Plant Mol Biol (2010) 73:271–281
DOI 10.1007/s11103-010-9613-8
SacRALF1, a peptide signal from the grass sugarcane (Saccharum
spp.), is potentially involved in the regulation of tissue expansion
Fabiana B. Mingossi • Juliana L. Matos • Ana Paula Rizzato
Ane H. Medeiros • Maria C. Falco • Marcio C. Silva-Filho •
Daniel S. Moura
•
Received: 10 May 2009 / Accepted: 30 January 2010 / Published online: 11 February 2010
Ó Springer Science+Business Media B.V. 2010
Abstract Rapid alkalinization factor (RALF) is part of a
growing family of small peptides with hormone characteristics in plants. Initially isolated from leaves of tobacco
plants, RALF peptides can be found throughout the plant
kingdom and they are expressed ubiquitously in plants. We
took advantage of the small gene family size of RALF
genes in sugarcane and the ordered cellular growth of the
grass sugarcane leaves to gain information about the
function of RALF peptides in plants. Here we report
the isolation of two RALF peptides from leaves of sugarcane plants using the alkalinization assay. SacRALF1 was
the most abundant and, when added to culture media,
Electronic supplementary material The online version of this
article (doi:10.1007/s11103-010-9613-8) contains supplementary
material, which is available to authorized users.
J. L. Matos D. S. Moura (&)
Departamento de Ciências Biológicas, Escola Superior
de Agricultura Luiz de Queiroz, Universidade de São Paulo,
C.P. 9, Piracicaba, SP 13400-970, Brazil
e-mail: [email protected]
F. B. Mingossi A. P. Rizzato A. H. Medeiros M. C. Silva-Filho
Departamento de Genética, Escola Superior de Agricultura
Luiz de Queiroz, Universidade de São Paulo, C.P. 83,
Piracicaba, SP 13400-970, Brazil
M. C. Falco
Centro de Tecnologia Canavieira, CTC, C.P. 162,
Piracicaba, SP 13400-970, Brazil
Present Address:
A. P. Rizzato
Alellyx Applied Genomics, Rod. Anhanguera, Km 104
(Techno Park), Campinas, SP 13067-850, Brazil
inhibited growth of microcalli derived from cell suspension
cultures at concentrations as low as 0.1 lM. Microcalli
exposed to exogenous SacRALF1 for 5 days showed a
reduced number of elongated cells. Only four copies of
SacRALF genes were found in sugarcane plants. All four
SacRALF genes are highly expressed in young and
expanding leaves and show a low or undetectable level of
expression in expanded leaves. In half-emerged leaf blades,
SacRALF transcripts were found at high levels at the basal
portion of the leaf and at low levels at the apical portion.
Gene expression analyzes localize SacRALF genes in
elongation zones of roots and leaves. Mature leaves, which
are devoid of expanding cells, do not show considerable
expression of SacRALF genes. Our findings are consistent
with SacRALF genes playing a role in plant development
potentially regulating tissue expansion.
Keywords Alkalinization assay Cell suspension Development Peptide hormone
Introduction
Rapid alkalinization factor (RALF) was isolated from
leaves of tobacco, tomato and alfalfa using the alkalinization assay (Pearce et al. 2001a, b). The newly discovered
peptide can be found throughout the plant kingdom and its
gene is expressed ubiquitously in the plant. RALF peptide
rapidly causes an increase in the external pH of cell suspension cultures and also induces the activation of a MAP
kinase protein (Pearce et al. 2001a). Scheer et al. (2005)
showed that RALF peptide interacts with two integral
membrane proteins of 25 and 120 kDa that may be components of a receptor complex in membranes of tomato
cells. Haruta et al. (2008) reported that a RALF isoform
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from Arabidopsis induces Ca2? influx across the plasma
membrane as well as release of Ca2? from intracellular
reserves. RALF peptides are derived from precursors of
over 100 amino acids. PreproRALF proteins have an
N-terminal signal sequence, a non-conserved region
downstream the signal sequence, and a well-conserved
C-terminal region that covers the active peptide (Pearce
et al. 2001a). A conserved dibasic site just upstream of the
active peptide was shown to be essential for proper RALF
processing similarly to animal and yeast peptide hormone
processing (Matos et al. 2008). Recently, Srivastava et al.
(2009) identified a subtilase AtS1P from Arabidopsis as
responsible for preproRALF23 processing. Using a virus
expression based system to study protein transport in
tobacco, Escobar et al. (2003) showed that a RALF-GFP
fusion protein was targeted to the secretory pathway.
RALF peptide was found first in the endoplasmic reticulum
(ER) and 24 h later in the ER and the cell wall. After two
more days, RALF-GFP fusion protein was found solely in
the cell wall. Using hybrid poplar leaf protein extracts,
Haruta and Constabel (2003) isolated five RALF peptides
(PtdRALF). Two cDNAs were isolated, PtdRALF1 and
PtdRALF2, and gene expression analyses revealed the
presence of PtdRALF transcripts in all tissues tested.
PtdRALF2 gene was down regulated by methyl jasmonate
in cell suspension cultures. Auxin and cytokinin treatments
did not cause significant changes in PtdRALF gene
expression. The properties of RALF peptides suggest that it
is a powerful receptor-mediated signal that may have a role
in regulating growth and development.
In Solanum chacoense, Germain et al. (2005) isolated
five cDNAs from libraries made of fertilized ovule and
ovary tissues. Wounding and treatments with growth hormones did not cause variation in mRNA levels of ScRALF
transcripts. RALF-like genes have also been identified in
reproductive tissues in other species (Becker et al. 2003;
McCubbin et al. 2006). RALF genes have also being
pointed out as having a potential function in legume nodules and seeds (Silverstein et al. 2006).
Arabidopsis has over 30 RALF/RALF-like genes (Olsen
et al. 2002) and this large family size compounds the difficulty of using normal genetic approaches to unravel gene
function in this model plant. Native tobacco, Nicotiana
attenuata, has only one copy of the RALF gene (NaRALF)
and when it was silenced (irRALF) showed longer roots
when compared to wild type (Wu et al. 2007). Wu et al.
(2007) reported that a longer elongation zone and a rapid
root growth could account for the longer roots observed in
irRALF lines. In addition, irRALF lines produced trichoblasts that formed abnormal root hairs.
Here, we report the isolation of RALF peptides from
leaves of the grass sugarcane (Saccharum spp.) using the
alkalinization assay. SacRALF1 peptide showed a half
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maximal activity of 1.5 nM and was able to inhibit growth
of cell suspension-derived microcalli at concentrations as
low as 10-7 M. Microcalli cells treated with SacRALF1
peptide failed to elongate or showed delayed entrance into
the elongation phase. Four SacRALF cDNAs were identified and SacRALF1 transcripts were the most abundant.
SacRALF1 transcripts were found in elongation zones of
roots and in the basal parts of leaf blades. While young
elongating leaves showed abundance of SacRALF transcripts, mature leaves were devoid of them. Our results are
consistent with SacRALF1 peptide playing a role in plant
development and suggest its potential involvement in regulating tissue expansion.
Materials and methods
Plant material and plant growth
Sugarcane plants (Saccharum hybrid cultivar SP-80-3280)
were kindly provided by Centro de Tecnologia Canavieira
(CTC), Piracicaba, SP, Brazil. Sugarcane plants were
obtained from vegetative stalk cuttings called setts (nodal
buds). One-eyed setts were planted in 200 mL plastic cups
containing a commercial planting mix (Plantmax, Eucatex)
and cultivated in a greenhouse for 20 days and at temperatures ranging from 18°C (night) to 34°C (day). For protein
extraction, sugarcane setts were planted directly in soil and
were watered and cultivated accordingly. Leaves were
harvested from 30-day-old sugarcane plants. Each collection was made from approximately 3,000 plants that were
cut near the soil. After cutting the aerial part of the plants,
new tillers were produced from underground nodal buds
and were allowed to re-growth to be harvested again.
Sugarcane cell suspension culture and alkalinization
assay
Embryogenic calli were induced from sugarcane (Saccharum hybrid cultivar SP83-2847) using young transversal
leaf discs as described (Falco et al. 1996). Approximately
2 g of calli were inoculated into 40 mL of liquid medium
[4.43 g L-1 Murashige and Skoog basal medium with
vitamins, 3 mg L-1 2,4-D, 30 g L-1 sucrose, 50 mL L-1
of industrial coconut water, 2 g L-1 casein acid hydrolysate, and adjusted to pH 5.8 with KOH] in a 250 mL
Erlenmeyer flask. Cultures were placed under agitation
(120 rpm) in the dark at constant temperature (28 ± 1°C).
Cell suspensions were subcultured every 3 days by
allowing the cells to decant for a few seconds and transferring 15 mL of the intermediate phase to 35 mL of fresh
medium. After 6 months, a rapidly growing suspension
culture of embryogenic cells, small cell aggregates with a
Plant Mol Biol (2010) 73:271–281
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dense cytoplasm, was obtained. Cell suspension cultures
were 3 year-old at the time the experiments were made and
have been maintained by diluting 5 mL of a 5-day-old
culture in 45 mL of fresh medium. For the alkalinization
assay, one milliliter of a 5-day-old cell suspension culture
were transferred into each well of 24-well cell-culture
plates and allowed to equilibrate on an orbital shaker at
140 rpm for 1 h at room temperature. Small aliquots of
chromatographic fractions (1–10 lL) were added to the
cells and the pH of the medium was monitored over time
(Thermo ORION perfHect Meter ROSS).
TFA at 1 mL/min flow rate. Further purification for trypsin
digestion and LC/MS/MS analysis was performed using
narrow-bore column at 0.2 mL/min flow rate (Source
5RPC, 2.1 9 150 mm, Amersham). SacRALF1 peptide
was separated in an 15% acrylamide SDS–PAGE gel
(Laemmli 1970) and stained for total protein using a
solution of 0.1% (w/v) Comassie Blue in 40% methanol
and 10% acetic acid. Trypsin digestion and LC/MS/MS
analyses were made at The Yale Cancer Center Mass
Spectrometry Resource and the W. M. Keck Foundation
Biotechnology Resource Laboratory.
SacRALF peptide purification
Microcalli growth assay
Leaves of sugarcane plants were cut, immediately frozen in
liquid N2, transported to the laboratory in dry ice and stored
at -80°C. Frozen leaves were ground to a fine powder
using a mortar and pestle. Powdered leaves were mixed
with extraction buffer, 1% trifluoroacetic acid (TFA) in a
4 L blender for approximately 5 min. The mixture was
filtered through eight layers of cheesecloth and one layer of
miracloth (Calbiochem). The filtrate was centrifuged at
10,000g for 20 min at 4°C. The clarified extract was loaded
onto a C-18 flash column (BakerBond resin, 16.0 9 3 cm)
using compressed N2. The resin was equilibrated and
washed with 0.1% TFA and eluted with 60% methanol,
0.1% TFA. Methanol was removed using a rotary evaporator followed by lyophilization. The freeze-dried protein
was dissolved in 0.1% TFA and loaded onto a G-25 column
by gravity (Sephadex, Amersham, 44.5 9 2.5 cm). The
resin was equilibrated with 0.1% TFA. Eight-milliliter
fractions were collected and evaluated using the alkalinization assay described above. Active fractions were
pooled and lyophilized. Eighty milligrams of dried-protein
were dissolved in 0.1% TFA and injected into a reversed
phase Resource 3 mL HPLC column (Amersham) previously equilibrated with 0.1% TFA. A 90-min gradient from
0 to 40% acetonitrile (CH3CN) in 0.1% TFA was applied
after sample injection in the HPLC system at 2 mL/min
flow rate (ÄKTA Purifier, GE/Amersham). Fractions
(2 mL) were collected and assayed in sugarcane cell suspension cultures. Active fractions were pooled and lyophilized. Dried protein was dissolved in 5 mM potassium
phosphate pH 3.0/25% CH3CN and injected into a strong
cation exchange column (Zorbax 300-SCX, 4.6 9
250 mm, Agilent). After injection, a 60-min gradient from
0 to a 100% of 5 mM K phosphate pH 3.0/25% CH3CN/
500 mM KCl was applied at 1 mL/min flow rate. Fractions
were checked for activity using the alkalinization assay.
Active fractions were desalted and further purified using a
Zorbax 300SB-C18 HPLC column (4.6 9 250 mm, Agilent) previously equilibrated with 0.1% TFA and eluted
with a 90-min gradient from 0 to 50% methanol in 0.05%
Aliquots of a 4-day-old cell suspension culture (100 lL)
were plated on 1 mL of semi-solid culture medium
[4.43 g L-1 Murashige and Skoog basal medium with
vitamins (Phytotechnology), 3 mg L-1 2,4-D, 30 g L-1
sucrose, 50 mL L-1 of industrial coconut water, 2 g L-1
casein acid hydrolysate, 7 g L-1 Phytagar and adjusted to
pH 5.8 with KOH] in each well of 12-well cell-culture
plates. SacRALF1 peptide purified from sugarcane leaves
was added to the medium at 50°C and to the 100 lLsamples of suspension cultures to obtain the desired
concentrations. Water was used as control. After the cell
suspension aliquots were absorbed by the medium, plates
were incubated in the dark at 30°C. Microcalli growth in
semi-solid medium was analyzed every 24 h by visualization
under a stereoscope (microNal). Visualization after 5 days
was performed using a light microscope (Olympus BX50).
Hypocotyl elongation
Hypocotyl measurements were taken as described in
Weigel and Glazebrook (2002). After cold treatment (48 h
at 4°C), seeds were transferred to a growth room [16 h day,
24°C, 150 lE m-2 s-1 (E, Einstein; 1 E = 1 mol of photons)] and maintained until the end of the experiment.
Seeds were placed in liquid MS media (half-strength) with
gentle agitation on a rotary shaker, exposed to light for 4 h
before germination, and kept in the dark. Measurements
were taken 2, 4 and 6 days after germination. Seeds were
examined 2 days after light treatment for uniform germination. RALF peptide (10 lM) was added 2 days after
germination. For this experiment was used a recombinant
RALF peptide (AtRALF1, locus at1g02900) that shares
over 80% similarity with SacRALF1 and shows the same
activity in the alkalinization assay (data not shown).
RNA isolation and cDNA synthesis
Total RNA of sugarcane tissues was isolated using TRIZOLÒ reagent (Invitrogen), followed by deoxyribonucleic
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acid removal by treatment with 2 units of DNAseI Rnase
free (Fermentas) for 20 min at 37°C. After DNAse
treatment, RNA was re-extracted using TRIZOL. Total
RNA was quantified and quality was checked by spectrophotometer and agarose gel. First strand synthesis
was performed in a volume of 20 lL, containing 2 lg of
total RNA with 2.5 lM of poly-(dT) primer and 1 lL
of reverse transcriptase following recommendations of
the manufacturer (Improm-IITM Reverse Transcriptase,
Promega).
Real-time PCR gene expression and data analysis
Real-time quantitative PCR was performed using the Rotor
Gene 3000 machine (Corbett Life Science) using SYBR
Green to monitor double-strand DNA synthesis. Genespecific primers were designed (SacRALF1-FwdATC GGT
TGC TGT GCT AGC TT, SacRALF1-Rev CCA CTG
CAC CAC AAT AAT CG, SacRALF2-Fwd CAA CGC
AAG GTG TTG CTA AA, SacRALF2-Rev ACA TCT
TGC ACT GCC AAC AG, SacRALF4-Fwd TCC ACC
GAG AGG AAG AGA AG, SacRALF4-Rev ACC ACC
ACC AAT CAT TCT CG, SacRALF3-Fwd TTC TTC
TGC CTA CCG CTA CC, SacRALF3-Rev GAA TCC
ATC CAT CTC CCC TAA, GADPH-Fwd TTT GAA TGG
CAA GCT CAC TG and GADPH-Rev GGT GGA AAC
CAA ATC CTC CT) using a stringent set of criteria,
including predicted melting temperature of 60 ± 3°C,
primer lengths of 20–22 nucleotides, guanine-cytosine
content of 40–70% and product size of 100–270 bp. Primer
specificity was confirmed by analysis on 2% agarose gel,
by melting curve analysis and by sequence verification
through cloning of PCR products in pCRÒ2.1-TOPOÒ
vector (Invitrogen). GAPDH was used as internal control
(Iskandar et al. 2004). PCR reactions contained 4 lL of the
cDNA (in the appropriate dilution), 10 lL of PlatinumÒ
SYBRÒ Green qPCR SuperMix-UDG (Invitrogen), 5.2 lL
of nuclease-free water and 0.2 lM of each primer in a final
volume of 20 lL. Non-template control reactions were
performed with 4 lL of nuclease-free water. PCR conditions were as follows: 50°C for 2 min, 95°C for 2 min, and
40 cycles of 95°C for 15 s and 62°C for 30 s. The fluorescence signal was captured at the end of each cycle and
melting curve analysis was performed from 72 to 99°C,
holding 45 s on the first step and holding 5 s on next steps.
Data analysis was performed using the Pfaffl method
(Pfaffl 2001). GAPDH expression was used to normalize
the transcript level in each sample. The threshold was
manually defined as 0.1 of the normalized fluorescence.
Statistical analyses were performed using MedCalc for
Windows, version 9.5.2.0 (MedCalc Software, Mariakerke,
Belgium).
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Results
SacRALF peptide purification
Rapid alkalinization factor peptides were isolated from
protein extracts of sugarcane leaves using the alkalinization
assay (Pearce et al. 2001b). A crude peptide extract was
separated using a series of chromatographic columns as
described in materials and methods. A typical protein
extraction was made with 2 kg of powdered frozen leaves
in 4 L of extraction buffer. After clarifying the extract, it
was loaded onto a C-18 flash chromatography. A 60%
methanol eluate yielded 2.62 g of protein after freezedrying. Combined dried proteins were resuspended in
5 mL of 0.1% trifluoroacetic acid (TFA) and passed
through a G-25 size exclusion column, approximately 3 g
of protein in each run. An average of 12 G-25 runs were
needed to process the proteins extracted from each collection of 3,000 plants. Fractions from the G-25 were
checked for alkalinization activity and the activity was
found at or near the void volume. Active fractions were
combined, freeze-dried, dissolved in 0.1% TFA and
injected into a RESOURCE-3 mL HPLC reversed phase
column (GE/Amersham). Sequential runs of 80 mg each
were made and active fractions yielded 47 mg of freezedried protein per kg of frozen leaves. Figure 1a demonstrates the protein profile and the alkalinization activity of
the fractions eluted from the RESOURCE-3 mL column.
The complex mixture of proteins showed a major peak of
activity in fractions corresponding to 70–80 mL of elution
volume. The activity peak showed in Fig. 1a was separated
in two peaks using a strong cation exchange column.
Further purification of these two peaks in a narrow-bore
reversed phase column resulted in two peptides with similar activities (Fig. 1b, peaks I and II). The most abundant
peptide was peak number II. An aliquot of peak number II
was loaded on an SDS–PAGE gel and showed the purified
peptide as a single band, which estimated molecular weight
(Mr) is 5,000 (Fig. 1c). Mass spectrometric analyses of
peaks I and II revealed the presence of a 5198.202 and a
5302.061 Da peptides, respectively. Both peptides were
subjected to Edman degradation for N-terminal sequence
determination, and the lack of results suggested that both
peptides were blocked. Because SacRALF1 peptide was
much more abundant than its homologue SacRALF3, we
used only SacRALF1 for activity assays. Using a dilution
series of the purified peptide showed in peak II, we estimated its half maximal activity in the alkalinization assay
as 1.5 nM (Fig. 1d).
A search in the available databases including SUCEST,
a sugarcane EST database (Vettore et al. 2003), showed the
existence of four cDNAs coding for precursors of sugar-
Plant Mol Biol (2010) 73:271–281
(A)
275
(B)
II
6.0
5.1
(C)
I
4
60
3
2
30
1
0
100
Volume (mL)
pH
pH
60
45.0kDa
31.0kDa
21.5kDa
14.4kDa
4.5
40
20
0
50
(D)
CH3CN (%)
ABS280nm(AU)
4.5
ABS214nm(mAU)
pH
25
35
45
Fractions (min)
5.9
5.7
5.5
5.3
5.1
4.9
4.7
4.5
0 2
4 6
8 10 12 14 16 18 20 22 24 26 28 30
SacRALF1 (nM)
Fig. 1 Purification of SacRALF peptides from sugarcane leaf protein
extracts. a Initial reversed phase HPLC separation of 80 mg of
sugarcane crude protein extract (lower panel) and the alkalinization
assay of 10 lL of each HPLC fraction in 1 mL of cell suspension
cultures (upper panel). b Narrow-bore HPLC separation of
SacRALF1 (peak II) and SacRALF3 (peak I). Equal amounts of both
purified peptides were loaded to show the activity in the alkalinization
assay (dotted line). The pH of the media was measured after 15 min
of adding 2 lL of each fraction in 1 mL of cell suspension cultures.
c SacRALF1 peptide (2.5 mg) was loaded on an SDS–PAGE gel
(15%). Arrow indicates SacRALF1 peptide and molecular weight
standards are shown on the right. d Alkalinization response of
sugarcane cell suspension culture to purified SacRALF1 peptide.
Different concentrations of the peptide were added to 1 mL of cell
suspension and the pH was measured after 15 min
cane RALF peptide homologs (SacRALF1 to 4, Fig. 2). All
four putative RALF precursors code for proteins with a
non-conserved N-terminal portion and a well-conserved
C-terminal portion that covers the active RALF peptide.
The similarities between SacRALF sequences and the
original tobacco RALF range from 62 to 86%. At first,
none of the SacRALF peptides matched the masses
obtained from the peptides isolated from sugarcane leaves.
However, two of them, SacRALF1 and SacRALF3 have
theoretical masses of 5319.8 and 5216.8 Da respectively,
that are similar to the ones obtained experimentally. Both
SacRALF1 and SacRALF3 deduced RALF peptides have a
glutamine residue at the N-terminus. A very common
N-terminus transformation in proteins is the cyclization of
glutamine in pyroglutamic acid (pyrrolid-2-one-5-carboxylic acid). This modification could be physiologic,
making a protein more stable, or could be a result of protein purification (Wold 1981). In the transformation process, glutamine loses ammonia (17.0306 Da). If such a
reduction is taking into account, the masses calculated
for both SacRALF1 and SacRALF3 are 5302.76 and
Fig. 2 Alignment of the four deduced preproproteins coded by
SacRALF genes in sugarcane. The deduced tobacco preproprotein
(RALF) is shown for comparison. GenBank accession numbers are
CA182793 for SacRALF1, CA245303 for SacRALF2, CA144056 for
SacRALF3 and CA156164 for SacRALF4. Arrows connected by
traced lines indicate the peptides that were sequenced after trypsin
digestion
5199.76 Da, respectively. Both in close agreement to the
peptides purified from sugarcane leaves. To positively
identify the most abundant peptide, we sent out a
SacRALF1 sample for trypsin digestion and LC MS/MS
analysis. Three tryptic peptides were identified spanning
the residues 41–49, 41–50 and 23–40 of SacRALF1
(arrows in Fig. 2; Supplemental Fig. S1). These three
tryptic peptides cover 28 of 50 amino acids of SacRALF1.
SacRALF1 effect on cell elongation
SacRALF1 peptide purified from sugarcane leaves was
added to the growth medium in order to investigate its
effects on the development of microcalli derived from
sugarcane cell suspension cultures. In Fig. 3a are the
results of microcalli grown on media containing different
concentrations of SacRALF1. Concentrations of 10, 5, 1
and 0.1 lM were all effective in halting microcalli growth.
Microcalli after 5 days of exposure to SacRALF1 were
examined under the microscope and a reduced number of
elongated cells were observed (Fig. 3b, arrow heads).
When compared to untreated controls, SacRALF1 was
found to induce the formation of smaller cells on the borders of the microcallus. After 5 days in the media containing SacRALF1, microcalli were transferred to control
media and resumed growth. In a separate experiment, using
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a recombinant RALF peptide (AtRALF1, locus at1g02900)
that shares over 80% similarity with SacRALF1 and shows
the same activity in the alkalinization assay (data not
shown), a large number of microcalli treated with RALF
peptide was evaluated. Significant differences (t-test,
P \ 0.05) in the number of microcalli with elongated cells
were observed at two different concentrations after 5 and
7 days of treatment (Supplemental Table 1). After a week
of being exposed to 5 lM RALF, microcalli growing in
media without RALF showed more than double the number
of microcalli with elongated cells, 26.8 and 12.66,
respectively (t-test, P \ 0.0001). In order to further
investigate the role of RALF peptide in regulating cell
elongation, we also exposed dark-growing Arabidopsis
seedlings to the peptide and evaluated elongation of
hypocotyls. Arabidopsis hypocotyls have a fixed number of
cells (Cheng et al. 1995; Gendreau et al. 1997) and hypocotyl growth is considered to be a reflection of cell elongation (Gendreau et al. 1997). As shown in Fig. 4, addition
of 10 lM of RALF peptide to the medium of Arabidopsis
seeds germinating in darkness inhibited hypocotyl length.
After 6 days in the dark, the hypocotyls lengths of control
seedlings averaged 14.8 mm, while hypocotyls of RALFtreated seedlings, averaged less than 10 mm in length.
Hypocotyl elongation resumed when RALF-treated seedlings were transferred to medium lacking RALF (data not
shown), similar to seedlings grown in light/dark regimes
that were treated with RALF peptide and then rinsed with
water (Pearce et al. 2001a).
SacRALF gene expression in young sugarcane plants
Young sugarcane plants form a leaf roll, a set of young
non-elongated and elongating leaf blades encircled by
external old leaf sheaths. New leaves emerge from the leaf
roll and continue to expand until reaching full size. All four
SacRALF genes are expressed in roots, leaf rolls, and
expanded leaves (Fig. 5). SacRALF1 and SacRALF3
showed a similar level of expression between roots and leaf
rolls, while SacRALF4 was mainly expressed in leaf rolls,
and SacRALF2 showed no detectable expression in roots.
Without exception, SacRALF genes showed a low level of
transcripts in fully expanded leaves. When the levels of
expression of all four SacRALF genes in leaf rolls and
roots were compared with each other, SacRALF1 was
predominant. None of the three other SacRALF genes
reached 10% of the expression level of SacRALF1 in both
leaf rolls and roots: 3.3, 9.7 and 7.9% for SacRALF2, 3,
and 4, respectively.
Root tips are characteristically separated into meristematic, elongating and differentiating zones. For the purpose of our study, we evaluated two regions of the root tip:
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Plant Mol Biol (2010) 73:271–281
(A)
0
5 th day
Ctr
10µM
5µM
1µM
0.1µM
(B)
Ctr
SacRALF1
Fig. 3 Growth assay of microcalli derived from sugarcane cell
suspension culture. a An aliquot of cell suspension culture (100 lL)
was placed on top of media containing different concentrations (10, 5,
1 and 0.1 lM) of SacRALF1 peptide. Water was used as control.
Cells were incubated at 28°C in the dark. Pictures were taken at day
zero and at the 5th day after starting the experiment with the help of a
stereoscope (magnification 109). b After 5 days in control media and
in media containing 5 lM SacRALF1, microcalli were visualized
under a microscope (magnification 4009). Arrows indicate elongated
cells in control microcalli
Plant Mol Biol (2010) 73:271–281
277
expression in the root tips (not statistically different, t-test,
P = 0.19). Transcripts of SacRALF2 were not detected
along the root zones evaluated.
Hypocotyl length
(mm)
20
15
RALF
10
SacRALF gene expression in blade
and sheath of sugarcane leaves
5
0
0
2
4
6
Time (days)
(A)
(B)
1.5
1.5
1.0
1.0
0.5
0.5
0.0
0.0
(A)
Root
b
30
Root
Leaf Roll Exp. Leaf
Leaf Roll Exp. Leaf
20
(C)
(D)
1.5
1.5
1.0
1.0
0.5
0.5
0.0
0.0
10
a
0
Root
Leaf Roll Exp. Leaf
Root
Leaf Roll Exp. Leaf
Fig. 5 Analysis of relative SacRALF genes expression using realtime quantitative PCR. Different tissues (roots, leaf roll and expanded
leaf) of young sugarcane plants were evaluated. Relative quantification of mRNA was performed with efficiency correction and GAPDH
as reference gene. A value of 1 for each SacRALF gene was
arbitrarily attributed to the expression level found in the leaf roll.
a SacRALF1. b SacRALF2. c SacRALF3. d SacRALF4. Values are
means of three replicates ± SD
the meristematic zone, composed of the first 4 mm of the
root tip (tip-4 mm); and the elongation zone, the root tissue
behind 4 mm ([4 mm). The end of the elongation zone
was established by the presence of root hairs (Moore 1987).
SacRALF1 and SacRALF3 showed different expression
levels at both root zones (t-test, P \ 0.001, Fig. 6).
SacRALF1 gene showed a high level of transcripts in the
elongation zone ([4 mm). The expression of SacRALF1 in
the elongation zone was over 20 times higher than the level
found in the meristematic zone. SacRALF3 followed the
same trend whereas SacRALF4 showed minor changes in
Relative mRNA levels
Relative mRNA levels
Fig. 4 RALF inhibition of seedling hypocotyl elongation. Hypocotyl
elongation of Arabidopsis seedlings germinated and grown in the dark
was measured. After 48 h, 10 lM RALF (open square) or an equal
volume of water (filled squares) was added to the medium and
hypocotyl length was measured at times indicated. Values are
means ± SD of 90 seedlings. Arrow indicates the addition of RALF
peptide
Similarly to roots, grass leaves are appropriate for studies
on developmental processes because of their unidirectional
growth. Maturation of leaf cells occurs basipetally, that is
from the apex to the base, confining the elongation to the
basal region of the blade (Dickison 2000). As in other
grasses, sugarcane leaf blade elongates first followed by the
leaf sheath that is enclosed in a whorl of young leaf sheaths
(Moore 1987). We took advantage of such ordered cellular
growth to perform gene expression analyses in leaves of
different developmental stages. Figure 7A shows a typical
plant used in our studies. Leaves are numbered according
to Moore (1987), from the oldest to the youngest, ?3 to
-1, respectively. Leaves were detached and sectioned at
the junction blade-sheath, called dewlap, and they were
(B)
Tip-4mm
>4mm
6
4
2
b
a
0
(C)
Tip-4mm
>4mm
a
a
Tip-4mm
>4mm
6
4
2
0
Fig. 6 Analysis of relative SacRALF genes expression in root tips
of young sugarcane plants using real-time quantitative PCR.
A SacRALF1. B SacRALF3. C SacRALF4. Root tips were separated
into two regions: meristematic zone, composed of the first 4 mm of
the root tip (tip-4 mm) and elongation zone, the root tissue behind
4 mm ([4 mm). The end of the elongation zone was established by
the presence of root hairs. Relative quantification of mRNA was
performed with efficiency correction and GAPDH as reference gene.
A value of 1 for each SacRALF gene was arbitrarily attributed to the
expression level found in the meristematic zone. Values are means of
three replicates ± SD. Letters on top of each column were generated
by the t-test. Columns followed by the same letter are not statistically
different at P \ 0.05
123
278
evaluated separately (Fig. 7B). SacRALF1 showed very
low levels of expression in old leaf blades, such as ?3, ?2
and ?1, and the expression increased in young leaves, 0
and -1 (Fig. 7C). The youngest leaf blade (-1) showed a
level of SacRALF1 transcripts over 24 times higher than
the level found in the oldest leaf (?3). A similar trend was
observed in leaf sheaths, that is high gene expression in
young leaf sheaths and low in old ones. In the leaf ?1 that
shows a fully expanded leaf blade and an expanding leaf
sheath, the level of SacRALF1 transcripts was almost 30fold higher in the leaf sheath than in the blade. The same
gene expression profile was seen for SacRALF2, 3 and 4,
with the exception of the gene expression profile in leaf
sheaths of the SacRALF3 that showed an expected reduction at the ?2 leaf sheath (Fig. 7D–F).
Plant Mol Biol (2010) 73:271–281
(A)
-1
Discussion
Rapid alkalinization factor peptides were initially isolated
as strong and RALFs from leaf extracts of tobacco plants
(Pearce et al. 2001a). Although originally extracted from
leaves, effects of exogenous RALF have only been shown
in roots (Pearce et al. 2001a).
An alkalinization assay based on sugarcane cell suspension cultures was developed and two SacRALF peptides were purified from sugarcane leaf protein extracts
(Fig. 1a–d). We adopted a purification scheme similar to
the ones described by Pearce et al. (2001a) and Pearce and
Ryan (2003), and comparable results regarding the peptide
123
+1
+2
+3
+3
+2
+1
45
30
15
0
-1
0
(C)
SacRALF gene expression in half-emerged leaves
of sugarcane plants
c’
d
b’
c
ab
+3B
a
b
+2B
+1B
0B
a’
-1B
+3S
+2S
a’
a’
+1S
(D)
Relative mRNA levels
To investigate even further the specifics of SacRALF gene
expression in expanding leaves of sugarcane, we cut the
leaf number zero into three parts of equal length: apical,
medial and basal. The blade of the leaf number zero is half
emerged and its sheath has not yet initiated elongation. At
the time of the tissue collection, leaf zero had half of the
blade exposed to light and half covered by the sheaths of
old leaves. The sheath of leaf zero was not analyzed due to
its small size. Figure 8 shows the result of the relative
expression analyses of SacRALF genes in the half-emerged
blade of leaf number zero. SacRALF1 gene shows a high
expression at the base of the leaf, near fivefold the level
found at the middle, and over 50 times the level at the apex
(Fig. 8A). SacRALF3 also showed significant differences
from the base to the apex of the expanding leaf: 10 times
higher than the level found in the medial portion and 22
times higher than the level at the apical portion (Fig. 8C).
Both SacRALF2 and 4 genes showed a similar trend
(Fig. 8B, D).
(B)
0
9
6
3
0
b
c
+1B
0B
ab
ab
a
+2B
+1B
0B
a
+3B
+2B
c’
d
-1B
+3S
c
b’
+2S
+1S
(E)
9
6
3
0
+3B
-1B
a’
+3S
+2S
a’
a’
b’
+1S
(F)
9
6
3
0
c
b
a
b
+3B
+2B
+1B
b
0B
-1B
b’
+3S
+2S
+1S
Fig. 7 Young sugarcane plant (30-day-old) used in this study (A)
and leaves used for gene expression analyses (B). The leaves ?3, ?2
and ?1 were separated into blades and sheaths. For leaves 0 and -1,
only the blades were analyzed. Leaves are numbered from the oldest
to the youngest, ?3 to -1, respectively. C–F Analysis of relative
SacRALF genes expression in different leaves of young sugarcane
plants using real-time quantitative PCR. Relative quantification of
mRNA was performed with efficiency correction and GAPDH as
reference gene. A value of 1 for each SacRALF gene was arbitrarily
attributed to the expression level found in ?3B (C, F) and ?2B (D,
E). C SacRALF1. D SacRALF2. E SacRALF3. F SacRALF4. Values
are means of three replicates ± SD. Leaves are numbered from the
oldest, ?3 to the youngest, -1. B = blade, S = sheath. Letters on top
of each column were generated by the t-test. Columns followed by the
same letter are not statistically different at P \ 0.05
chromatographic behavior were obtained. All SacRALF
peptides eluted in the void volume of a G-25 size exclusion
column and also showed a similar elution time in reversed
Plant Mol Biol (2010) 73:271–281
(B)
(A)
60
279
10
a
Relative mRNA levels
8
40
20
6
4
b
c
0
BASAL MEDIAL APICAL
10
8
6
a
20
0
b
(D)
(C)
10
b
0
BASAL MEDIAL APICAL
30
a
2
b
c
BASAL MEDIAL APICAL
4
2
0
a
a
b
BASAL MEDIAL APICAL
Fig. 8 Analysis of relative SacRALF genes expression in the leaf
zero of young sugarcane plants using real-time quantitative PCR. Leaf
zero was separated in three parts of equal length: basal, medial and
apical. Relative quantification of mRNA was performed with
efficiency correction and GAPDH as reference gene. A value of 1
for each SacRALF gene was arbitrarily attributed to the expression level found in apical region. A SacRALF1. B SacRALF2.
C SacRALF3. D SacRALF4. Values are means of three replicates ± SD. Letters on top of each column were generated by the
t-test. Columns followed by the same letter are not statistically
different at P \ 0.05
phase columns. Different SacRALF peptides were only
separated when injected into a strong cation exchange
HPLC column.
SacRALF1 peptide effects on cell suspension cultures
and cell suspension culture-derived microcalli were evaluated. RALF has been shown to inhibit growth and
development of tomato and Arabidopsis roots (Pearce et al.
2001a). Recently, Wu et al. (2007) also showed that
NaRALF gene, a RALF homolog from Nicotiana attenuata, is essential for root hair formation. Because leaf cells
are not amenable to peptide treatments, we have tested
SacRALF1 peptide effect on cell suspension cultures. Cell
suspension-derived microcalli grown on media containing
SacRALF1 peptide showed an inhibition of growth, which
correlates to the SacRALF1 concentration that was used
(Fig. 3). After 5 days, control cells showed a noticeable
growth, while SacRALF1-treated cells did not. Inhibition
of growth was observed with concentrations as low as
10-7 M. A closer look into the possible effect of microcalli
direct exposure to exogenous SacRALF1 revealed that the
elongation of cells was arrested or delayed (Fig. 3b; Supplemental Table 1). Cells treated with SacRALF1 peptide
seemed to delay entrance in the expansion and elongation
phases, both characteristics of the early stationary phase of
cell suspension cultures. In addition, the presence of the
RALF peptide caused reduced elongation of hypocotyls of
seedlings grown in darkness, where hypocotyl length is
primarily a reflection of cell elongation rather than cell
division (Fig. 4). A similar effect was observed when
Arabidopsis plants were exposed to exogenous RALF
peptide (Pearce et al. 2001a) and plants overexpressing a
RALF root isoform also showed a semi-dwarf phenotype
(Matos et al. 2008). Wu et al. (2007) generated transgenic
N. attenuata plants silenced for NaRALF gene, and they
observed that their roots grew longer. An elongation zone
significantly longer was also observed in NaRALF silenced
plants, suggesting that NaRALF peptide may have a role in
regulating cell elongation in native tobacco too. N. attenuata with a silenced NaRALF gene had also a significantly higher maximal expansion rate of cells. Curiously,
N. attenuata has only one RALF homolog and the silenced
plants showed normal above-ground parts. One possible
explanation for this event could be the residual levels of
NaRALF mRNA found in silenced plants (10%), hypothetically enough to generate normal aerial parts. More
recently, Srivastava et al. (2009) overexpressed another
RALF isoform, preproRALF23, and obtained shorter and
bushier plants. In the same report, the authors also found
that overexpression of AtRALF23 impairs BL-induced
hypocotyls elongation in seedlings.
In addition to being ubiquitous in the plant kingdom,
RALF transcripts have also been found in all plant tissues
(Pearce et al. 2001a). In our study, SacRALF transcripts
were detected in roots, leaf rolls and expanded leaves
(Fig. 5a–d). All four RALF genes showed a high level of
expression in young tissues, such as roots and expanding
leaves (leaf rolls), when compared to mature tissues such as
fully expanded leaves.
Searching over 245,000 ESTs available in the public
databases, only four SacRALF genes were identified. In
addition, our gene expression analyses showed that only
one transcript, SacRALF1, was dominant. Considering that
sugarcane plants are complex polyploids (Grivet and Arruda 2002), the other three SacRALF genes that showed
very low levels of expression could be paralogous of
SacRALF1 that would have a similar role. In this regard,
sugarcane plants seem to be similar to N. attenuata, which
has only one RALF gene (Wu et al. 2007). Additionally,
they do not follow the model plant Arabidopsis, or poplar
hybrids, or Solanum chacoense that show more than one
copy of RALF gene with similar levels of expression in
their genomes (Olsen et al. 2002; Haruta and Constabel
2003; Germain et al. 2005).
SacRALF transcripts analyses of root tissues showed a
lower expression in the root meristem than in the elongation zone just behind it (Fig. 6). Sugarcane roots show
characteristic division, elongation and maturation zones
(Moore 1987). We hypothesized that if SacRALF genes
were involved in cell division and not cell elongation, a
high level of expression would be restricted to the meristematic zone. However, our results showed that SacRALF
genes are expressed in the elongation zone of roots,
123
280
suggesting a role in cell elongation and not in cell division.
Recently, Haruta et al. (2008) showed a promoter analysis
of an Arabidopsis root specific isoform that demonstrates
lack of expression in the meristematic zone too.
Sugarcane leaves expand in such orderly manner that, at
any given time, during the vegetative growth, the leaf with
the top visible dewlap (leaf ?1) has a fully expanded blade
and an elongating sheath. Leaf ?2 (older than ?1) has both
leaf blade and sheath fully elongated, and leaf zero
(younger than ?1) has both leaf blade and sheath elongating (Moore 1987). Our analyses of SacRALF transcripts
in leaves of different developmental stages (?3, ?2, ?1, 0
and -1) showed a high level of transcripts in young and
elongating leaf blades or sheaths (Fig. 7). RALF genes in
poplar hybrids also showed a high level of expression in
young petioles and young leaves, when compared to old
petioles and old leaves. The same expression trend was
observed for bark and wood tissues in poplar (Haruta and
Constabel 2003). Analyses of RALF gene expression in
reproductive tissues of Solanum chacoense also showed
that ScRALF genes expression declines with fruit maturation (Germain et al. 2005).
We took advantage of the grass unidirectional cell
expansion in leaves to evaluate SacRALF gene expression.
Our rationale is that if SacRALF genes were involved in cell
elongation, its gene expression should be confined into
elongation zones. The expression should also be nearly
absent in plant regions, such as the apex of a grass leaf, where
cells are mature and have already ceased the expansion
process. Our data showed that SacRALF transcripts are
mainly found in young elongating cells, and they are almost
absent in mature differentiated cells (Fig. 8). It is unlikely
that SacRALF genes are involved in cell division in leaves
because we have found SacRALF transcripts beyond the
meristematic region, not only in leaves but also in roots.
SacRALF genes seem to be involved in the regulation of cell
elongation process, since they are not expressed in mature
cells and they show a decreased expression in the middle and
tip portions of leaves.
A developmental role for RALF peptides has been
suggested since its discovery (Pearce et al. 2001a), and
several other authors have provided evidence in support of
that hypothesis (Haruta and Constabel 2003; Olsen et al.
2002; Germain et al. 2005; Wu et al. 2007). The data
presented here show that exogenously applied SacRALF1
inhibited microcalli development and SacRALF1-treated
microcalli did not show typical elongated cells. Our analyses of gene expression localize SacRALF genes in regions
of young elongating cells and in elongation zones of roots
and leaves. Mature leaves that are devoid of elongating
cells do not show considerable expression of SacRALF
genes. As cells are pushed away from the meristem, they
elongate and differentiate gradually. A still unknown
123
Plant Mol Biol (2010) 73:271–281
mechanism controls this gradual cell expansion until cells
assume their final cell size. We are proposing that
SacRALF peptides, and in particular SacRALF1, are
potentially involved in the regulation of cell expansion in
roots and leaves of sugarcane plants. Our data suggest a
scenario where concentrations in the micro molar range
would exacerbate the effect, inhibiting almost completely
the expansion process, similar to the overexpression of the
RALF root isoform in transgenic Arabidopsis plants
(Matos et al. 2008). The levels of expression detected in
young expanding tissues are probably enough to control the
expansion rate and not to inhibit completely. The development of a method to precisely quantify the amount of
RALF peptides would assist in this matter.
Acknowledgments The authors thank Gregory Pearce (Washington
State University, Pullman, WA) for helpful discussions on peptide
purification. Prof. Antonio Figueira (CENA/USP, Piracicaba, Brazil)
for Real-time PCR facilities. Dr. Kathy Stone and Dr. Tom Abbott
from the Yale Cancer Center Mass Spectrometry Resource and W. M.
Keck Foundation Biotechnology Resource Laboratory Keck facility
for LC MS/MS analysis. This research was supported by Fundação de
Amparo a Pesquisa do Estado de São Paulo, FAPESP projects 02/
08661-1, 08/11109-5 and the Bioenergy Program (08/52067-3)BIOEN. F.B.M. was supported by graduate fellowship from CAPES.
J.L.M., A.P.R. and A.H.M. were supported by fellowships from
FAPESP. M.C.S.F. is a research fellow of CNPq. D.S.M. is recipient
of a Young Researcher Grant from FAPESP.
References
Becker JD, Boavida LC, Carneiro J, Haury M, Feijo JA (2003)
Transcriptional profiling of arabidopsis tissues reveals the unique
characteristics of the pollen transcriptome. Plant Physiol 133:
713–725
Cheng J, Seeley KA, Sung ZR (1995) RML7 and RML2, Arabidopsis
genes required for cell proliferation at the root tip. Plant Physiol
107:365–376
Dickison WC (2000) Integrative plant anatomy. Academic Press,
New York
Escobar NM, Haupt S, Thow G, Boevink P, Chapman S, Oparka K
(2003) High-throughput viral expression of cDNA-green fluorescent protein fusions reveals novel subcellular addresses and
identifies unique proteins that interact with plasmodesmata. Plant
Cell 15:1507–1523
Falco MC, Tulmann Neto A, Mendes BMJ, Zapata-Arias FJ (1996)
Isolation and cultivation of sugarcane protoplasts. Braz J Plant
Physiol 8:175–179
Gendreau E, Traas J, Desnos T, Grandjean O, Caboche M, Höfte H
(1997) Cellular basis of hypocotyl growth in Arabidopsis
thaliana. Plant Physiol 114:295–305
Germain H, Chevalier E, Caron S, Matton DP (2005) Characterization
of five RALF-like from Solanun chacoense provides support for
a developmental role in plants. Planta 220:447–454
Grivet L, Arruda P (2002) Sugarcane genomics: depicting the
complex genome of an important tropical crop. Curr Opin Plant
Biol 5:122–127
Haruta M, Constabel CP (2003) Rapid alkalinization factors in poplar
cell cultures. Peptide isolation, cDNA cloning, and differential
Plant Mol Biol (2010) 73:271–281
expression in leaves and methyl jasmonate-treated cells. Plant
Physiol 131:814–823
Haruta M, Monshausen G, Gilroy S, Sussman MR (2008) A
Cytoplasmic Ca2? functional assay for identifying and purifying
endogenous cell signaling peptides in Arabidopsis seedlings:
identification of AtRALF1 peptide. Biochemistry 47:6311–6321
Iskandar HM, Simpson RS, Casu RE, Bonnett GD, Maclean DJ,
Manners JM (2004) Comparison of reference genes for quantitative real-time polymerase chain reaction analysis of gene
expression in sugarcane. Plant Mol Biol Rep 22:325–337
Laemmli UK (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227:680–685
Matos JM, Fiori CS, Silva-Filho MC, Moura DS (2008) A conserved
dibasic site is essential for correct processing of the peptide
hormone AtRALF1 in Arabidopsis thaliana. FEBS Lett 582:
3343–3347
McCubbin AG, Lee C, Hetrick A (2006) Identification of genes
showing differential expression between morphs in developing
flowers of Primula vulgaris. Sex Plant Reprod 19:63–72
Moore PH (1987) Anatomy and morphology. In: Heinz DJ (ed)
Sugarcane improvement through breeding. Elsevier Science,
New York, pp 85–143
Olsen AN, Mundy J, Skriver K (2002) Peptomics, identification of
novel cationic Arabidopsis peptides with conserved sequence
motifs. In Silico Biol 2:441–451
Pearce G, Ryan CA (2003) Systemic signaling in tomato plants for
defense against herbivores. Isolation and characterization of
three novel defense-signaling glycopeptide hormones coded in a
single precursor gene. J Biol Chem 278:30044–30050
Pearce G, Moura DS, Stratmann J, Ryan CA (2001a) RALF, a 5-kDa
ubiquitous polypeptide in plants, arrests root growth and
development. Proc Natl Acad Sci USA 98:12843–12847
Pearce G, Moura DS, Stratmann J, Ryan CA (2001b) Production of
multiple plant hormones from a single polyprotein precursor.
Nature 411:817–820
281
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:2001–2007
Scheer JM, Pearce G, Ryan CA (2005) LeRALF, a plant peptide that
regulates root growth and development, specifically binds to 25
and 120 kDa cell surface membrane proteins of Lycopersicon
peruvianum. Planta 221:667–674
Silverstein KA, Graham MA, VandenBosch KA (2006) Novel
paralogous gene families with potential function in legume
nodules and seeds. Curr Opin Plant Biol 9:142–146
Srivastava R, Liu J, Guo H, Yin Y, Howell SH (2009) Regulation and
processing of a plant peptide hormone, AtRALF23, in Arabidopsis. Plant J 59:930–939
Vettore AL, da Silva FR, Kemper EL, Souza GM, da Silva AM, Ferro
MI, Henrique-Silva F, Giglioti EA, Lemos MV, Coutinho LL,
Nobrega MP, Carrer H, França SC, Bacci Júnior M, Goldman MH,
Gomes SL, Nunes LR, Camargo LE, Siqueira WJ, Van Sluys MA,
Thiemann OH, Kuramae EE, Santelli RV, Marino CL, Targon
ML, Ferro JA, Silveira HC, Marini DC, Lemos EG, MonteiroVitorello CB, Tambor JH, Carraro DM, Roberto PG, Martins VG,
Goldman GH, de Oliveira RC, Truffi D, Colombo CA, Rossi M, de
Araujo PG, Sculaccio SA, Angella A, Lima MM, de Rosa Júnior
VE, Siviero F, Coscrato VE, Machado MA, Grivet L, Di Mauro
SM, Nobrega FG, Menck CF, Braga MD, Telles GP, Cara FA,
Pedrosa G, Meidanis J, Arruda P (2003) Analysis and functional
annotation of an expressed sequence tag collection for tropical
crop sugarcane. Genome Res 13:2725–2735
Weigel D, Glazebrook J (2002) Arabidopsis: a laboratory manual.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Wold F (1981) In vivo chemical modification of proteins (posttranslational modification). Annu Rev Biochem 50:783–814
Wu J, Kurten EL, Monshausen G, Hummel GM, Gilroy S, Baldwin IT
(2007) NaRALF, a peptide signal essential for the regulation of
root hair tip apoplastic pH in Nicotiana attenuata, is required for
root hair development and plant growth in native soils. Plant J
52:877–890
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