Plant Cell Rep (2015) 34:1561–1568 DOI 10.1007/s00299-015-1808-7
ORIGINAL PAPER
Functional characterization of PhGR and PhGRL1 during flower senescence in the petunia Weiyuan Yang1,2 • Juanxu Liu1 • Yinyan Tan1 • Shan Zhong1 • Na Tang1 Guoju Chen2 • Yixun Yu1,2
•
Received: 3 April 2015 / Revised: 5 May 2015 / Accepted: 11 May 2015 / Published online: 19 May 2015 Ó Springer-Verlag Berlin Heidelberg 2015
Abstract Key message Petunia PhGRL1 suppression accelerated flower senescence and increased the expression of the genes downstream of ethylene signaling, whereas PhGR suppression did not. Abstract Ethylene plays an important role in flowers senescence. Homologous proteins Green-Ripe and Reversion to Ethylene sensitivity1 are positive regulators of ethylene responses in tomato and Arabidopsis, respectively. The petunia flower has served as a model for the study of ethylene response during senescence. In this study, petunia PhGR and PhGRL1 expression was analyzed in different organs, throughout floral senescence, and after exogenous ethylene treatment; and the roles of PhGR and PhGRL1 during petunia flower senescence were investigated. PhGRL1 suppression mediated by
Communicated by M. C. Jordan. W. Yang and J. Liu contributed equally to this work.
virus-induced gene silencing accelerated flower senescence and increased ethylene production; however, the suppression of PhGR did not. Taken together, these data suggest that PhGRL1 is involved in negative regulation of flower senescence, possibly via ethylene production inhibition and consequently reduced ethylene signaling activation. Keywords Petunia GR-Like Ethylene response Flower senescence RTE1
Introduction The plant hormone ethylene controls various processes in the plant life cycle, including seed germination, fruit ripening, leaf and flower senescence, and a multitude of biotic and abiotic stresses (Abeles et al. 1992). Studies on Arabidopsis thaliana have revealed a universally conserved set of components in the ethylene signaling pathway that are present in higher plants. Ethylene is detected by members of the ethylene receptor family, which are
Electronic supplementary material The online version of this article (doi:10.1007/s00299-015-1808-7) contains supplementary material, which is available to authorized users. & Yixun Yu [email protected]
Na Tang [email protected]
Weiyuan Yang [email protected] Juanxu Liu [email protected]
Guoju Chen [email protected] 1
Guangdong Key Laboratory for Innovative Development and Utilization of Forest Plant Germplasm, College of Forestry and Landscape Architecture, South China Agricultural University, Guangzhou 510642, China
2
College of Horticulture, South China Agricultural University, Guangzhou 510642, China
Yinyan Tan [email protected] Shan Zhong [email protected]
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negative regulators of the ethylene response (Chang et al. 1993; Hua and Meyerowitz 1998; Qu et al. 2007). In the absence of ethylene, the receptors are in an active state and constitutively activate CONSTITUTIVE TRIPLE RESPONSE1 (CTR1), which acts as a negative regulator in the pathway. CTR1 phosphorylates the central regulator ETHYLENE-INSENSITIVE2 (EIN2), a member of the N-Ramp family of metal-transporters, to control ethylene hormone signaling from the endoplasmic reticulum (ER) membrane to the nucleus (Ju et al. 2012). Under-phosphorylated EIN2 is processed for proteolytic cleavage and triggers its endoplasmic reticulum (ER)–to–nucleus translocation (Qiao et al. 2012). The carboxyl end of EIN2 (CEND) is a trafficking signal for translocation from the ER membrane to the nucleus and ethylene signaling is transmitted to the EIN3 family of transcription factors (Chao et al. 1997; Wen et al. 2012; Ji and Guo 2013). EIN3/EIL proteins activate the transcription of ETHYLENE RESPONSE FACTORs (ERFs), another type of transcription factor that regulates the expression of genes involved in the ethylene response (Solano et al. 1998; Potuschak et al. 2003; Xiao et al. 2013). The membrane protein GREEN RIPE (GR)/REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1) negatively regulates the ethylene response (Resnick et al. 2006; Barry and Giovannoni 2006). In Arabidopsis, RTE1 interacts with ethylene resistant1 (ETR1), and they co-localize in the ER (Dong et al. 2008; 2010). A newly published study suggested that cytochrome b5 interacts with RTE1 in Arabidopsis (Chang et al. 2014). In most eudicots, the RTE family includes two to three members (Ma et al. 2012; Tan et al. 2014). The petunia flower has served as a model for the study of ethylene biosynthesis and signaling during senescence (Underwood et al. 2005). Several petunia genes involved in ethylene biosynthesis and signal transduction have been identified, e.g., PhACO1, PhETR1, PhEIN2, PhEIN3/EIL and PhERFs (Chen et al. 2004; Shibuya et al. 2004; Liu et al. 2011). In addition, three fulllength GR-like cDNAs, PhGR (accession no. JQ659030), PhGRL1 (accession no. JQ659031) and PhGRL2 (accession no. KJ451475), have been cloned (Ma et al. 2012). Recently, our study suggested that the PhGRL2 protein interacts with PhACO1 and is involved in flower senescence in petunia (Tan et al. 2014), but the function of the other two members of GR family, PhGR and PhGRL1, is unknown. In this study, the roles of PhGR and PhGRL1 during petunia flower senescence were investigated. VIGS-mediated suppression of PhGRL1 accelerated flower senescence and increased the ethylene production; however, the suppression of PhGR did not. Taken together, these data
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suggest that PhGRL1 is involved in negative regulation of flower senescence in petunia.
Results Phylogenetic analysis of GR-like proteins Previous reports showed that GR-like proteins contain two conserved regions, CR1 and CR2, and two putative transmembrane domains close to the C-terminus, TM1 and TM2 (Dong et al. 2008; Zhang et al. 2012). The phylogenetic tree generated with GR-like sequences showed that the GR/RTE family of dicot species includes two groups, the RTE group and the RTH group (Tan et al. 2014). Based on the consensus regions mentioned above, we further classified the GR-like proteins of Arabidopsis, tomato, petunia, and carnation, Prunus persica and Vitis vinifera into two sub-types as shown in Fig. 1: (1) RTE group proteins with two small conserved motifs, the (L/ F)WPL motif in the non-conserved region NCR1 of the N-terminus, the (G/R/S) GSM motif in the conserved region CR2 of the C-terminus, and a conserved region, CR3, in the non-conserved region NCR2 (AtRTE1, SlGR, SlGRl1, PhGR, PhGRL1, DcRTE1, PpGRL1, and VvGRL1); (2) RTH group proteins, which lack the (L/ F)WPL motif, (G/R/S)GSM motif and CR3 conserved region (AtRTH, SlGRL2, PhGRL2, DcRTH1, PpGRL2, and VvGRL2). Petunia PhGR and PhGRL1 expression in different organs and throughout floral senescence The expression patterns of a particular gene in different plant organs often provide important clues regarding the physiological function of the gene. To assist with the determination of the function of PhGR and PhGRL1 in ethylene-regulated developmental processes such as petunia flower senescence, the expression patterns of PhGR and PhGRL1 were examined in different plant organs and at various stages of flower senescence by quantitative realtime PCR (qRT-PCR). The expression of PhGR was high in leaves and was moderate in corollas and ovaries; however, the transcription levels of PhGRL1 were high in corollas and very low in roots and stems (Fig. 2a). During natural flower senescence, the PhGR transcriptional level remained almost unchanged before day 4, increased rapidly from days 4 to 5 and then gradually decreased. The PhGRL1 mRNA levels decreased from days 0 to 3, remained unchanged from days 3 to 5, and peaked on day 6 (Fig. 2b).
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NCR1
A
CR1 (L/F)WPL
MSRGRGVPMMDLKRSYDVEDRVVSVSIPSIIEADEADLWPLPEIDTKKSKFPCCIVWTPLPVVSWLAPFIGHIGLCREDGVILDFA MLPRR.YPQMDANPSNGGER.......DNALRGILQDLWPLDEIDPSTQKFPCCLVWTPLPVISWLAPFVGHVGICREDGTIVDFS MPSGR.RSLMDLEPAYKMES.......IRAVQDIQHEFWPLDEINGENAKFPCCLVWTPLPVVSWLAPFIGHVGICREDGSAVAFS MLPRR.LPMMEVDGRYVIDR.......SRALRVIRDDLWPLEEIDPTKEKFPCCLVWTPLPVVSWLAPFIGHVGICREDGTIIDFS .........MSTN...............DEAEFLQGELWPLVDVDPKKARFPCCLVWTPLPVVAWLAPFIGHFGICQEDGSILDFS .........MDLEPAYKVES.......IRTVQDIQHDFWPLDEIDPGNAKFPCCLVWTPLPVVSWLAPFIGHVGICTEDGNAVGFS MQHIR.FPVMEVKEAYDVEH.......MRSTQSIQHELWPLDEIDPKKAKFPCCLVWTPLPIVSWLAPFIGHVGICREDGAILDFA MSPRR.VPKMELNAINDVED.......MSTARRTQHELWPLDEIDPKKAKLPCCLVWTPLPVVSWLAPFIGHVGICREDGAILDFA ........MGETATDSE...........HRMMIG...LSDPMKIDPKRDRFPCCIVWTPLPFISWLVPFIGHVGICREDGVILDFA .........MVPEVDAD...........HALMIEE.NYPNPMLIDPKRDRFPCCIVWSPLPVLSWFIPFIGHIGICREDGVILDFA .........MVPEVEPD...........HALMIEE.DSRDTMQIDPKGDRFPCCIVWSPLPVLSWFLPFIGHIGICRQDGVILDFA .........METNVDIE...........EQSVISDRPVETTMHIDPERGRFPFLFVWAPLPVLTWLIPFIGHIGICREDGVILCFT .........METSASPE...........HNLMIDG.SASPTLQIDPRRARFPCCIVWTPLPVISWLIPFIGHIGICREDGVILDFA .........MDSNADPE...........DHMMIER.SVSQMMQIDPRRARFPCCIVWTPLPVISWLIPFVGHIGICREDGVILDFA
NCR2
GSNFINVDDFAFGPPARYLQLDRT.KCCLPPNMGGHTCKYGFKHTDFG...TARTWDNALSSSTRSFEHKTYNIFTCNCHSFVANC GDSMIHFGQLFYGTVAKYYQVDRQ.QCCFARNFGGHTCRKGYEHVVFG...TAVSWDDAVQLFRRTFENRNFKVFSCNGHSFAADC GSNFINIDDFALGSVAKYLQLDRK.QCCFPRNLAAHTCKHGYKHTEFG...SAITWDDAIQSSVRHFEHKSYNIFTCNSYSFVANC GSNMINVGNLTYGAVARYYQLDRL.QCCFPPHLAGHTCKDGYQHAEFG...TAVNWDDALRSSTCSYEHRSFNPFTCNGHSFVANC GSNLVSIDDFAFGSVIRFFISSHLLKCCFPPNLSAHSCKHGYKHHEYG...TALSWDDALHTCKHEYETKTYNLFTCNCHSFVANC GSNFINVDDFAFGSVAKYLQLDRK.QCYFPRNLAAHTCKHGYKHTEFG...SAITWDDAIQSSIRHFEHKSYNLFTCNSYSFLAHC GSNFVNADDFAFGAVARYLQLDRT.QCCFAANLGGHTCKSGYKHAEFG...TAITWDDALQSSSRHFEHKTYNLFTCNCHSFVANC GSNFVNVNDFAFGAVARYLQLDRE.KCCFPPNLAGHTCKNGYKHAENG...TAVTWDDALLSSSHHFGHKSYNLFTCNCHSFVANC GPNFVCVDNFAFGAVSRYIQINKE.MESSRSSSSGMFNGERRYEQEEDSHEKEPTWDDALRKSTQEYQHHSYNILTCNCHSFVANN GPNFVSVDNFTFGAPTCYFQLSRE.QCCCLSPYSAEPTGEYVENHD.ESGGNVDTWESAIRKSIQEFQHQSYSIFTCNCHSFVANG GPNFVSVDNFTFGAPTRYIEISKE.QCCCISPYPAEHTSEYVQNHDDESGRNVDTWDAALRKSIQEFQHQSYSIFTCNCHSFVANG TSYFVFVDDFGYFSVTRYLQIDEK.LCRAISSLPSDKNEERHQNSE...ENKIVSWDHGLQKSILEYQHHSYNLLTCNCHSFVANS GPNFVCVDNFAFGAATRYIQISKE.KCHSIPN.PSVCQSEDQYRQD.EPGRDIMTWDDALRKGTQEFQHRSYNILTCNCHSFVANN GPNFVCVDNFAFGAVTRYIQISKE.KCCISPHHPAPYRRENGRGQD.ETEIDILTWDDALRKSTQEFQHQSYNLFTCNCHSFVANN
CR2 AtRTE1 SlGR SlGRL1 PhGR DcRTE1 PhGRL1 PpGRL1 VvGRL1 AtRTH SlGRL2 PhGRL2 DcRTH1 PpGRL2 VvGRL2
CR2
CR3
TM1
(G/R/S)GSM
TM2
LNRLCYGGSMEWNMVNVAILLMIKGKWINGSSVVRSFLPCAVVTSLG...VVLVGWPFLIGLSSFSLLLFAWFIIATYCFKNIIT LNLLSFRGSMRWNMINVGALIMFEGKWVSRWSMLRSFLPFIGILCFG...YLMIGWMFPIGLLSFVIGTFGWYVMICYCCKIEDD LNRLCYGGSMDWNMINVGALLLFKGHWVDNMSILRSFSPFMLVVCFG...IFMVGWPFMVALLAFSLLLLAWFIFGTYCLKNLLD LNRLSYGGSMSWNVVNVEVLILFKGHWVDRSSILRSFMPFIAMVCFG...VSMVGWEFLVGILSYFLLLAGCYLLAAYCVK..DD LNRLCYGGSMRWNMINVAILVLCQGHWVDTISVLRSFLPFVVVLCLG...VYMVGWPFVVAWFLFASLLVTWFVVGTYFVKDFLK LNRLCYGGSMDWNMINVGALLLFKGHWVDDISILRSFLPCILVVCLG...IFMVGWPFLVALLAFSVLLLAWFIFGTYCVKSLLD LNRIFYSGSMSWNMINVAGLVLLKGQWVDSMSVLRSFLPFLLVLSLG...VFMVGWPFLVALLSFSLLLLVWFLLGSYCFKTLLE LNRLAYGGSMGWNMINVCALILFKGRWVDSMSIFRSFSPFMLVLCLG...VFMAGWPFLIGLLSFSLLLIGWFLLGTYCFKNVLE LNRLSIK.SGGWNVVNLATLVLFKGRWVNKTAIVKSLLPPLIVYTIG...ILLGGWTFIASCSILVVLLTGWFIIGTYCFKKLIQ LNRLGFQ.SGGWNVVNLAIFIFLKGRWVNRTAMVKTYLPPLVVLGLG...LIFGGGTFLTYLLIFMFVLIGWFLLGTYCFKKLIH LNRLGFQ.AGGWNVVNLAIFIFLKGRWVNKTSIIKTYLPPLVVLGLG...LIFGGVTFLTYLLIFMFVLIGWFLLGTYCFKKLIH LNHLGFC.GGSWNVVSVAVLILLKGRWVDRVSMVKSYLPFAILFLICVFTVFYVSWDFLQFWVIFVVELVGWFLFGSYCCKGLVR LNRLGFC.SGGWNVVNLAALIFLKGKWVSTASMVRSFLPFLIAFGLG...VAFAGSTFLTYLGFFTAFLVGWFLLGTYCFKSLIH LNRLGFY.DGGWNVVNLAALIFLKGRWVSTTSMIKSFLPFAIVSALG...LFFGGLTFLTFLAFFTFLLVGWFLLGTYCFKNLIH
RTE group
AtRTE1 SlGR SlGRL1 PhGR DcRTE1 PhGRL1 PpGRL1 VvGRL1 AtRTH SlGRL2 PhGRL2 DcRTH1 PpGRL2 VvGRL2
B
RTH group
AtRTE1 SlGR SlGRL1 PhGR DcRTE1 PhGRL1 PpGRL1 VvGRL1 AtRTH SlGRL2 PhGRL2 DcRTH1 PpGRL2 VvGRL2
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Fig. 1 Predicted amino acid sequence alignments (a) and phylogenetic analysis (b) of 3PhGR-like proteins in Arabidopsis (AtRTE1, NP_180177; AtRTH, NP_190673), tomato (SlGR, ABD34613; SlGRL1, ABD34616; SlGRL2, ABD34617), and carnation (DcRTE1, ADW80941; DcRTH1, ADW80942), Prunus persica (PpGRL1, XP_007226043; PpGRL2, XP_007202517) and Vitis vinifera (VvGRL1, XM_002279795; VvGRL2, XM_002274070). The GR-
like proteins contain two conserved regions and two putative transmembrane domains close to the C-terminus. The phylogenetic tree shows that the GR/RTE family includes two groups, the RTE group and the RTH group. The RTE group possesses the (L/F) WPL and (G/R/S) GSM motif, and CR3 conserved region. CR conserved region, NCR non-conserved region, TM transmembrane domain
The expression of petunia PhGR and PhGRL1 in corollas treated with exogenous ethylene
VIGS-mediated suppression of PhGRL1 accelerated petunia flower senescence
To investigate whether the expression of PhGR and PhGRL1 is regulated by ethylene, qRT-PCR was used to test the relative mRNA accumulation in response to shortterm treatment with exogenous ethylene. After ethylene treatment, the mRNA levels of PhGR did not change significantly before hour 4, but increased from hours 4 to 12, and then decreased. The PhGRL1 mRNA levels decreased before hour 4, rapidly increased from hour 4 to 8, and then decreased again until hour 24 (Fig. 3).
To suppress PhGR and PhGRL1 in the petunia, we utilized the VIGS system, in which PhCHS served as the reporter gene (Spitzer-Rimon et al. 2007; Tan et al. 2014). The silencing of PhGR, PhGRL1, and PhCHS (control) were tested by constructing pTRV2-PhCHS-PhGR and pTRV2PhCHS-PhGRL1 vectors, which contained inserts of approximately 250 bp from the 30 untranslated sequences of the petunia PhGR and PhGRL1 cDNAs, respectively. About 25 plants were vaccinated for each vector and white
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Relative expression levels
A
B
5.0 4.0 3.0 2.0 1.0 0.0
PhGR
30
PhGR
20
R
S
L
C
O
PhGRL1
3.0 2.0
Relative expression levels
Fig. 2 Expression of PhGR and PhGRL1 as determined by quantitative real-time PCR in different organs (a) and in corollas during natural flower senescence (b). R roots, L leaves, S stems, C corollas, O ovaries. Relative expression levels are shown as fold change values. Data represent mean ± SD (n = 3)
10 0 0 2.0
3
4
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7
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PhGRL1
1.5 1.0
1.0 0.5
0.0
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S
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C
O
0.0
0
3
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5
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7
day
7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
18.0 16.0 14.0 12.0 10.0 8.0 6.0 4.0 2.0 0.0
PhCHS suppression
ethylene treatment
PhCHS/PhGR suppression
PhGR
0
2
4
8
12
24
PhGRL1
0
2
PhCHS/PhGRL1 suppression
5.0 4.0 3.0 2.0 1.0 0.0
PhGR expression
PhGRL1 expression
PhGRL2 expression
Fig. 4 Effects of TRV2-CHS/PhGR and TRV2-CHS/PhGRL1 treatment on the expression of PhGR, PhGRL1, and PhGRL2 in white flowers on day 4 after opening by quantitative real-time PCR, respectively. Relative expression levels are shown as fold change values. Data represent mean ± SD (n = 3)
4
8
12
24
hour
Fig. 3 Effects of exogenous ethylene on the expression of PhGR and PhGRL1 in corollas by quantitative real-time PCR in petunia corollas. Relative expression levels are shown as fold change values. Data represent mean ± SD (n = 3)
flowers were obtained in approximately 90 % of vaccinated plants. The results from qRT-PCR analysis showed that the PhGR and PhGRL1 mRNA levels were decreased to less than 15 % of the control (TRV PhCHS) in white flowers treated with TRV PhCHS/PhGR and TRV PhCHS/ PhGRL1, respectively, whereas the expression of PhGRL2 was almost unchanged (Fig. 4). The longevity of white flowers treated with TRV PhCHS/PhGRL1 was reduced compared to that of purple flowers from the same plants, white flowers from TRV PhCHS plants, and control flowers (Fig. 5). The longevity of PhCHS/PhGRL1 suppression flowers was 5.28 ± 0.56 days; however, the longevity of the PhCHS suppression flowers (control) was 6.58 ± 0.62 days. The
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Relative expression levels
Relative expression levels
control
longevity of white flowers treated with TRV PhCHS/PhGR was 6.84 ± 0.67 days, which was not significantly different from that of flowers subjected to TRV PhCHS treatment (Table 1). The expression of the genes downstream of ethylene signaling increased in the corollas of PhGRL1 suppression The expression of PhEIN2, PhEIL1, and PhERF3, which are downstream components of the ethylene signaling pathway, was examined by qRT-PCR in flowers in which PhGRL1 was suppressed. After the flower was open for 2 days, PhCHS suppression, and PhCHS/PhGRL1 suppression white flowers were cut, capped and treated with 10 lL L-1 ethylene for 8 h. The PhEIN2, PhEIL1, and PhERF3 mRNA levels were measured by qRT-PCR. As shown in Fig. 6, ethylene treatment increased PhEIN2, PhEIL1, and PhERF3 expression in PhCHS suppression flowers; however, at both 0 h and 8 h after ethylene
Plant Cell Rep (2015) 34:1561–1568 Fig. 5 Effects of PhCHS suppression, PhCHS/PhGR suppression, and PhCHS/ PhGRL1 suppression on the longevity of petunia flowers. Petunia plants were infected with TRV CHS, TRV PhCHS/ PhGR, or TRV PhCHS/ PhGRL1. Flowers showing the white silencing phenotype and purple flowers from uninfected plants were excised and photographed every day after opening
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Control
CHS Suppression
PhGR Suppression
PhGRL1 Suppression
Table 1 The effects of PhGR and PhGRL1 suppression on the longevity of petunia flowers Wild type Longevity of flowers (days)
6.76 ± 0.76
PhCHS suppression a
a
PhCHS/PhGR suppression a
6.58 ± 0.60
PhCHS/PhGRL1 suppression 5.28 ± 0.56b
6.84 ± 0.68
Values represent the mean ± SE; means followed by different letters are significantly different according to LSD-test (P = 0.05). All measurements were performed in three replicates
PhCHS suppression
PhCHS/PhGRL1 suppression
PhEIL1
1.0
PhEIN2
0.8
0.25
0.8
0.6
0.20
0.6
0.4
0.4
0.2
0.2
0.0
0.0
0
8
PhERF3
0.30
1.0
0.15 0.10 0.05 0
8
0.00
0
8
Ethylene treatment (hour)
Fig. 6 Induction of PhEIL1, PhEIN2, and PhERF3 mRNA by ethylene in PhCHS and PhCHS/PhGRL1 suppression flowers. Flowers were detached on the day before anthesis and treated with 10 lL L-1 ethylene for 8 h. Total RNA was isolated from white
Ethylene Production (nL g-1Fw h-1)
50 40
PhCHS suppression
flower tissues. PhEIL1, PhEIN2, and PhERF3 mRNA levels were determined by quantitative real-time PCR. Relative expression levels are shown as fold change values. Data represent mean ± SD (n = 3)
treatment, PhEIN2, PhEIL1, and PhERF3 expression were significantly higher in white flowers with PhCHS/PhGRL1 suppression.
PhCHS/PhGR suppression PhCHS/PhGRL1 suppression
30 20
Ethylene production increased in flowers with PhGRL1 suppression
10 0
2
3
4
Day
Fig. 7 Ethylene production in PhGR and PhGRL1 suppression flowers. Ethylene production was measured for white flower corollas of PhCHS/PhGR, PhCHS/PhGRL1, and PhCHS (control) suppression plants after the flowers were open for 2, 3, and 4 days. Ethylene was collected for 2 h and subsequently measured using a gas chromatograph. Ethylene production rates were calculated based on tissue FW. Mean ± SE values were determined from five samples
We next interrogated the effects of PhGR and PhGRL1 suppression on ethylene production. After the flowers were open for 2, 3, and 4 days, the white flowers of PhCHS, PhCHS/PhGR, and PhCHS/PhGRL1 suppression were cut, then capped for 2 h, before ethylene in the headspace was measured by gas chromatography. As shown in Fig. 7, after the flowers were open for 3 and 4 days, PhCHS/
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PhGRL1 suppression white flowers produced more ethylene than PhCHS suppression white flowers, whereas there were no significant differences between ethylene production in the white flowers of PhCHS/PhGR suppression and PhCHS suppression after the flowers were open for 2, 3 and 4 days,
Discussion Different copy numbers of the GR family are present in different species: although most eudicot species contain two members, petunia, tomato and other species of the Solanaceae family possess three copies of GR-like genes (Ma et al. 2012; Tan et al. 2014). The GR/RTE1 gene family in dicot species can be subdivided into two groups, the RTE group and the RTH group (Tan et al. 2014). PhGR and PhGRL1, together with Arabidopsis RTE1 and tomato SlGR and SlGRL1, encode proteins belonging to the RTH group. Previous research has revealed that GR/RTE1 and its homologs have discrete and specific expression patterns in Arabidopsis, tomato, Rosa hybrida and carnation (Resnick et al. 2006; Barry and Giovannoni 2006; Yu et al. 2010, 2011; Ma et al. 2012). In this study, both PhGR and PhGRL1 mRNA were present at different levels in different tissues and in corollas during flower senescence (Fig. 1), indicating that PhGR and PhGRL1 expression are spatially and temporally regulated. Ethylene induces flower senescence in the petunia, and reduced ethylene sensitivity results in a significant delay in flower senescence in the petunia (Wilkinson et al. 1997). The expression of GR-like genes is also regulated by ethylene in a tissue-specific manner in Arabidopsis, tomato, R. hybrida and carnation (Resnick et al. 2006; Barry and Giovannoni 2006; Yu et al. 2010, 2011; Ma et al. 2012). PhGRL1 mRNA level was the highest in corollas, changed with flower senescence, and increased after an 8 h ethylene treatment, suggesting a probable role during flower senescence. Down-regulation of PhGRL1 expression in response to ethylene would have the effect of stimulating the response pathway, and the subsequent upregulation might be a response to the signaling cascade increasing the feedback control of ethylene perception. The members of the RTE group, Arabidopsis RTE1 and tomato SlGR, negatively regulate ethylene responses (Barry and Giovannoni 2006; Resnick et al. 2006). In addition, the members of the RTE group have evolved the ability to influence different subsets of ethylene responses or were involved in tissue-specific ethylene responses (Ma et al. 2012). In rice, OsRTH1 negatively regulates ethylene signaling (Zhang et al. 2012). In this study, the longevity of
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flowers in which VIGS-mediated suppression of PhGRL1 occurred was reduced compared to the longevity of the control. The expression of PhGR was the highest in leaves and is regulated by exogenous ethylene treatment (Figs. 1b, 2); however, the longevity of PhGR suppression flowers did not significantly change compared to that of the control. It is possible that PhGR is involved in other processes, such as fruit maturation, which are controlled by ethylene. After all, SlGR overexpression delays fruit maturation in the tomato (Barry and Giovannoni 2006). The expression of the genes downstream of ethylene signaling, namely PhEIN2, PhEIL1 and PhERF3, increased in the corollas of VIGS-induced PhGRL1 suppression (Fig. 5). It is noteworthy that suppression of PhGRL2 also increased the expression of PhEIN2, PhEIL1 and PhERF3 (Tan et al. 2014). Overexpression of Arabidopsis AtRTE1 also represses ethylene responses in the wild type plant but not in the loss-of-function etr1-7 mutant (Resnick et al. 2006). RTE1 promotes ETR1 signaling through a conformational effect on the ethylene-binding domain (Zhou et al. 2007; Resnick et al. 2008). Genetic analysis indicates that AtRTE1 functions at or upstream of the ethylene receptors, but acts independently of the RESPONSIVE TO ANTAGONIST1 copper transporter (Resnick et al. 2006, 2008). These data suggested that members of the RTE group, such as PhGRL1 and RTE1, are involved in ethylene signaling suppression. PhGRL1 suppression increased ethylene production after the flowers were open for 3 and 4 days, which suggests it may exert negative control over ethylene production. It would be possible that it was the onset of early ethylene evolution burst that resulted in early flower senescence and increasing of expression PhEIN2, PhEIL1 and PhERF3 in the VIGS-induced PhGRL1 suppression. Overall, PhGRL1 is involved in flower senescence and is a negative regulator of the ethylene response. In addition, further studies are required to assess the biological significance of the response of PhGR to ethylene treatment.
Materials and methods Plant material Petunia (Petunia hybrida ‘Ultra’) plants were grown in the greenhouse under 25 °C/20 °C day/night temperatures and natural photoperiod (Liu et al. 2011). RNA extraction Total RNA was extracted was performed according to previously described protocols (Liu et al. 2011).
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Quantitative real-time PCR assays Petunia total RNA was extracted with the Tri-Reagent kit (Molecular Research Center) and treated with RNase-free DNase (Fermentas) followed by reverse transcription (RT) according to the manufacturer’s instructions. First strand cDNA was synthesized using 0.1 mg total RNA, oligo(dT) primer, and Reverse Transcriptase ImProm-II (Promega). Quantitative real-time PCR (qRT-PCR) was performed as described by Tan et al. (2014). For real-time PCR, oligonucleotide primers were designed according to the untranslated region of each gene with Primer 5 software. All primers were tested with melting peaks and dissociation curves to confirm that only one product was generated for each pair of primers. To verify that the primers generated specific amplicons of the target genes, all PCR products were purified and resequenced. The sequences of all primers used for real-time PCR analysis are described in Supplemental Table 1. Petunia actin was used as the housekeeping gene to quantify cDNA abundance. Flower longevity Flower Longevity was measured according to previously described protocols (Tan et al. 2014). All measurements were performed in three replicates. The data were analyzed using the ANOVA function of SAS 8.02 (Cary, NC, USA) to compare differences between genotypes. The Tukey’s honestly significant difference mean-separation test was used to calculate the mean separation at the 5 % level (HSD0.05). Ethylene treatment Petunia flowers were treated with ethylene according to previously described protocols (Tan et al. 2014). Petunia flowers were harvested at anthesis, and their stems were recut to 5 cm, placed in flasks with distilled water, and subsequently treated with 2 ll l-1 ethylene for 0, 2, 4, 8, 12 and 24 h. Corollas from 8 to 10 flowers were collected at each time point, immediately frozen in liquid nitrogen, and stored at -80 °C for subsequent RNA extraction. Agroinoculation of TRV vectors To generate pTRV2-CHS containing the 30 untranslated region of PhGR and PhGRL1 (TRV2-CHS: PhGR and TRV2-CHS: PhGRL1), the two genes sequences of approximately 250 bp were amplified by PCR using forward primers and reverse primers (Supplemental Table 2), and the PCR products were inserted upstream of PhCHS. Agrobacterium tumefaciens (strain GV3101) transformed with pTRV1 and pTRV2 derivatives were prepared as
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previously described (Spitzer-Rimon et al. 2012; Tan et al. 2014). About 25 plants were vaccinated for each vector. Author contribution statement Yu Y, designed the research; Yang W, Tan Y, Zhong S and Tang N performed the research; Yang W, Yu Y and Liu J wrote the manuscript. Acknowledgments This study was supported by the National Natural Science Foundation of China (31270736, 31170653, and 31470700). Conflict of interest of interest.
The authors declare that they have no conflict
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ORIGINAL PAPER
Functional characterization of PhGR and PhGRL1 during flower senescence in the petunia Weiyuan Yang1,2 • Juanxu Liu1 • Yinyan Tan1 • Shan Zhong1 • Na Tang1 Guoju Chen2 • Yixun Yu1,2
•
Received: 3 April 2015 / Revised: 5 May 2015 / Accepted: 11 May 2015 / Published online: 19 May 2015 Ó Springer-Verlag Berlin Heidelberg 2015
Abstract Key message Petunia PhGRL1 suppression accelerated flower senescence and increased the expression of the genes downstream of ethylene signaling, whereas PhGR suppression did not. Abstract Ethylene plays an important role in flowers senescence. Homologous proteins Green-Ripe and Reversion to Ethylene sensitivity1 are positive regulators of ethylene responses in tomato and Arabidopsis, respectively. The petunia flower has served as a model for the study of ethylene response during senescence. In this study, petunia PhGR and PhGRL1 expression was analyzed in different organs, throughout floral senescence, and after exogenous ethylene treatment; and the roles of PhGR and PhGRL1 during petunia flower senescence were investigated. PhGRL1 suppression mediated by
Communicated by M. C. Jordan. W. Yang and J. Liu contributed equally to this work.
virus-induced gene silencing accelerated flower senescence and increased ethylene production; however, the suppression of PhGR did not. Taken together, these data suggest that PhGRL1 is involved in negative regulation of flower senescence, possibly via ethylene production inhibition and consequently reduced ethylene signaling activation. Keywords Petunia GR-Like Ethylene response Flower senescence RTE1
Introduction The plant hormone ethylene controls various processes in the plant life cycle, including seed germination, fruit ripening, leaf and flower senescence, and a multitude of biotic and abiotic stresses (Abeles et al. 1992). Studies on Arabidopsis thaliana have revealed a universally conserved set of components in the ethylene signaling pathway that are present in higher plants. Ethylene is detected by members of the ethylene receptor family, which are
Electronic supplementary material The online version of this article (doi:10.1007/s00299-015-1808-7) contains supplementary material, which is available to authorized users. & Yixun Yu [email protected]
Na Tang [email protected]
Weiyuan Yang [email protected] Juanxu Liu [email protected]
Guoju Chen [email protected] 1
Guangdong Key Laboratory for Innovative Development and Utilization of Forest Plant Germplasm, College of Forestry and Landscape Architecture, South China Agricultural University, Guangzhou 510642, China
2
College of Horticulture, South China Agricultural University, Guangzhou 510642, China
Yinyan Tan [email protected] Shan Zhong [email protected]
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negative regulators of the ethylene response (Chang et al. 1993; Hua and Meyerowitz 1998; Qu et al. 2007). In the absence of ethylene, the receptors are in an active state and constitutively activate CONSTITUTIVE TRIPLE RESPONSE1 (CTR1), which acts as a negative regulator in the pathway. CTR1 phosphorylates the central regulator ETHYLENE-INSENSITIVE2 (EIN2), a member of the N-Ramp family of metal-transporters, to control ethylene hormone signaling from the endoplasmic reticulum (ER) membrane to the nucleus (Ju et al. 2012). Under-phosphorylated EIN2 is processed for proteolytic cleavage and triggers its endoplasmic reticulum (ER)–to–nucleus translocation (Qiao et al. 2012). The carboxyl end of EIN2 (CEND) is a trafficking signal for translocation from the ER membrane to the nucleus and ethylene signaling is transmitted to the EIN3 family of transcription factors (Chao et al. 1997; Wen et al. 2012; Ji and Guo 2013). EIN3/EIL proteins activate the transcription of ETHYLENE RESPONSE FACTORs (ERFs), another type of transcription factor that regulates the expression of genes involved in the ethylene response (Solano et al. 1998; Potuschak et al. 2003; Xiao et al. 2013). The membrane protein GREEN RIPE (GR)/REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1) negatively regulates the ethylene response (Resnick et al. 2006; Barry and Giovannoni 2006). In Arabidopsis, RTE1 interacts with ethylene resistant1 (ETR1), and they co-localize in the ER (Dong et al. 2008; 2010). A newly published study suggested that cytochrome b5 interacts with RTE1 in Arabidopsis (Chang et al. 2014). In most eudicots, the RTE family includes two to three members (Ma et al. 2012; Tan et al. 2014). The petunia flower has served as a model for the study of ethylene biosynthesis and signaling during senescence (Underwood et al. 2005). Several petunia genes involved in ethylene biosynthesis and signal transduction have been identified, e.g., PhACO1, PhETR1, PhEIN2, PhEIN3/EIL and PhERFs (Chen et al. 2004; Shibuya et al. 2004; Liu et al. 2011). In addition, three fulllength GR-like cDNAs, PhGR (accession no. JQ659030), PhGRL1 (accession no. JQ659031) and PhGRL2 (accession no. KJ451475), have been cloned (Ma et al. 2012). Recently, our study suggested that the PhGRL2 protein interacts with PhACO1 and is involved in flower senescence in petunia (Tan et al. 2014), but the function of the other two members of GR family, PhGR and PhGRL1, is unknown. In this study, the roles of PhGR and PhGRL1 during petunia flower senescence were investigated. VIGS-mediated suppression of PhGRL1 accelerated flower senescence and increased the ethylene production; however, the suppression of PhGR did not. Taken together, these data
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suggest that PhGRL1 is involved in negative regulation of flower senescence in petunia.
Results Phylogenetic analysis of GR-like proteins Previous reports showed that GR-like proteins contain two conserved regions, CR1 and CR2, and two putative transmembrane domains close to the C-terminus, TM1 and TM2 (Dong et al. 2008; Zhang et al. 2012). The phylogenetic tree generated with GR-like sequences showed that the GR/RTE family of dicot species includes two groups, the RTE group and the RTH group (Tan et al. 2014). Based on the consensus regions mentioned above, we further classified the GR-like proteins of Arabidopsis, tomato, petunia, and carnation, Prunus persica and Vitis vinifera into two sub-types as shown in Fig. 1: (1) RTE group proteins with two small conserved motifs, the (L/ F)WPL motif in the non-conserved region NCR1 of the N-terminus, the (G/R/S) GSM motif in the conserved region CR2 of the C-terminus, and a conserved region, CR3, in the non-conserved region NCR2 (AtRTE1, SlGR, SlGRl1, PhGR, PhGRL1, DcRTE1, PpGRL1, and VvGRL1); (2) RTH group proteins, which lack the (L/ F)WPL motif, (G/R/S)GSM motif and CR3 conserved region (AtRTH, SlGRL2, PhGRL2, DcRTH1, PpGRL2, and VvGRL2). Petunia PhGR and PhGRL1 expression in different organs and throughout floral senescence The expression patterns of a particular gene in different plant organs often provide important clues regarding the physiological function of the gene. To assist with the determination of the function of PhGR and PhGRL1 in ethylene-regulated developmental processes such as petunia flower senescence, the expression patterns of PhGR and PhGRL1 were examined in different plant organs and at various stages of flower senescence by quantitative realtime PCR (qRT-PCR). The expression of PhGR was high in leaves and was moderate in corollas and ovaries; however, the transcription levels of PhGRL1 were high in corollas and very low in roots and stems (Fig. 2a). During natural flower senescence, the PhGR transcriptional level remained almost unchanged before day 4, increased rapidly from days 4 to 5 and then gradually decreased. The PhGRL1 mRNA levels decreased from days 0 to 3, remained unchanged from days 3 to 5, and peaked on day 6 (Fig. 2b).
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NCR1
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CR1 (L/F)WPL
MSRGRGVPMMDLKRSYDVEDRVVSVSIPSIIEADEADLWPLPEIDTKKSKFPCCIVWTPLPVVSWLAPFIGHIGLCREDGVILDFA MLPRR.YPQMDANPSNGGER.......DNALRGILQDLWPLDEIDPSTQKFPCCLVWTPLPVISWLAPFVGHVGICREDGTIVDFS MPSGR.RSLMDLEPAYKMES.......IRAVQDIQHEFWPLDEINGENAKFPCCLVWTPLPVVSWLAPFIGHVGICREDGSAVAFS MLPRR.LPMMEVDGRYVIDR.......SRALRVIRDDLWPLEEIDPTKEKFPCCLVWTPLPVVSWLAPFIGHVGICREDGTIIDFS .........MSTN...............DEAEFLQGELWPLVDVDPKKARFPCCLVWTPLPVVAWLAPFIGHFGICQEDGSILDFS .........MDLEPAYKVES.......IRTVQDIQHDFWPLDEIDPGNAKFPCCLVWTPLPVVSWLAPFIGHVGICTEDGNAVGFS MQHIR.FPVMEVKEAYDVEH.......MRSTQSIQHELWPLDEIDPKKAKFPCCLVWTPLPIVSWLAPFIGHVGICREDGAILDFA MSPRR.VPKMELNAINDVED.......MSTARRTQHELWPLDEIDPKKAKLPCCLVWTPLPVVSWLAPFIGHVGICREDGAILDFA ........MGETATDSE...........HRMMIG...LSDPMKIDPKRDRFPCCIVWTPLPFISWLVPFIGHVGICREDGVILDFA .........MVPEVDAD...........HALMIEE.NYPNPMLIDPKRDRFPCCIVWSPLPVLSWFIPFIGHIGICREDGVILDFA .........MVPEVEPD...........HALMIEE.DSRDTMQIDPKGDRFPCCIVWSPLPVLSWFLPFIGHIGICRQDGVILDFA .........METNVDIE...........EQSVISDRPVETTMHIDPERGRFPFLFVWAPLPVLTWLIPFIGHIGICREDGVILCFT .........METSASPE...........HNLMIDG.SASPTLQIDPRRARFPCCIVWTPLPVISWLIPFIGHIGICREDGVILDFA .........MDSNADPE...........DHMMIER.SVSQMMQIDPRRARFPCCIVWTPLPVISWLIPFVGHIGICREDGVILDFA
NCR2
GSNFINVDDFAFGPPARYLQLDRT.KCCLPPNMGGHTCKYGFKHTDFG...TARTWDNALSSSTRSFEHKTYNIFTCNCHSFVANC GDSMIHFGQLFYGTVAKYYQVDRQ.QCCFARNFGGHTCRKGYEHVVFG...TAVSWDDAVQLFRRTFENRNFKVFSCNGHSFAADC GSNFINIDDFALGSVAKYLQLDRK.QCCFPRNLAAHTCKHGYKHTEFG...SAITWDDAIQSSVRHFEHKSYNIFTCNSYSFVANC GSNMINVGNLTYGAVARYYQLDRL.QCCFPPHLAGHTCKDGYQHAEFG...TAVNWDDALRSSTCSYEHRSFNPFTCNGHSFVANC GSNLVSIDDFAFGSVIRFFISSHLLKCCFPPNLSAHSCKHGYKHHEYG...TALSWDDALHTCKHEYETKTYNLFTCNCHSFVANC GSNFINVDDFAFGSVAKYLQLDRK.QCYFPRNLAAHTCKHGYKHTEFG...SAITWDDAIQSSIRHFEHKSYNLFTCNSYSFLAHC GSNFVNADDFAFGAVARYLQLDRT.QCCFAANLGGHTCKSGYKHAEFG...TAITWDDALQSSSRHFEHKTYNLFTCNCHSFVANC GSNFVNVNDFAFGAVARYLQLDRE.KCCFPPNLAGHTCKNGYKHAENG...TAVTWDDALLSSSHHFGHKSYNLFTCNCHSFVANC GPNFVCVDNFAFGAVSRYIQINKE.MESSRSSSSGMFNGERRYEQEEDSHEKEPTWDDALRKSTQEYQHHSYNILTCNCHSFVANN GPNFVSVDNFTFGAPTCYFQLSRE.QCCCLSPYSAEPTGEYVENHD.ESGGNVDTWESAIRKSIQEFQHQSYSIFTCNCHSFVANG GPNFVSVDNFTFGAPTRYIEISKE.QCCCISPYPAEHTSEYVQNHDDESGRNVDTWDAALRKSIQEFQHQSYSIFTCNCHSFVANG TSYFVFVDDFGYFSVTRYLQIDEK.LCRAISSLPSDKNEERHQNSE...ENKIVSWDHGLQKSILEYQHHSYNLLTCNCHSFVANS GPNFVCVDNFAFGAATRYIQISKE.KCHSIPN.PSVCQSEDQYRQD.EPGRDIMTWDDALRKGTQEFQHRSYNILTCNCHSFVANN GPNFVCVDNFAFGAVTRYIQISKE.KCCISPHHPAPYRRENGRGQD.ETEIDILTWDDALRKSTQEFQHQSYNLFTCNCHSFVANN
CR2 AtRTE1 SlGR SlGRL1 PhGR DcRTE1 PhGRL1 PpGRL1 VvGRL1 AtRTH SlGRL2 PhGRL2 DcRTH1 PpGRL2 VvGRL2
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LNRLCYGGSMEWNMVNVAILLMIKGKWINGSSVVRSFLPCAVVTSLG...VVLVGWPFLIGLSSFSLLLFAWFIIATYCFKNIIT LNLLSFRGSMRWNMINVGALIMFEGKWVSRWSMLRSFLPFIGILCFG...YLMIGWMFPIGLLSFVIGTFGWYVMICYCCKIEDD LNRLCYGGSMDWNMINVGALLLFKGHWVDNMSILRSFSPFMLVVCFG...IFMVGWPFMVALLAFSLLLLAWFIFGTYCLKNLLD LNRLSYGGSMSWNVVNVEVLILFKGHWVDRSSILRSFMPFIAMVCFG...VSMVGWEFLVGILSYFLLLAGCYLLAAYCVK..DD LNRLCYGGSMRWNMINVAILVLCQGHWVDTISVLRSFLPFVVVLCLG...VYMVGWPFVVAWFLFASLLVTWFVVGTYFVKDFLK LNRLCYGGSMDWNMINVGALLLFKGHWVDDISILRSFLPCILVVCLG...IFMVGWPFLVALLAFSVLLLAWFIFGTYCVKSLLD LNRIFYSGSMSWNMINVAGLVLLKGQWVDSMSVLRSFLPFLLVLSLG...VFMVGWPFLVALLSFSLLLLVWFLLGSYCFKTLLE LNRLAYGGSMGWNMINVCALILFKGRWVDSMSIFRSFSPFMLVLCLG...VFMAGWPFLIGLLSFSLLLIGWFLLGTYCFKNVLE LNRLSIK.SGGWNVVNLATLVLFKGRWVNKTAIVKSLLPPLIVYTIG...ILLGGWTFIASCSILVVLLTGWFIIGTYCFKKLIQ LNRLGFQ.SGGWNVVNLAIFIFLKGRWVNRTAMVKTYLPPLVVLGLG...LIFGGGTFLTYLLIFMFVLIGWFLLGTYCFKKLIH LNRLGFQ.AGGWNVVNLAIFIFLKGRWVNKTSIIKTYLPPLVVLGLG...LIFGGVTFLTYLLIFMFVLIGWFLLGTYCFKKLIH LNHLGFC.GGSWNVVSVAVLILLKGRWVDRVSMVKSYLPFAILFLICVFTVFYVSWDFLQFWVIFVVELVGWFLFGSYCCKGLVR LNRLGFC.SGGWNVVNLAALIFLKGKWVSTASMVRSFLPFLIAFGLG...VAFAGSTFLTYLGFFTAFLVGWFLLGTYCFKSLIH LNRLGFY.DGGWNVVNLAALIFLKGRWVSTTSMIKSFLPFAIVSALG...LFFGGLTFLTFLAFFTFLLVGWFLLGTYCFKNLIH
RTE group
AtRTE1 SlGR SlGRL1 PhGR DcRTE1 PhGRL1 PpGRL1 VvGRL1 AtRTH SlGRL2 PhGRL2 DcRTH1 PpGRL2 VvGRL2
B
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AtRTE1 SlGR SlGRL1 PhGR DcRTE1 PhGRL1 PpGRL1 VvGRL1 AtRTH SlGRL2 PhGRL2 DcRTH1 PpGRL2 VvGRL2
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Fig. 1 Predicted amino acid sequence alignments (a) and phylogenetic analysis (b) of 3PhGR-like proteins in Arabidopsis (AtRTE1, NP_180177; AtRTH, NP_190673), tomato (SlGR, ABD34613; SlGRL1, ABD34616; SlGRL2, ABD34617), and carnation (DcRTE1, ADW80941; DcRTH1, ADW80942), Prunus persica (PpGRL1, XP_007226043; PpGRL2, XP_007202517) and Vitis vinifera (VvGRL1, XM_002279795; VvGRL2, XM_002274070). The GR-
like proteins contain two conserved regions and two putative transmembrane domains close to the C-terminus. The phylogenetic tree shows that the GR/RTE family includes two groups, the RTE group and the RTH group. The RTE group possesses the (L/F) WPL and (G/R/S) GSM motif, and CR3 conserved region. CR conserved region, NCR non-conserved region, TM transmembrane domain
The expression of petunia PhGR and PhGRL1 in corollas treated with exogenous ethylene
VIGS-mediated suppression of PhGRL1 accelerated petunia flower senescence
To investigate whether the expression of PhGR and PhGRL1 is regulated by ethylene, qRT-PCR was used to test the relative mRNA accumulation in response to shortterm treatment with exogenous ethylene. After ethylene treatment, the mRNA levels of PhGR did not change significantly before hour 4, but increased from hours 4 to 12, and then decreased. The PhGRL1 mRNA levels decreased before hour 4, rapidly increased from hour 4 to 8, and then decreased again until hour 24 (Fig. 3).
To suppress PhGR and PhGRL1 in the petunia, we utilized the VIGS system, in which PhCHS served as the reporter gene (Spitzer-Rimon et al. 2007; Tan et al. 2014). The silencing of PhGR, PhGRL1, and PhCHS (control) were tested by constructing pTRV2-PhCHS-PhGR and pTRV2PhCHS-PhGRL1 vectors, which contained inserts of approximately 250 bp from the 30 untranslated sequences of the petunia PhGR and PhGRL1 cDNAs, respectively. About 25 plants were vaccinated for each vector and white
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Relative expression levels
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Relative expression levels
Fig. 2 Expression of PhGR and PhGRL1 as determined by quantitative real-time PCR in different organs (a) and in corollas during natural flower senescence (b). R roots, L leaves, S stems, C corollas, O ovaries. Relative expression levels are shown as fold change values. Data represent mean ± SD (n = 3)
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PhGRL2 expression
Fig. 4 Effects of TRV2-CHS/PhGR and TRV2-CHS/PhGRL1 treatment on the expression of PhGR, PhGRL1, and PhGRL2 in white flowers on day 4 after opening by quantitative real-time PCR, respectively. Relative expression levels are shown as fold change values. Data represent mean ± SD (n = 3)
4
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Fig. 3 Effects of exogenous ethylene on the expression of PhGR and PhGRL1 in corollas by quantitative real-time PCR in petunia corollas. Relative expression levels are shown as fold change values. Data represent mean ± SD (n = 3)
flowers were obtained in approximately 90 % of vaccinated plants. The results from qRT-PCR analysis showed that the PhGR and PhGRL1 mRNA levels were decreased to less than 15 % of the control (TRV PhCHS) in white flowers treated with TRV PhCHS/PhGR and TRV PhCHS/ PhGRL1, respectively, whereas the expression of PhGRL2 was almost unchanged (Fig. 4). The longevity of white flowers treated with TRV PhCHS/PhGRL1 was reduced compared to that of purple flowers from the same plants, white flowers from TRV PhCHS plants, and control flowers (Fig. 5). The longevity of PhCHS/PhGRL1 suppression flowers was 5.28 ± 0.56 days; however, the longevity of the PhCHS suppression flowers (control) was 6.58 ± 0.62 days. The
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Relative expression levels
Relative expression levels
control
longevity of white flowers treated with TRV PhCHS/PhGR was 6.84 ± 0.67 days, which was not significantly different from that of flowers subjected to TRV PhCHS treatment (Table 1). The expression of the genes downstream of ethylene signaling increased in the corollas of PhGRL1 suppression The expression of PhEIN2, PhEIL1, and PhERF3, which are downstream components of the ethylene signaling pathway, was examined by qRT-PCR in flowers in which PhGRL1 was suppressed. After the flower was open for 2 days, PhCHS suppression, and PhCHS/PhGRL1 suppression white flowers were cut, capped and treated with 10 lL L-1 ethylene for 8 h. The PhEIN2, PhEIL1, and PhERF3 mRNA levels were measured by qRT-PCR. As shown in Fig. 6, ethylene treatment increased PhEIN2, PhEIL1, and PhERF3 expression in PhCHS suppression flowers; however, at both 0 h and 8 h after ethylene
Plant Cell Rep (2015) 34:1561–1568 Fig. 5 Effects of PhCHS suppression, PhCHS/PhGR suppression, and PhCHS/ PhGRL1 suppression on the longevity of petunia flowers. Petunia plants were infected with TRV CHS, TRV PhCHS/ PhGR, or TRV PhCHS/ PhGRL1. Flowers showing the white silencing phenotype and purple flowers from uninfected plants were excised and photographed every day after opening
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Control
CHS Suppression
PhGR Suppression
PhGRL1 Suppression
Table 1 The effects of PhGR and PhGRL1 suppression on the longevity of petunia flowers Wild type Longevity of flowers (days)
6.76 ± 0.76
PhCHS suppression a
a
PhCHS/PhGR suppression a
6.58 ± 0.60
PhCHS/PhGRL1 suppression 5.28 ± 0.56b
6.84 ± 0.68
Values represent the mean ± SE; means followed by different letters are significantly different according to LSD-test (P = 0.05). All measurements were performed in three replicates
PhCHS suppression
PhCHS/PhGRL1 suppression
PhEIL1
1.0
PhEIN2
0.8
0.25
0.8
0.6
0.20
0.6
0.4
0.4
0.2
0.2
0.0
0.0
0
8
PhERF3
0.30
1.0
0.15 0.10 0.05 0
8
0.00
0
8
Ethylene treatment (hour)
Fig. 6 Induction of PhEIL1, PhEIN2, and PhERF3 mRNA by ethylene in PhCHS and PhCHS/PhGRL1 suppression flowers. Flowers were detached on the day before anthesis and treated with 10 lL L-1 ethylene for 8 h. Total RNA was isolated from white
Ethylene Production (nL g-1Fw h-1)
50 40
PhCHS suppression
flower tissues. PhEIL1, PhEIN2, and PhERF3 mRNA levels were determined by quantitative real-time PCR. Relative expression levels are shown as fold change values. Data represent mean ± SD (n = 3)
treatment, PhEIN2, PhEIL1, and PhERF3 expression were significantly higher in white flowers with PhCHS/PhGRL1 suppression.
PhCHS/PhGR suppression PhCHS/PhGRL1 suppression
30 20
Ethylene production increased in flowers with PhGRL1 suppression
10 0
2
3
4
Day
Fig. 7 Ethylene production in PhGR and PhGRL1 suppression flowers. Ethylene production was measured for white flower corollas of PhCHS/PhGR, PhCHS/PhGRL1, and PhCHS (control) suppression plants after the flowers were open for 2, 3, and 4 days. Ethylene was collected for 2 h and subsequently measured using a gas chromatograph. Ethylene production rates were calculated based on tissue FW. Mean ± SE values were determined from five samples
We next interrogated the effects of PhGR and PhGRL1 suppression on ethylene production. After the flowers were open for 2, 3, and 4 days, the white flowers of PhCHS, PhCHS/PhGR, and PhCHS/PhGRL1 suppression were cut, then capped for 2 h, before ethylene in the headspace was measured by gas chromatography. As shown in Fig. 7, after the flowers were open for 3 and 4 days, PhCHS/
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PhGRL1 suppression white flowers produced more ethylene than PhCHS suppression white flowers, whereas there were no significant differences between ethylene production in the white flowers of PhCHS/PhGR suppression and PhCHS suppression after the flowers were open for 2, 3 and 4 days,
Discussion Different copy numbers of the GR family are present in different species: although most eudicot species contain two members, petunia, tomato and other species of the Solanaceae family possess three copies of GR-like genes (Ma et al. 2012; Tan et al. 2014). The GR/RTE1 gene family in dicot species can be subdivided into two groups, the RTE group and the RTH group (Tan et al. 2014). PhGR and PhGRL1, together with Arabidopsis RTE1 and tomato SlGR and SlGRL1, encode proteins belonging to the RTH group. Previous research has revealed that GR/RTE1 and its homologs have discrete and specific expression patterns in Arabidopsis, tomato, Rosa hybrida and carnation (Resnick et al. 2006; Barry and Giovannoni 2006; Yu et al. 2010, 2011; Ma et al. 2012). In this study, both PhGR and PhGRL1 mRNA were present at different levels in different tissues and in corollas during flower senescence (Fig. 1), indicating that PhGR and PhGRL1 expression are spatially and temporally regulated. Ethylene induces flower senescence in the petunia, and reduced ethylene sensitivity results in a significant delay in flower senescence in the petunia (Wilkinson et al. 1997). The expression of GR-like genes is also regulated by ethylene in a tissue-specific manner in Arabidopsis, tomato, R. hybrida and carnation (Resnick et al. 2006; Barry and Giovannoni 2006; Yu et al. 2010, 2011; Ma et al. 2012). PhGRL1 mRNA level was the highest in corollas, changed with flower senescence, and increased after an 8 h ethylene treatment, suggesting a probable role during flower senescence. Down-regulation of PhGRL1 expression in response to ethylene would have the effect of stimulating the response pathway, and the subsequent upregulation might be a response to the signaling cascade increasing the feedback control of ethylene perception. The members of the RTE group, Arabidopsis RTE1 and tomato SlGR, negatively regulate ethylene responses (Barry and Giovannoni 2006; Resnick et al. 2006). In addition, the members of the RTE group have evolved the ability to influence different subsets of ethylene responses or were involved in tissue-specific ethylene responses (Ma et al. 2012). In rice, OsRTH1 negatively regulates ethylene signaling (Zhang et al. 2012). In this study, the longevity of
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flowers in which VIGS-mediated suppression of PhGRL1 occurred was reduced compared to the longevity of the control. The expression of PhGR was the highest in leaves and is regulated by exogenous ethylene treatment (Figs. 1b, 2); however, the longevity of PhGR suppression flowers did not significantly change compared to that of the control. It is possible that PhGR is involved in other processes, such as fruit maturation, which are controlled by ethylene. After all, SlGR overexpression delays fruit maturation in the tomato (Barry and Giovannoni 2006). The expression of the genes downstream of ethylene signaling, namely PhEIN2, PhEIL1 and PhERF3, increased in the corollas of VIGS-induced PhGRL1 suppression (Fig. 5). It is noteworthy that suppression of PhGRL2 also increased the expression of PhEIN2, PhEIL1 and PhERF3 (Tan et al. 2014). Overexpression of Arabidopsis AtRTE1 also represses ethylene responses in the wild type plant but not in the loss-of-function etr1-7 mutant (Resnick et al. 2006). RTE1 promotes ETR1 signaling through a conformational effect on the ethylene-binding domain (Zhou et al. 2007; Resnick et al. 2008). Genetic analysis indicates that AtRTE1 functions at or upstream of the ethylene receptors, but acts independently of the RESPONSIVE TO ANTAGONIST1 copper transporter (Resnick et al. 2006, 2008). These data suggested that members of the RTE group, such as PhGRL1 and RTE1, are involved in ethylene signaling suppression. PhGRL1 suppression increased ethylene production after the flowers were open for 3 and 4 days, which suggests it may exert negative control over ethylene production. It would be possible that it was the onset of early ethylene evolution burst that resulted in early flower senescence and increasing of expression PhEIN2, PhEIL1 and PhERF3 in the VIGS-induced PhGRL1 suppression. Overall, PhGRL1 is involved in flower senescence and is a negative regulator of the ethylene response. In addition, further studies are required to assess the biological significance of the response of PhGR to ethylene treatment.
Materials and methods Plant material Petunia (Petunia hybrida ‘Ultra’) plants were grown in the greenhouse under 25 °C/20 °C day/night temperatures and natural photoperiod (Liu et al. 2011). RNA extraction Total RNA was extracted was performed according to previously described protocols (Liu et al. 2011).
Plant Cell Rep (2015) 34:1561–1568
Quantitative real-time PCR assays Petunia total RNA was extracted with the Tri-Reagent kit (Molecular Research Center) and treated with RNase-free DNase (Fermentas) followed by reverse transcription (RT) according to the manufacturer’s instructions. First strand cDNA was synthesized using 0.1 mg total RNA, oligo(dT) primer, and Reverse Transcriptase ImProm-II (Promega). Quantitative real-time PCR (qRT-PCR) was performed as described by Tan et al. (2014). For real-time PCR, oligonucleotide primers were designed according to the untranslated region of each gene with Primer 5 software. All primers were tested with melting peaks and dissociation curves to confirm that only one product was generated for each pair of primers. To verify that the primers generated specific amplicons of the target genes, all PCR products were purified and resequenced. The sequences of all primers used for real-time PCR analysis are described in Supplemental Table 1. Petunia actin was used as the housekeeping gene to quantify cDNA abundance. Flower longevity Flower Longevity was measured according to previously described protocols (Tan et al. 2014). All measurements were performed in three replicates. The data were analyzed using the ANOVA function of SAS 8.02 (Cary, NC, USA) to compare differences between genotypes. The Tukey’s honestly significant difference mean-separation test was used to calculate the mean separation at the 5 % level (HSD0.05). Ethylene treatment Petunia flowers were treated with ethylene according to previously described protocols (Tan et al. 2014). Petunia flowers were harvested at anthesis, and their stems were recut to 5 cm, placed in flasks with distilled water, and subsequently treated with 2 ll l-1 ethylene for 0, 2, 4, 8, 12 and 24 h. Corollas from 8 to 10 flowers were collected at each time point, immediately frozen in liquid nitrogen, and stored at -80 °C for subsequent RNA extraction. Agroinoculation of TRV vectors To generate pTRV2-CHS containing the 30 untranslated region of PhGR and PhGRL1 (TRV2-CHS: PhGR and TRV2-CHS: PhGRL1), the two genes sequences of approximately 250 bp were amplified by PCR using forward primers and reverse primers (Supplemental Table 2), and the PCR products were inserted upstream of PhCHS. Agrobacterium tumefaciens (strain GV3101) transformed with pTRV1 and pTRV2 derivatives were prepared as
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previously described (Spitzer-Rimon et al. 2012; Tan et al. 2014). About 25 plants were vaccinated for each vector. Author contribution statement Yu Y, designed the research; Yang W, Tan Y, Zhong S and Tang N performed the research; Yang W, Yu Y and Liu J wrote the manuscript. Acknowledgments This study was supported by the National Natural Science Foundation of China (31270736, 31170653, and 31470700). Conflict of interest of interest.
The authors declare that they have no conflict
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