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Plant Signaling & Behavior
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In silico analysis of 3 expansin gene promoters reveals 2 hubs controlling light and cytokinins response during bud outgrowth
Hanaé Roman, Tiffanie Girault, José Le Gourrierec & Nathalie Leduc
To cite this article: Hanaé Roman, Tiffanie Girault, José Le Gourrierec & Nathalie Leduc (2017) In silico analysis of 3 expansin gene promoters reveals 2 hubs controlling light and cytokinins response during bud outgrowth, Plant Signaling & Behavior, 12:2, e1284725, DOI:
10.1080/15592324.2017.1284725
To link to this article: http://dx.doi.org/10.1080/15592324.2017.1284725
Accepted author version posted online: 13 Feb 2017.
Published online: 13 Feb 2017.
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ARTICLE ADDENDUM
In silico analysis of 3 expansin gene promoters reveals 2 hubs controlling light and cytokinins response during bud outgrowth
Hana e Roman , Tiffanie Girault, Jos e Le Gourrierec , and Nathalie Leduc
IRHS, Universite d’Angers, INRA, AGROCAMPUS-Ouest, SFR 4207 QUASAV, Beaucouze cedex, FranceARTICLE HISTORY Received 4 January 2017 Revised 10 January 2017 Accepted 16 January 2017 ABSTRACT
Bud outgrowth is under the intricate control of environmental and endogenous factors. In a recent paper,1 we demonstrated that light perceived byRosabuds triggers cytokinins (CK) synthesis within 3 hours in the adjacent node followed by their transport to the bud. There, CK control expression of a set of major genes (strigolactones-, auxin-, sugar sink strength-, cells division and elongation-related genes) leading to bud outgrowth in light. Conversely, under dark condition, CK accumulation and transport to the bud are repressed and no bud outgrowth occurs. In this paper, we show that the 3 expansin genesRhEXPA1,2,3are under the control of both light and CK during bud outgrowth.In silicoanalysis of promoter sequences highlights 2 regions enriched in light and CKcis-regulatory elements as well as a specific cis-element in pRhEXPA3, potentially responsible for the expression patterns observed in response to CK and light.
KEYWORDS
Abiotic factor; branching; cell elongation; EXPA; hormones;
Rosa; signaling
In rose, our previous
findings showed that darkness represses leafgrowth and meristem organogenic activity in buds leading to full inhibition of bud outgrowth.
2Our molecular analysis revealed that light acts in buds through transcriptional control of genes involved in major mechanisms of bud outgrowth (strigolac- tones-, auxin-, gibberellins-, sugar sink strength-, cells division and elongation-related genes).
1,3-8Recently, we demonstrated that cytokinins (CK) are early targets of the light signal perceived by buds.
1Upon light perception by buds, CK synthesis in adja- cent node and transport toward the buds is triggered within 3 hours where they control several bud outgrowth genes.
To get a more precise insight in the role of light and CK on organogenesis during bud outgrowth, we further studied the response of cell elongation to both these actors. Exposure of rose plants to white light (WL) as described in
1causes sus- tained and significant elongation of the 3 outermost bud’s young leaves after decapitation (Fig. 1A). Observations using electronic microscopy 7 d after decapitation show that WL pro- motes leaves elongation through increasing cell elongation (Fig. 1B). This confirms our molecular data
1showing that light exerts a strong control on expansin gene RhEXPA3 (named RhEXP in
1) involved in cell elongation during bud outgrowth.
In Rosa, 3 expansins genes were described
9: RhEXPA1 (AB370116), RhEXPA2 (AB370117) and RhEXPA3 (AB370118).
To further study the impact of light and CK on these genes, their expressions in bud were studied from 3 h to 48 h of light treatment (WL vs Darkness) and of CK treatments (mock under WL and 10 mM of Benzylaminopurine (BAP) or mock under Darkness) as described in.
1Expression of the 3 genes RhEXPA1, RhEXPA2 and RhEXPA3 are all promoted by WL but different patterns of response were obtained (Fig. 2A). For example, strong promotion
of RhEXPA3 was observed at 24 h but only later for RhEXPA1 (48 h). No such temporal regulation occurred for RhEXPA2 for which a constant level of expression was measured under WL throughout the experiment. Concerning CK effect, expression of all 3 genes was promoted by CK supply under darkness. RhEXPA1 was earlier promoted (3 h) than the 2 other expansin genes. After 3 h, little difference was then observed between the levels of expres- sion of the 3 genes (Fig. 2B). The differences in the response pat- terns of the 3 expansin genes to light and CK could re
flect a differential regulation at the promoter level by these 2 factors. We therefore sequenced their promoters to identify cis-acting elements involved in light and/or CK regulations.
Based on the similarity with Fragaria vesca (genome data- base on
www.rosaceae.org10), 1066 bp, 959 bp and 1069 bp fragments of the RhEXPA1/2/3 promoters were respectively isolated. In silico analysis performed with PlantPan 2.0 program (http://plantpan2.itps.ncku.edu.tw/index.html
11), identified putative cis-elements involved in the regulation by light and CK (Table 1).
Many common light-responsive elements were identified between the 3 promoters as previously observed in tobacco expan- sin promoters.
22The high occurrence of these motifs (40/33/26) is consistent with the high transcript levels of the 3 genes in response to light treatment (Fig. 2A). Motifs are evenly spread between the plus (17/18/14) and minus (23/15/12) strands of pRhEXPA1,2,3.
Interestingly, E-box (also called PBE for PIF-binding E-box
23) but no G-box were found in rose expansin promoters (Table 1). The opposite situation was reported for tobacco and strawberry expan- sin promoters.
22,24Additionally, the promoter regions of RhEXPA1,2,3 harbored a total of 17, 9 and 10 CK responsive ele- ments respectively, in agreement with increased expansin
CONTACT Nathalie Leduc nathalie.leduc@univ-angers.fr IRHS, Campus du Vegetal, 42 rue Georges Morel, 49071 Beaucouze, France.
Addendum to: Roman H, Girault T, Barbier F, Peron T, Brouard N, Pencık A, Novak O, Vian A, Sakr S, Lothier J, et al. Cytokinins are initial targets of light in the control of bud outgrowth. Plant Physiol 2016; 172:489-509; PMID:274620851284725; http://dx.doi.org/10.1104/pp.16.00530.
© 2017 Taylor & Francis Group, LLC
http://dx.doi.org/10.1080/15592324.2017.1284725
expression genes in response to CK treatment in darkness (Fig. 2B).
Beside the most abundant cis-element GARP-ARRB (14, 7 and 9 copies in pRhEXPA1,2,3), CBPCSPOR
19and 3 different octameric sequence motifs (TATATATA, CATATATA, TATATTTA) recently found enriched in cytokinin-responsive promoters
21were also identified. Finally, we found a total of 7, 4 and 3 GATA-box cis-elements reported to be involved in light-CK crosstalk,
12,13sup- porting evidence to the combined and integrated roles of these 2 factors in RhEXPA1,2,3 regulation (Table 1).
Cytokinin- and light-responsive elements reveal equally distributed between proximal and distal regions in the 3 promoters. Comparative promoter analysis also highlights 2 similar light-CK motifs-enriched-regions (gray zones in
Fig. 3). The first one is located between
¡600 and
¡800 bp and found in RhEXPA1,3 promoters only. It displays in a same arrangement the same types of LREs (Box-4, I-box, E- boxes, GT-elements), CK-responsive elements (CBPCSPOR, GARP-ARRB) together with a GATA-box predicted to be involved in light-CK crosstalk (Fig. 3 and
Table 1). Interest-ingly, this zone also exhibits several major shoot-meristem specific elements (LFY
25and WUS
26), in line with the expression and the biologic role of expansin genes in leaf primordia expansion and meristem organogenesis during bud outgrowth
2(Fig. 3). The second shared region is found in all 3 promoters and presents GT-elements, GARP-ARRB and E-boxes. As suggested for tobacco pNtEXP,
22these 2 regions may act as hub recruiting different light- and CK- signaling actors that would work in a synergistic manner to promote strong response as observed in
Fig. 2.Of interest, are also speci
fic regions/elements which may explain the different temporal expression patterns of the 3 genes in response to light. For example, 3 FHY3/FAR1 binding sites of transcription factors involved in phyA sig- naling were only found in RhEXPA3 promoter (Fig. 3). Fur- ther investigations on this element would be valuable to assess its functional role since fhy3 mutants show repressed axillary bud outgrowth.
27In conclusion, this study reveals several putative shared but also speci
fic light- and CK- responsive elements in rose
Figure 1.Effect of light condition on the elongation of thefirst leaf primordia in the axillary buds ofRosa hybrida‘Radrazz’. (A) Lengths of the 3 outermost bud leaves (F1, F2 and F3) just after decapitation (Tdecap), followed by 24 h of darkness (T0) and 24 h to 72 h exposure to white light or darkness. (B) Epidermal cells of F1 leaf from buds of plants grown 7 d under white light or darkness. Cell lengths from the apical zone (2) and from the basal zone (1) of the leaves were measured.
White bars represent 10mm. Data are means§standard errors, with nD7 to 11 F1 leaves. Asterisks show significant differences between light and dark conditions for one timing (p<0.001).
Figure 2.Expression of expansin genes in bud from 3 to 48 h after T0 under WL or dark treatment (A) or following bud treatment with BAP (10 mM) under darkness (B).
Changes in transcript levels are indicated by color codes. Green indicates promotion of gene expression by WL (in A) or by BAP treatment (in B). The color log scales are included. Same biologic samples and analysis as in1were used.
e1284725-2 H. ROMAN ET AL.
expansin promoters. Biological relevance of these regions will be further determined through deletion of promoter fragments and fusion to reporter gene followed by transient expression assay.
Disclosure of potential con fl icts of interest
No potential conflicts of interest were disclosed.
Acknowledgments
We thank Angers Loire Metropole and the Ministere de l’Enseignement superieur for thefinancial support of the PhD grant of HR and of TG; the platform SCIAM of Angers University; Benedicte Dubuc and Nathalie Brouard for technical assistance; and IRHS INEM platform for rose cutting production and plant care.
ORCID
Hanae Roman http://orcid.org/0000-0002-8475-2976 Jose Le Gourrierec http://orcid.org/0000-0002-6200-0116 Nathalie Leduc http://orcid.org/0000-0002-5323-0892
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Table 1.Light- and cytokinin-responsivecis-elements found in the promoter sequences ofRhEXPA1, RhEXPA2, RhEXPA3.
RhEXPA1 RhEXPA2 RhEXPA3
Fonction Cis-element name Sequence Reference C ¡ C ¡ C ¡
Light and CK responsive element GATA-box WGATAR 12,13 1 6 2 1 1 3
Light responsive element FAR1/FHY3 CACGCG 14 0 0 0 0 2 1
E-Box CANNTG 15 8 8 8 8 3 3
I-Box GATAA 16 1 5 2 1 1 2
Box-4 ATTAAT 17 1 1 2 2 1 1
GT-element GRWAAW 18 7 9 6 4 7 5
Total 17 23 18 15 14 12
CK responsive element CBPCSPOR TATTAG 19 0 0 1 0 0 1
GARP-ARRB RGATY 20 8 6 3 4 4 5
TATATATA 1 1 0 0 0 0
CATATATA 21 0 0 1 0 0 0
TATATTTA 1 0 0 0 0 0
Total 10 7 5 4 4 6
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