the contrast of L1, and was designed to identify differentially expressed genes in the late-flowering, vegetative
SAM (SAMLV). Libraries L3 and L4 were constructed
using only the early-flowering cultivar at two different
developmental phases of SAM, in July 2005 (vegetative
SAM) and February 2006 (reproductive SAM). L3 was
designed to be enriched in transcripts from earlyflowering reproductive SAM (SAMEVR) and L4 is the
inverse of L3, with transcripts from vegetative earlyflowering SAM (SAMERV).
Total RNA was isolated from 300 to 500 mg of SAM (corresponding to 3 meristems, which included some developing
leaves) using the TRI reagent (Ambion). Five micrograms of
total RNA was treated with TURBO DNaseI kit (Ambion).
After digestion, two micrograms of total RNA were used to
synthesize cDNA using the Super SMART PCR cDNA
Synthesis kit (Clontech). The subtractive library was synthesized with PCR-Select cDNA Subtraction kit (Clontech),
cloned into pGEMT-Easy (Promega), transformed into
E . c o l i s t r ai n D H 10 B , an d s e q ue nc ed u s i ng t h e
DYEnamic ET Dye Terminator kit (GE Healthcare Life
Sciences) in a GE MEgaBace 1000 sequencer. The sequences
were queried against the DFCI Sugarcane database version
3.0 (E v a l u e 10e 1 0 ) (ftp://occams.dfci.harvard.
edu/pub/bio/tgi/data/Saccharum_officinarum/SOGI.
release_3.zip) and further categorized using the Gene
Ontology Biological Process tool (www.geneontology.org).

In order to understand the phylogenetic relationships of the

sugarcane 143-3 transcript identified in the subtractive libraries, unrooted phylogenetic tree was constructed using the
maximum-likelihood method, as implemented by MEGA v.5
(Tamura et al. 2011). The nucleotide sequences used were:
from sugarcane: TC143247, TC143749, and TC106943;
Arabidopsis: At3g02520, At1g35160, At1g78300,
At5g65430, At510450, At1g26480, At1g34760, At1g22300,
and At1g78220; rice: Os01g11110, Os02g36974,
Os03g50290, Os04g38870, Os08g33370, Os08g37490, and
Os11g34450; and sorghum: Sb05g024160, Sb07g025680,
Sb07g020990, Sb05g021020, Sb07g029110, and
Sb06g019100. The alignment of the Sc143-3 transcript
encompassed 688 nucleotides. Branch support was calculated by bootstrap analysis using 1000 replicates
(Felsenstein 1985).
Quantitative RT-qPCR
Total RNA was extracted as described above from four SAM
biological samples in a pool of three SAM per extraction.
After that, 10 g of RNA from each biological sample (early-flowering (SP813250) and late-flowering (RB75126)
SAM were treated with DNaseI. The absence of genomic
DNA was confirmed by RT-qPCR using primers for the reference gene EF1 using DNaseI-treated RNA as template (for
primer sequences, see Suppl. Table 1). RNA integrity was
determined using Bioanalyzer (Agilent) and quantified with
ND-1000 spectrophotometer (NanoDrop). cDNA was synthesized from 3 g total RNA using the High Capacity cDNA
Amplification kit (Life Technologies) and random primers for
first-strand reverse transcription. In order to determine cDNA
synthesis efficiency, two primer sets amplifying fragments of
the 5 end of the cDNA coding for the EF1 reference gene
(EF1T1 and EF1T2 - TC72754) were expected to provide
nearly similar quantification values. The distance between the
amplicon regions was 60 nucleotides. Only cDNA preparations with a CT 3 between these two set primers, which
denotes the RNA used was not degraded and cDNA synthesis
was sufficiently efficient, were used for RT-qPCR
(Czechowski et al. 2004; Udvardi et al. 2008). For data shown
in Figs. 1 and 4, RT-qPCR was carried out in 48-well optical
plates using StepOne Real-Time PCR System (Life
Technologies). Reactions were performed using 5 L 2X
SYBR Green Master Mix, 0.5 L 10-fold dilution of original
cDNA, and 1 L 0.5 M each gene-specific primer (cf. Suppl.
Table 1) in a 10-L reaction. For the data shown in Fig.2, RTqPCR was carried out in 384-well optical plates with an ABI
PRISM 7900HT Sequence Detection System (Life
Technologies). Reactions used 2.5 L 2X SYBR Green
Master Mix (Life Technologies), 0.5 L 10-fold dilution of

Tropical Plant Biol.

cDNA, and 1 L 0.5 M each gene-specific primer (cf. Suppl.

Table 1) in 5-L reactions. Two reference genes were used in
each plate: Ubiquitin (UBQ - TC72899) and EF1
(TC72754) (Suppl. Table 1). The Ct values were consistent
among the plates. Expression values of genes of interest were
normalized to EF1 (TC72754). For both experiments, the
following standard thermal profile was used: 50 C for
2 min; 95 C for 10 min; 40 cycles of 95 C for 15 s, and
60 C for 1 min, followed by a melting curve. The experimental design involved four biological replicates (RNA
pools derived from three SAM different plants) and four
technical replicates for each reaction. PCR efficiencies (E)
were calculated using LinRegPCR (Ramakers et al. 2003),
threshold cycles (C T ) were normalized for E = 2
[CT = CT x (log2 E/log2 2)], and errors bars corresponds
to standard errors calculated using biological and technical replicates (Pant et al. 2008). The data obtained were
analyzed using One-way ANOVA and Tukeys test with
p 0.05 in GraphPad Prism data analysis software version 5.0 (www.graphpad.com).
In Silico Interactome
Nucleotide sequences were translated using Expasy Translate
tool (web.expasy.org/translate). The putative sugarcane
protein was compared for similarity against the STRING
(string-db.org) using the Arabidopsis database.
BAC Sequencing, Assemble and Sequence Analyses
Two 143-3 homologs were identified in the sugarcane
SHCRBa BAC library by macroarray hybridization using sugarcane membrane set from Clemson Universitys Genomics
Institute (Tomkins et al. 1999) and hybridization was performed
according to the providers instructions (www.genome.
clemson.edu/cgi-bin/orders?page=serviceSearch&service=
bacrc&libtype=BAC&action=search). The BACs identified,
SHCRBa_121_P14 and SHCRBa_268_K21, were sequenced
using the 454/Roche platform as described in Setta et al. (2014).
BAC gene content was evaluated via microcollinearity
(microsynteny) analysis against sorghum and rice genomes,
protein annotation data and by tBLASTx (Evalue < 10e10) at
Phytozome v.8.0 (www.phytozome.net).

Acknowledgments This research was supported by the Brazilian

Council for Research and Development (CNPq 552722/2007-3;
478029/2012-8) and the So Paulo Research Foundation (FAPESP
2008/52074-0). N.S. was supported by a scholarship from FAPESP.
A.L.M., C.M.F., F.S.L., V.S.S. were supported by scholarships from
CNPq and the Coordination for the Improvement of Higher Education
Personnel (CAPES).

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