Solanum lycopersicum cytokinin response factor (SlCRF) genes: characterization of CRF domain-containing ERF genes in tomato.

Abstract

Cytokinin is an influential hormone in growth and developmental processes across many plant species. While several cytokinin-regulated genes have been well characterized in Arabidopsis, few have been identified in tomato, Solanum lycopersicum. Here a tomato family of 11 highly related cytokinin response factor genes designated as SlCRF1-SlCRF11 (Solanum lycopersicum cytokinin response factor) are identified and characterized. SlCRFs are AP2/ERF transcription factors and generally orthologous to Arabidopsis CRF clade members (AtCRFs). Some SlCRF genes lack a direct Arabidopsis orthologue and one SlCRF has a unique protein domain arrangement not seen in any other CRF protein. Expression analysis of SlCRF1-SlCRF11 revealed differential patterns and levels across plant tissues examined (leaf, stem, root and flower). Several SlCRFs show induction by cytokinin to various degrees, similar to AtCRFs. Additionally it is shown that some SlCRFs can be regulated by other factors, including NaCl, ethylene, methyl jasmonate, and salicylic acid. Examination of SlCRF proteins in transient Agrobacterium infiltration experiments indicates they can be nuclear localized in planta. Using a bimolecular fluorescence complementation (split-yellow fluorescent protein) system, it is also shown that SlCRF proteins can interact to form homo- and heterodimers. Overall this work indicates that some SlCRFs resemble previously identified CRFs in terms of structure, expression, and cytokinin regulation. However, SlCRFs have novel CRF protein forms and responses to abiotic factors, suggesting they may have a diverse set of roles in stress and hormone regulation in tomato.

Full text

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Journal of Experimental Botany, Vol. 63, No. 2, pp. 973–982, 2012
doi:10.1093/jxb/err325 Advance Access publication 7 November, 2011
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER
Solanum lycopersicum cytokinin response factor (SlCRF)
genes: characterization of CRF domain-containing ERF
genes in tomato
Xiuling Shi*, Sarika Gupta* and Aaron M. Rashotte†
Department of Biological Sciences, Auburn University, Auburn, AL 36849, USA
* These authors contributed equally to this work
yTo whom correspondence should be addressed. E-mail: rashotte@auburn.edu
Received 24 May 2010; Revised 13 September 2011; Accepted 16 September 2011
Abstract
Cytokinin is an influential hormone in growth and developmental processes across many plant species. While
several cytokinin-regulated genes have been well characterized in Arabidopsis, few have been identified in tomato,
Solanum lycopersicum. Here a tomato family of 11 highly related cytokinin response factor genes designated as
SlCRF1–SlCRF11 (Solanum lycopersicum cytokinin response factor) are identified and characterized. SlCRFs are
AP2/ERF transcription factors and generally orthologous to Arabidopsis CRF clade members (AtCRFs). Some SlCRF
genes lack a direct Arabidopsis orthologue and one SlCRF has a unique protein domain arrangement not seen in any
other CRF protein. Expression analysis of SlCRF1–SlCRF11 revealed differential patterns and levels across plant
tissues examined (leaf, stem, root and flower). Several SlCRFs show induction by cytokinin to various degrees,
similar to AtCRFs. Additionally it is shown that some SlCRFs can be regulated by other factors, including NaCl,
ethylene, methyl jasmonate, and salicylic acid. Examination of SlCRF proteins in transient Agrobacterium infiltration
experiments indicates they can be nuclear localized in planta. Using a bimolecular fluorescence complementation
(split-yellow fluorescent protein) system, it is also shown that SlCRF proteins can interact to form homo- and
heterodimers. Overall this work indicates that some SlCRFs resemble previously identified CRFs in terms of
structure, expression, and cytokinin regulation. However, SlCRFs have novel CRF protein forms and responses to
abiotic factors, suggesting they may have a diverse set of roles in stress and hormone regulation in tomato.
Key words: CRF, cytokinin, cytokinin response factor, SlCRF, tomato.
Introduction
Cytokinin is an essential plant hormone known to be
involved in numerous plant growth and developmental
processes (Mok and Mok, 2001; Werner and Schmu ¨lling,
2009). Over the last decade, a model of cytokinin signalling
in plants resembling bacterial two-component systems has
become well established (To and Kieber, 2008; Werner and
Schmu ¨lling, 2009). In this model, the binding of a sensor
histidine kinase-like receptor to cytokinin initiates a multi-
step phosphorelay. Upon autophosphorylation, the receptor
transfers the phosphoryl group to a histidine-containing
phosphotransfer protein (HPt), which then transfers the
phosphate to one of two types of response regulators (RRs)
localized in the nucleus. Type-B RRs, transcription factors,
then activate the expression of their target genes mediating
cytokinin-regulated growth and developmental processes or
other aspects of plant life, whereas type-A RRs act as part
of a feedback control loop to regulate this process (To and
Kieber, 2008).
Recently the cytokinin response factors (CRFs) were
identified as several highly related AP2/ERF transcription
factors induced by cytokinin from global expression analy-
ses in Arabidopsis (Hoth et al., 2003; Rashotte et al., 2003;
2006; Brenner et al., 2005; Kiba et al., 2005; Hirose et al.,
2007). CRFs appear to form a branch pathway of the
ª 2011 The Author(s).
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cytokinin signalling pathway and may regulate downstream
cytokinin targets independently or in conjunction with type-
B response regulators (Rashotte et al., 2006; Werner and
Schmu ¨lling, 2009). CRFs form a unique group of ERF
proteins containing a clade-specific CRF domain that is
always accompanied by an AP2/ERF DNA-binding do-
main. Furthermore, CRF domain-containing proteins are
present in all land plants, but not in green algae, indicating
that they may play important roles specific to land plants
(Rashotte and Goertzen, 2010). Mutant analyses in Arabi-
dopsis have implicated CRFs in the development of
cotyledons, leaves, and embryos, as indicated by reduced
size of cotyledons of the crf1,2,5 triple mutant and the
embryo-lethal phenotype of the crf5,6 double mutant
(Rashotte et al., 2006). In general, little is known of the
function of CRFs outside of Arabidopsis, and very few CRF
genes from other species have been examined in any detail.
The genes that have been studied, PTI6/SlCRF1 and TSI1,
are linked to processes other than cytokinin regulation,
including disease resistance and stress responses (Zhou
et al., 1997; Park et al., 2001; Gu et al., 2002). This study
was conducted to completely identify and characterize all
CRF genes in tomato Solanum lycopersicum, which here are
designated as SlCRF genes. Eleven SlCRF genes were
identified through a combination of existing sequence
comparisonandrapidamplification
(RACE)-PCR. Once SlCRF genes were identified, their
expression was examined in different plant tissues, as was
regulation by cytokinin, salt, and other hormones. In
addition, the cellular localization of SlCRF genes in planta
and the ability of SlCRF proteins to form homo- and
heterodimers with each other was determined. Together this
study generates a first complete picture of all CRF genes in
any species, suggesting a broader function for CRF beyond
cytokinin regulation and allowing functional parallels to be
made between related clades of CRFs across species.
ofcDNA ends
Materials and methods
Plant materials and growth conditions
The tomato dwarf cultivar Micro-Tom was used for all experi-
ments. Plants were grown in Sunshine Mix #8 soil under a 16:8 h
light:dark photoperiod at 150 lE, with a 26 ?C day (light), 22 ?C
night (dark) temperature.
RNA isolation, cDNA synthesis, and expression analysis
Leaves, stems, flowers, and roots were harvested from 52-day-old
Micro-Tom plants, and immediately flash-frozen in liquid nitro-
gen. RNA was extracted using a Qiagen RNeasy Kit according to
the manufacturer’s instructions. A 500 ng aliquot of the total
RNA was used for each tissue type in the subsequent reverse
transcription with Qiagen qScript cDNA supermix. The first
strand of cDNA was diluted 10 or 20 times before it was used in
the reverse transcription-PCR (RT-PCR). PCR conditions were
initiated for 2 min at 95 ?C, followed by cycles of 30 s at 94 ?C,
a 30 s annealing step, a 35 s extension at 72 ?C, and a 5 min final
extension at 72 ?C. RT-PCR was conducted for SlCRF1–SlCRF5,
SlCRF11, and TIP41 over 29 cycles with a 56 ?C annealing
temperature step, and for SlCRF6–SlCRF10 over 35 cycles with
a 54 ?C annealing temperature step. The SlCRF-specific primers
used in the RT-PCR are as follows: SlCRF1forward, 5#-GGAAA
ATTCAGTTCCGGTGA-3#; SlCRF1reverse, 5#-AAAATTGG-
TAACGGCGTCAG-3#; SlCRF2 forward, 5#-TGCCGGTCCTA-
GAGTTGTAA-3#; SlCRF2 reverse, 5#-CAGTGGCTGCTCTGC
TCTAT-3#;
SlCRF3forward,
GAACC-3#;
SlCRF3reverse,
CAA-3#; SlCRF4 forward, 5#-TGAATCCCTCTGTTCCAAGG-
3#;
SlCRF4reverse,5#-GTTTTGCCATTTCCACTGCT-3#;
SlCRF5forward,5#-ACGATGACGACGAGAGGAAT-3#;
SlCRF5 reverse, 5#-CTGACACCGCGAAACTTTTT-3#; SlCRF6
forward, 5#-GGTAATGGGAAGAAGCGAGTA-3#; SlCRF6 re-
verse, 5#-GAAGGAAACGTCTGTGGGTAAG-3#; SlCRF7 for-
ward, 5#-GCTTCACGAAAATGAGGTTG-3#; SlCRF7 reverse,
5#-GGTTGATGGGGTCGATTTC-3#;
CCACCAAGGATGAGCTAAAG-3#;
GTGGCACGGTGTTGATGG-3#; SlCRF9 forward, 5#-TGAG-
GAAATGGGGGAAATATG-3#; SlCRF9 reverse, 5#-TGTCAT-
CAAAGCCTAGAAGTT-3#; SlCRF10 forward 5#-TGATGATG
AAGGGGT TGATGTA-3#; SlCRF10 reverse, 5#-TGCTGGA-
GATGTGTGTGAAGTA-3#; SlCRF11 forward, 5#-AAGTGCC
TGAGTTGGCTATG-3#; and SlCRF11 reverse, 5#-TCACCCTC-
GATCAGATAAAC-3#. All samples are compared with the
control gene TIP41 (Expo ´sito-Rodrı ´guez et al., 2008).
SlCRF gene expression in response to hormone or salt
treatment, as described below, was examined using RT-PCR
initiated with 2 min at 95 ?C, followed by 29–40 cycles of 30 s at
94 ?C, 45 s at 57 ?C, and 40 s at 72 ?C, and a 5 min final extension
at 72 ?C. RT-PCR at different cycle lengths was performed for
genes of varying intensities: SlCRF3 (29 cycles), SlCRF1, SlCRF2,
SlCRF4, SlCRF6, SlCRF10, and SlCRF11 (30 cycles), SlCRF5 (30
cycles for salt, 35 for other treatments), SlCRF7 [35 cycles for
methyljasmonate (MeJA), 40 for other treatments), and SlCRF8
and SlCRF9 (40 cycles). Primers used to examine SlCRF3–5 and
TIP41 were as noted above. RT-PCR primers for SlCRF1,
SlCRF2, and SlCRF6–11 are as follows: SlCRF1 forward, 5#-
AACGATGTCGCTTTGTCACC-3#; SlCRF1 reverse, 5#-GGGC
AAAATCGTCAAAGTCA-3#; SlCRF2 forward, 5#-ATGCTGCC
GGTCCTAGAGTT-3#; SlCRF2 reverse, 5#-GAGCAGTTTCCG
ACGATGAC-3#; SlCRF6 forward, 5#-AGATGAGCTTTTTGG
GCGTA-3#;
SlCRF6reverse,
CAC-3#; SlCRF7 forward, 5#-ACGTTGGTTGGGAAGTTTTG-
3#; SlCRF7 reverse, 5#-TAATGGTTGATGGGGTCGAT-3#; Sl
CRF8 forward, 5#-ACGTTGGTTGGGAACTTTTG-3#; SlCRF8
reverse, 5#-GTGTTGATGGGGTTGATTCC-3#; SlCRF9 for-
ward, 5#-GCGTTGCCTAAAGGAGTTAG-3#; SlCRF9 reverse,
5#-ACCAGGGCTCAAATTCTTAC-3#; SlCRF10 forward, 5#-CT
CAGAGTTTGGTCTCACATAC-3#; SlCRF10 reverse, 5#-AACA
TGTCCATCTCCGTATC-3#; SlCRF11 forward, 5#-AAGTGCC
TGAGTTGGCTATG-3#; and SlCRF11 reverse, 5#-TCACCCTC-
GATCAGATAAAC-3#. For characterizing SlCRF7 response to
ethephon and SlCRF8 response to MeJA, primers used are the
same as those utilized for examining the expression in different
organs as noted above.
For quantitative real-time PCR (qRT-PCR) analysis, total RNA
was extracted from cytokinin- or dimethylsulphoxide (DMSO)
control-treated leaves using the same reagents and protocol as
described for RT-PCR. A 500 ng aliquot of total RNA was
converted into cDNA with Qiagen qScript cDNA supermix.
A 2 ll aliquot of a 20-fold cDNA dilution was used for each reaction
in the following qPCR. qPCR was performed with the SYBR-
Green chemistry in a Eppendorf Mastercycler ep realplex with the
same set of primers used for examining salt or hormone responses
except SlCRF1 and SlCRF2. Primers for SlCRF1 and SlCRF2 are
the same as used in the first RT-PCR experiment. Each reaction
contains 9 ll of SYBR-Green supermix, 2 ll of cDNA template,
3 ll of 4 lM primers, and 3 ll of sterile water. The qPCR
program consists of one cycle at 95 ?C, followed by 40 cycles of
15 s at 95 ?C, 30 s at 56 ?C, and 35 s at 68 ?C. The relative
5#-AATGATGCAGTCGAG-
5#-CCTGGTCTTCCCATTCT-
SlCRF8
SlCRF8
forward,
reverse,
5#-
5#-
5#-TCGCTTCTTCCCATTAC-
974 | Shi et al.

Page 3

expression data used in the figure represent means6SE of two
biological replicates. All samples are compared with the control
gene TIP41 (Expo ´sito-Rodrı ´guez et al., 2008).
Hormone and salt treatments
For all hormone and salt (NaCl) treatments, plants were grown as
described above and then leaves or other tissues were excised from
15-day-old Micro-Tom plants, placed in water, and gently shaken
for 2 h prior to treatment. Then treatments or appropriate
controls were added to shaking tissue for various times as
indicated: 5 lM cytokinin (N6-benzyladenine; BA), 100 lM MeJA,
and 2 mM SA (salicylic acid), each with the carrier solvent
DMSO, and 200 mM NaCl and 1 mM Ethephon (of which
ethylene is a breakdown product) with the appropriate level water
controls. After designated treatment times (1 h or 3 h) leaves were
removed from solution, patted dry, and immediately flash-frozen
in liquid nitrogen, and stored at –80 ?C until RNA extraction.
Phylogenetic analysis
Full-length sequences of SlCRF genes were originally identified by
making use of existing sequence data from the four full-length
SlCRF genes (SlCRF1, SlCRF3, SlCRF4, and SlCRF5) that were
previously known either through 3# RACE-PCR analysis of partial
unigene constructs (SlCRF3, SlCRF4, and SlCRF5) or from an
existing gene sequence for SlCRF1, also known as PTI6. BLAST
analysis of the tomato unigene collection and now fully sequenced
tomato genome was conducted using these four SlCRF genes and
additional CRF sequences from other species, primarily Arabidop-
sis, at http://solgenomics.net using publicly available genome
sequence data from the International Tomato Genome Sequencing
Project and from the Kazusa Full-length Tomato cDNA Database
at http://www.pgb.kazusa.or.jp/kaftom. Searches were done pri-
marily using conserved AP2/ERF- or CRF-specific domain regions
of the known SlCRF genes in a manner similar to that done in the
identification of CRF genes in a wide range of plant species
(Rashotte and Goertzen, 2010). Once all full-length SlCRF gene
sequences were found, they were translated and aligned as proteins
in CLC Sequence Viewer v6.5.1 using default parameters.
A phylogenic cladogram was generated using the Neighbor–
Joining method via bootstrap analysis of full-length aligned
SlCRF proteins again in CLC Sequence Viewer v6.5.1 using
default parameters. Arabidopsis genes examined herein are desig-
nated as follows: CRF9 (At1g49120), CRF10 (At1g68550), CRF11
(At3g25890), and CRF12 (At1g25470); and were previously noted
as B-clade members of the CRF genes in Rashotte and Goertzen
(2010), CRF9¼CRF-B1, CRF10¼CRF-B3, CRF11¼CRF-B4, and
CRF12¼CRF-B2.
Protein examination
Vector construction: All plasmids for BiFC (bimolecular fluores-
cence complementation) were generated using the Invitrogen
GATEWAY? cloning system according to the manufacturer’s
instructions. Entry clones for SlCRF1, SlCRF2, SlCRF3, and
SlCRF5 were prepared/generated via a BP reaction using the
pDONR221 and the att-B PCR product containing att-B adaptor
sites and full-length cDNA sequence except the stop codon.
Through an LR reaction, coding sequence was transferred to
destination vectors pSAT4-DEST-n (1–174) EYFP-C1 and pSAT5-
DEST-c (175–end) EYFP-C1 which have N- and C-terminal parts
of the yellow fluorescent protein (YFP) gene, respectively. These
destination clones were later used to transform Micro-Tom proto-
plasts. To examine cellular localization in planta, SlCRF1, SlCRF2,
and SlCRF5 were transferred, through an LR reaction, to the
35S:SlCRF:GFP (green fluorescent protein) constitutive expression
destination vector pMDC84. These destination clones were later
used to transform Agrobacterium tumefaciens that was injected into
tobacco leaves. All destination vectors were obtained through the
ABRC at Ohio State University.
Protoplast isolation and transformation for BiFC analysis
For isolating leaf protoplasts, leaves were taken from 15-day-old
plants, cut into thin strips, and placed in enzyme solution [2%
Cellulase R10, 1% Macerozyme R10, 0.6 M mannitol, 20 mM
KCl, 25 mM MES solution, pH 5.7 which was heated at 55 ?C for
10 min, then cooled down to room temperature before adding
10 mM CaCl2 and 1% bovine serum albumin (BSA)] under
vacuum for 30 min. Next, leaf strips were gently shaken for 4 h or
overnight at 40–60 rpm before increased shaking at 90–100 rpm
for 10 min to release protoplasts. Enzyme solution containing the
protoplasts was filtered with a 40 lm cell sifter into a 50 ml conical
tube and spun at 100 g for 2 min to pellet the protoplasts. Pelleted
protoplasts were resuspended in 2 ml of cold wash solution (0.6 M
mannitol, 5 mM MES pH 5.7, 20 mM KCl, 10 mM CaCl2) and
spun again. Then the pellet was resuspended in wash solution to
obtain the final volume for electroporation and kept on ice until
transformation. Electroporation of protoplasts was performed as
in Rashotte et al. (2006) and then they left undisturbed in the dark
at room temperature overnight prior to microscopic observation.
Agrobacterium infiltration and transformation for in planta
examination of cellular location
Tobacco (Nicotiana tabacum) plants were grown under a long day
16 h light 26 ?C, 8 h dark 22 ?C cycle. Destination vectors used for
transformation (SlCRF genes in pMDC84, as described above) were
transformed into A. tumefaciens (C58-C1) by a method similar to
that used in Rashotte et al. (2006), leading to a floral dip. However,
once properly antibiotic-selected individual colonies were identified,
further grown up in liquid culture, and spun down, they were then
resuspended in infiltration media (10 mM MgCl2, 10 mM MES,
100 lM acetosyringone) and left at room temperature for 3 h similar
to the method of Liu et al. (2002). Agrobacterium was then infiltrated
into the abaxial side of 14- to 21-day-old plant leaves using a needle-
less 2 ml syringe. Plants were then examined for transient
transformation and GFP expression 48–72 h after injection using
epifluorescence microscopy as in Cutcliffe et al. (2011).
Epifluorescence microscopy
BiFC and Agrobacterium-infiltrated tobacco leaves were examined
using a Nikon Eclipse 80i epifluorescence microscope with a UV
source in transformed protoplast. A standard UV filter was used in
addition to 1 ng ml?1of Hoechst 33342 dye initially to observe and
identify nuclei in intact cells as a measure of the cell viability. A
YFP filter that blocks both chlorophyll fluorescence and Hoechst
33342 fluorescence was used to examine the localization of any split-
YFP fusions that occur due to BiFC between proteins. Cytokinin
(2 lM BA) was routinely added to protoplasts prior to examina-
tion. A GFP filter that blocks both chlorophyll fluorescence and
Hoechst 33342 fluorescence was used to examine cellular localiza-
tion of any cells expressing GFP in Agrobacterium-infiltrated
tobacco leaves. All photos were taken with a Qimaging Fast 1394
digital camera and are presented as composite images using Adobe
Photoshop CS3 without altering the original integrity of the picture.
Results
Identification of novel tomato CRF genes (SlCRF genes)
A family of 11 CRF genes from tomato, known as Solanum
lycopersicum cytokinin response factor genes or SlCRF1–
SlCRF11, have been identified and characterized (Fig. 1,
Table 1; Supplementary Table S1 available at JXB online)
Cytokinin response factors in tomato | 975

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These genes are members of the AP2/ERF transcription
factor family, specifically related to clade VI and VI-L of
the ERF subfamily of genes, known in Arabidopsis as
AtCRF genes (Sakuma et al., 2002; Nakano et al., 2006;
Rashotte and Goertzen, 2010). These genes were identified
from a combination of BLAST searches of emerging
tomato genome sequence resources using previously identi-
fied CRF genes in tomato, orthologous AtCRF sequences,
and 3’ RACE of incomplete expressed sequence tag (EST)
unigene builds of SlCRF genes. Previous work identified
transcription of four SlCRF sequences (SlCRF1, SlCRF3,
SlCRF4, and SlCRF5), including the existing PTI6 gene,
that has also been designated as SlCRF1 (Rashotte and
Goertzen, 2010). From this base, 10 novel full-length
expressed CRFs (SlCRF2–SlCRF11) have been identified,
comprising all proteins in tomato containing a CRF do-
main, a defining characteristic of CRF proteins (Fig. 1,
Table 1; Supplementary Table S1). In several cases 3#
RACE was used to generate full-length gene transcripts from
assembled unigenes lacking a 3# end region. Subsequent
genomeassemblageandsequenced
chromosome (BAC) contigs have verified the determined
bacterial artificial
sequence identified from 3# RACE experiments. Full-length
transcripts for SlCRF1–SlCRF11 are presented (Supplemen-
tary Table S1). SlCRFs at a protein level fall into three
classifications (Fig. 1A). One is a standard CRF protein
(SlCRF1,
SlCRF2,
SlCRF4–SlCRF6,
SlCRF11), which contains both a CRF and AP2 DNA-
binding domain in addition to a putative mitogen-activated
protein kinase (MAPK) phosphorylation motif, as seen in
a wide range of plant species (Rashotte and Goertzen, 2010).
The second is a shortened CRF protein (SlCRF7 and
SlCRF8), which contains the CRF and AP2 DNA-binding
domain, but lacks the 3’ third of the protein and the
phosphorylation motif, as is also seen in other species such
as Arabidopsis (CRF7 and CRF8). The final classification is
a unique CRF protein (SlCRF3), containing two CRF and
AP2 DNA-binding domains in an alternating pattern. This is
the only known CRF protein that contains more than a single
CRF domain and is expressed, from >250 identified CRF
proteins examined across all land plants. Interestingly its
chromosomal position is very close to the highly related
SlCRF8, only 9125 bp away, suggesting a possible gene
duplication event (Table 1).
and
SlCRF9–
Fig. 1. SlCRF protein form, alignment, and phylogenic relationships. (A) A model of SlCRF protein form including size, domains, and
motifs for all 11 SlCRFs. (B) Protein sequence alignment of the CRF domain for SlCRF1– SlCRF11 is shown with a sequence consensus,
including both SlCRF3 CRF domains. (C) Neighbor–Joining tree of SlCRF proteins based on alignment of the CRF domain with support
values shown out of 1000 bootstrap replicates. (D) Neighbor–Joining tree of SlCRF and Arabidopsis CRF (AtCRF) proteins based on
alignment of both the CRF and AP2 DNA-binding domains with support values shown out of 1000 bootstrap replicates.
976 | Shi et al.

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Alignment of these proteins revealed high similarity in
domain regions, such as the core conserved
DPDATDSSSD of the CRF domain (Fig. 1B), similar to
that seen in previous alignments of CRF proteins from
a wide range of land plants (Rashotte and Goertzen, 2010).
For ease of alignment and phylogenic analyses in this study,
the full-length SlCRF3 was split into N- and C-terminal
parts each containing a CRF and AP2 domain, although
a full-length version yielded similar results (data not
shown). Phylogenetic analysis based on similar domain
sequences indicates that some SlCRFs have a paired re-
lationship, suggesting an ancient duplication, as well as
most SlCRFs having an Arabidopsis orthologue (Fig. 1C;
D). Tomato and Arabidopsis do not have directly ortholo-
gous phylogenetic protein pairs since, in some cases, a single
SlCRF protein is grouped with two Arabidopsis proteins
(SlCRF2 with AtCRF1 and AtCRF2; SlCRF5 with
AtCRF5 and AtCRF6). Additionally, SlCRF1 has no
orthologous Arabidopsis gene partner (Fig. 1D), although it
is part of a related subclade of CRF proteins found in
a number of other species (Rashotte and Goertzen, 2010).
region
SlCRF genes are expressed in different plant tissues
Previous work identified four SlCRF genes (SlCRF1,
SlCRF3, SlCRF4, and SlCRF5) as expressed in leaf tissues
(Rashotte and Goertzen, 2010). Here it is shown that
SlCRF3– SlCRF11 are expressed in multiple different plant
tissues throughout the plant (leaf, stem, root, and flowers)
to varying degrees (Fig. 2). Generally, SlCRF expression
levels were consistent across plant tissues examined. How-
ever, some genes showed preferential tissue expression, as
seen for roots in SlCRF4 and SlCRF5 and for stems in
SlCRF8 and SlCRF11 (Fig. 2).
SlCRF transcript levels are regulated by cytokinin and
salt
Knowing that several CRFs in Arabidopsis have previously
been shown to be induced by cytokinin, the regulation of
SlCRF genes by cytokinin was examined. Tomato leaves
(15 d old) were treated with cytokinin (5 lM BA) or
DMSO as a vehicle control for 1 h and 3 h and examined
using real-time PCR. Three SlCRF genes (SlCRF2, SlCRF3,
and SlCRF5) were found that are strongly (4- to 6-fold)
induced by cytokinin (Fig. 3A). SlCRF2 showed rapid
induction by cytokinin at 1 h after treatment to 6-fold over
untreated levels and by 3 h was still induced, although at
this point only ;3.5-fold over control levels. Both SlCRF3
and SlCRF5 showed no induction at 1 h, but were highly
induced (4- to 5-fold) after 3 h of cytokinin treatment.
A few other SlCRF genes showed weaker levels (1.5- to 2-fold)
of induction at 3 h of cytokinin treatment (SlCRF1,
SlCRF6, SlCRF7, SlCRF8, and SlCRF9), whereas SlCRF4,
SlCRF10, and SlCRF11 showed no change in expression
(Fig. 3A). The results follow a pattern similar to that seen
for AtCRF genes whereby some, but not all, members of
this group are transcriptionally regulated by cytokinin
(Rashotte et al., 2006).
SlCRF genes were also examined for changes in response
to salt and other hormones in leaves treated at 1 h and 3 h
versus controls using RT-PCR. The results revealed expres-
sion changes in several genes, although many showed little
to no alterations (Fig. 3). Expression analysis of salt
treatment (200 mM NaCl) revealed induction of SlCRF1,
SlCRF4, and SlCRF6 at both 1 h and 3 h as well as a minor
induction of SlCRF2, SlCRF5, and SlCRF7 at 3 h (Fig.
3B). This suggests a new potential role for SlCRF genes in
stress regulation. Expression analysis of ethylene treatment
Fig. 2. SlCRF expression patterns in various tomato tissues. RT-
PCR analysis of SlCRF1– SlCRF11 in leaf, stem, root, and flower
tissues of 52-day-old plants is shown. The TIP41 gene serves as
an internal control.
Table 1. SlCRF gene description
Gene nameChromosome/position
(Build 2.40)
Gene model Size (amino
acids/bp)
SlCRF1/PTI6
SlCRF2
SlCRF3
SlCRF4
SlCRF5
SlCRF6
SlCRF7
SlCRF8
SlCRF9
SlCRF10
SlCRF11
Ch 6 (44654446–44653700)
Ch 8 (62045738–62046757)
Ch 1 (2911579–2910313)
Ch 3 (2016125–2014935)
Ch 1 (78502891–78503773)
Ch 6 (32043471–32044523)
Ch 1 (14595809–14596333)
Ch 1 (2901188–2900649)
Ch 3 (62191449–62190256)
Ch 5 (3622457–3621438)
Ch 4 (874453–875505)
Solyc06g082590
Solyc08g081960
Solyc01g008890
Solyc03g007460
Solyc01g095500
Solyc06g051840
Solyc01g014720
Solyc01g008880
Solyc03g119580
Solyc05g009450
Solyc04g007180
248/747
340/1023
344/1035
396/1191
293/882
350/1053
174/525
175/540
397/1194
339/1020
350/1053
Cytokinin response factors in tomato | 977