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DOI 10.1007/s12042-016-9175-2
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
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
Library
Subtraction
Number of clones
sequenced
L1 - SAMER
321
L2 - SAMLV
322
L3 - SAMEVR
L4 - SAMERV
241
335
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
L1a
L2a
L3a
L4a
Homology
143-3 protein
TC75568
1e-23
0
0
4
7
0
0
0
1
TC104861
TC82574
3e-10
9e-41
0
0
2
10
0
1
4
5
TC103395
TC77161
7e-33
2e-46
TC78689
4e-29
TC73046
0.0
32
TC78676
7e-68
DFCIb
e-valuec
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
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
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
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
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
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.
Discussion
The switch from vegetative to reproductive development is
extremely important for crop production and it is triggered
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
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
X
X
Endosperm expression
Light response
ARE
Box 4
X
X
Anoxy response
Light response
HSE
O2-site
TGACG-motif
CAAT-box
TATA-box
X
X
X
X
X
X
X
X
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
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).
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