Flower senescence: some molecular aspects
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- Shahri, W. & Tahir, I. Planta (2014) 239: 277. doi:10.1007/s00425-013-1984-z
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Some molecular aspects of flower senescence have been reviewed. The isolation, identification and characterization of different genes from various flowers (mainly from petals) associated with senescence have been discussed. The isolated genes were divided into different groups. A large proportion of genes have been found to be upregulated during flower senescence while some genes were also found to be downregulated indicating that there exists a complex interplay between the expression patterns of various genes. The genes involved in petal expansion are found to be upregulated during normal flower development from anthesis to open flower stage, but XTH (Xyloglucan endotransglucosylase hydrolase) is found to be involved in petal expansion as well as abscission. Cysteine proteases or the genes encoding cysteine proteases (assigned a central role in protein degradation) have been identified from various flower systems, but no cysteine protease has been identified from senescing Mirabilis jalapa flowers. In addition to proteases, the genes encoding ubiquitin (exhibiting proteasomal degradation by 26S proteasomes) have also been identified suggesting the two alternate pathways for protein degradation. Genes encoding specific nucleases have also been identified, but they displayed an early increase in transcript abundance before the senescence symptoms become evident and characterize the involvement of PCD during flower senescence. A range of transcription factors are described and their possible role in flower senescence has been discussed. A detailed description of genes involved in ethylene synthesis and the components involved in ethylene signaling have been presented.
KeywordsAbscission Cysteine proteases Ethylene Expansion Senescence PCD
Senescence, aging and death, conceived of in the past as inevitable, negative processes, are now considered an integral part of differentiation and development. Senescence involves a highly regulated gene expression and the presence of concerted mechanisms of cellular degradation (Yamada et al. 2007). The processes of senescence and senescence-induced PCD are regulated by a coordinated signaling pathway, which is consistent with the view that senescence involves PCD (Coupe et al. 2004). PCD is an active process that is regulated at both transcriptional and translational levels (Lawton et al. 1990; Nooden et al. 1997). Literature concerned with the physiology and biochemistry of flower senescence has been updated from time to time by various authors (Stead 1992; van Doorn and Stead 1997; Rubinstein 2000; van Doorn 2001; Zhou et al. 2005; Eason 2006; Rogers 2006; Tripathi and Tuteja 2007; van Doorn and Woltering 2008; Shahri and Tahir 2011). In our previous review (Shahri and Tahir 2011), we reported different strategies of flower senescence and some important events associated with it. The present review presents the information gathered from a number of recent research papers on isolation, characterization and identification of genes expressed during flower senescence with the intention to update the available literature on some molecular aspects of flower senescence, as in-depth understanding of the senescence and its regulation at molecular level is essential for bringing the possible improvement in floricultural crops like cut flowers and ornamentals.
Genes associated with flower senescence: an overview
An overview of the genes involved in flower senescence
Possible biological functions
EF1α (Elongation factor 1α), genes encoding metallothioneins, a receptor-like kinase, transcription factors (eG04 and lD10), GAPDH; (Glyceraldehyde-3-phosphate dehydrogenase), a rose homolog of the ArabidopsisAPETALA3 gene jD10 and a rose homolog of the BrassicaP8 gene
Protein and lipid turnover (protein synthesis), defense/stress, signal transduction, transcription, secondary metabolism (scent production), signaling role in programmed cell death or apoptosis and floral organ identity (petals)
Channelière et al. (2002)
Genes encoding serine and cysteine proteases
Proteolysis and remobilization
Sequences encoding Grap 2 and Cyclin D interacting protein, a MADS-domain transcription factor, a casein kinase and a nucleotide-gated ion channel-interacting protein
Regulation of flower senescence in Iris
van Doorn et al. (2003)
Homologs of a range of transcription factors (Ring Zinc-finger protein) and proteases (upregulated genes)
Protein turn over and degradation and transcriptional regulation
Xu et al. (2007a)
A homolog of CCA1 (a ‘clock gene’ identified in Arabidopsis thaliana), a Xa21 receptor-type protein kinase and an aspartyl protease. (downregulated genes)
Proteolysis and developmental control
Partial cDNA of the senescence-related gene Alstroemeria Defender Against Death 1 (ALSDAD1)
Regulation of flower senescence
Wagstaff et al. (2003)
A zinc-finger containing protein- LSD1 (lesion simulating disease: BoLSD1, BoLSD2), Bax inhibitor (BoBI-1, BoBI-2) and serine palmitoyltransferase (BoSPT1 and BoSPT2)
Suppression of cell death,
Regulation of sphingolipid signaling pathway
Alter intracellular Ca2+ flux control and lipid dynamics
Cell death control and/or stress management
Coupe et al. (2004)
Two cell wall related genes (one encoding an extensin and the other a caffeoyl-CoA-3-O-methyl transferase), a pectin acetyl asterase, genes homologous to alcohol dehydrogenase and three cysteine proteases, a leucine-rich repeat receptor protein kinase and a 14-3-3 protein (a protein kinase). Genes encoding putative SEC14 and ataxin-2
Growth and in disease resistance response
Cell wall degradation
Remobilization of essential nutrients
Golgi vesicle transport
Yamada et al. (2007)
Genes homologous to animal PCD genes [(Bax inhibitor-1 (BI-1), a vacuolar processing enzyme (VPE: homologous to caspases) and a monodehydroascorbate reductase [(MDAR: homologous to Apoptosis inducing factor (AIF)], vacuolar protein sorting 34 (VPS34) and Arabidopsis autophagy related proteins 4b and 8a (ATG4b and ATG8a)
Cell death suppression
Yamada et al. (2009)
Moreover, the identification of plant genes homologous to animal PCD (apoptosis) genes has been recently reported by Yamada et al. (2009) from the senescent petals of Ipomoea nil that included a Bax inhibitor-1 (BI-1), a vacuolar processing enzyme (VPE: homologous to caspases) and a monodehydroascorbate reductase [(MDAR: homologous to Apoptosis inducing factor (AIF)]. In addition, microarray screens and analyses of individual genes have revealed that a number of genes, generally considered to be stress-related, are also upregulated during petal senescence. These include metallothioneins, abscisic acid responsive genes and glutathione-S-transferases (Meyer et al. 1991; Channelière et al. 2002; Breeze et al. 2004; Price et al. 2008). In Ipomoea, genes-In12, In15 and In21 have been found to encode products related to stress responses (Yamada et al. 2007). Of the genes upregulated in wall flower petals specifically, 40 % have been found to encode chitinases, 23 % encode GSTs, 9 % are involved in reactive oxygen species (ROS)/stress responses, 9 % are involved in signaling, 6 % in remobilization/metabolism, 2 % in transcriptional regulation, 2 % in metal binding, and a further 9 % are of unknown function (Price et al. 2008). Of the GST genes identified in wall flower, two genes have been found to be most similar to AtGSTF2 and AtGSTF3 from Arabidopsis. Both AtGSTF2 and AtGSTF3 have a putative ethylene-responsive enhancer element in their promoter sequences similar to that of a petal senescence-enhanced GST from carnation (Itzhaki et al. 1994). Both genes are members of the phi (ϕ) class of GSTs from Arabidopsis and have been suggested to function as glutathione peroxidases (Wagner et al. 2002). It has been postulated that GST activity may protect a senescing cell from lipid hydroperoxides prior to the actual cell death (Meyer et al. 1991). An important observation during Alstroemeria senescence is that the pattern of gene expression induced by ambient dehydration stress has been found to be similar to that seen during developmental senescence, whereas the pattern elicited by cold stress is different, as has been confirmed in the case of three genes: a metallothionein and two genes related to remobilization and proteolysis, respectively, indicating that some processes such as remobilization and ubiquitin-mediated proteolysis, associated in Alstroemeria are being activated by stress treatment (Wagstaff et al. 2010).
To sum up, it can be concluded that the genes upregulated during normal developmental petal senescence relate to remobilization of nutrients (proteases, nucleases, lipases and transporters), regulatory genes (transcription factors: NAC-domain transcription factors, Zinc-finger proteins), stress-related genes (Metallothioneins, Abscisic acid responsive genes, Glutathione-S-transferases), signal transduction genes (various classes of protein kinases: Xa21 receptor-like protein kinase, casein kinase, leucine-rich receptor kinase, 14-3-3 protein kinase), genes encoding different types of proteases (Cysteine proteases, Serine proteases, Aspartic proteases), 26S-proteasome machinery genes (involved in protein ubiquitination by 26S proteasomes), Cell wall degrading genes (Pectin acetyl transferase), Bax inhibitor genes, genes encoding Vacuolar processing enzymes (VPE) and genes related to RNA metabolism (Ataxin-2). Similarly, the genes that were found to be downregulated included genes encoding MADS-domain transcription factors, MYB transcription factors, gibberellin-induced protein, Cytochrome P450, a homolog of ‘clock gene’ (CCA1), aspartyl protease (in senescing Mirabilis flowers) and Defender against Apoptotic Death (ALSDAD1). Thus, it is indicative of the fact that mechanism of flower senescence involves a continuous interplay of various genes that are differentially regulated in a spatio-temporal manner to bring the execution and advancement of events leading to senescence. Keeping in view the above summary, we will now discuss the various kinds of genes involved in flower senescence under the following headings.
Genes involved in cell wall expansion and abscission
Of the various genes involved in cell wall expansion, the genes encoding expansins are considered as primary regulators of cell enlargement in plants. Expansins are reported to be cell wall-located proteins and act in plant cell walls by disrupting non-covalent binding between matrix glycan cellulose microfibrils (Cosgrove 1999a, b, 2000b). Screening of the transcripts isolated from senescing carnation flowers has revealed the presence of at least three transcripts of which the most abundant one has been found to be strongly homologous to an Arabidopsis thaliana β-xylosidase gene (involved in senescence related to cell wall expansion; Goujon et al. 2003) while the two other transcripts homologous to expansin-encoding genes (involved in cell wall loosening during growth or disassembly; Cosgrove 2000a). Disassembly of the primary cell walls is regarded as an important process in the progression of chrysanthemum flower senescence (Elanchezhian and Srivastava 2001). While conducting the expression studies of two expansin genes (DcExp1 and DcExp2; sharing 76 % identity with each other) in carnation flowers, Song et al. (2007) reported the expression of DcExp2 in early flower development (as its expression decreased during senescence) while the expression of other transcript DcExp1 has not been detected at any stage of flower development. It is therefore suggested that DcExp2 might be involved in senescence progress of the cut carnation flowers at earlier stages and that DcExp1 might have some other developmental role which needs to be ascertained. Similar studies conducted by Yamada et al. (2007) have also identified a gene, In07 in Ipomoea that too encodes a putative extensin-like protein. Moreover, the expansin gene, GgEXPA1 (Gibberellic acid responsive gene in gladiolus) has been reported to be expressed prominently during phases of active tepal expansion and cell elongation in stamen filaments, gynoecium styles and expanding leaves but not in tissues where expansion had ceased and senescence had been initiated (Azeez et al. 2010). Recently, four cDNAs encoding xyloglucan endotransglucosylase/hydrolase (XTH) (DcXTH1-DcXTH4) and three cDNAs encoding expansin genes (DcEXPA1-DcEXPA3) have been cloned and characterized from petals of opening carnation flowers of which two XTH genes (DcXTH2 and DcXTH3) and two expansin genes (DcEXPA1 and DcEXPA2) have been reported to be associated with petal growth and development during flower opening (Harada et al. 2011b). Moreover, the analysis of five transcripts (RhCG1, RhCG2, RhCG4, RhCG6 and RHAG1) from two rose cultivars (‘Black magic’ and ‘Maroussia’) by Hajizadeh et al. (2011a) also revealed the presence of the transcripts that encode the products involved in cell wall expansion and degradation during senescence, e.g., RhCG6 has been found to share 65 % sequence similarity with the gene encoding apple β-galactosidase protein. van Doorn et al. (2003) and O’Donoghue et al. (2009) have demonstrated that there occurs an increase in galactosidase transcript abundance during Iris hollandica and Petunia petal senescence, that encodes an enzyme involved in cell wall degradation. Similarly RhAG1 homolog has been found to share 30 % similarity to Petunia arabinogalactan protein. Arabinogalactan-proteins (AGPs) are cell wall proteoglycans containing a high proportion of carbohydrate (typically > 90 %), widely distributed in plant species and are located at the plasma membrane and secondary cell wall and in the media of cell cultures. It has been suggested that certain AGPs contribute in cell expansion (Shi et al. 2003), seed germination, in vitro root regeneration (van Hengel and Roberts 2003), and response to abscisic acid (Johnson et al. 2003; van Hengel and Roberts 2003).
In addition to the genes involved in cell expansion, a number of transcripts or genes involved in abscission of flowers or floral parts have been identified and characterized from different flower systems, e.g., five ethylene-responsive cDNAs have been isolated from Rosa hybrida and identified as an ethylene-induced cDNA homologous to a laccase gene (RhLAC gene). Three cDNAs have been isolated from petioles and two from pedicels. The gene has been found to encode a putative protein of 573 amino acids containing three conserved domains characteristic of the multicopper oxidase family and has been found to be highly induced in the leaf abscission zone of petioles and the bud abscission zone of floral bud pedicels, suggesting that RhLAC might play an important role in senescence and abscission in roses (Ahmadi et al. 2008). Similarly, the expression of two XTH genes (RbXTH1 and RbXTH2; share 52 % amino acid identity and are conserved at the catalytic site) in Rosa bourboniana has been found to lead to petal abscission. Transcription of these genes has been found to be ethylene responsive, with the ethylene response being tissue-specific for RbXTH1 but largely tissue-independent for RbXTH2. The Expression of these genes have been found to correlate with an increase in xyloglucan endotransglucosylase (XET) action in petal abscission zones of both ethylene-treated and field abscising flowers and it has been suggested that changes brought about by the XET action might allow easier accessibility of the wall to other hydrolytic enzymes, thereby accelerating abscission (Singh et al. 2011). Moreover, the promoter of RbXTH1 has revealed the presence of the cis-element ATTTCAAA, present in the tomato ethylene-responsive E4 gene, the carnation ethylene-responsive GST1 gene, and the rose cysteine protease promoter (Montgomery et al. 1993; Itzhaki et al. 1994; Tripathi et al. 2009). However, RbXTH2 has not been found to contain any known ethylene-responsive elements, although sequences related to ATTTCAAA have been found, indicating that the ethylene-responsive expression in RbXTH2 might be conferred by cis-elements other than the GCC box and the ATTTCAAA elements or by the modified ATTTCAAA. In Arabidopsis, the BOP (BLADE-ON-PETIOLE2) gene has been shown to play an essential role in floral abscission by specializing the abscission zone (AZ) anatomy. A homolog of BOP gene from tobacco, NtBOP2 has been reported to be predominantly expressed at the base of the corolla in an ethylene-independent manner and that its antisense suppression has been found to cause a significant delay in corolla shedding (Wu et al. 2012).
From the above studies, it is evident that although the expression levels of expansin genes decline during senescence, they are important for the regulation of normal developmental program in different floral tissues eventually leading to the progression of senescence program. Furthermore, the role of XTH genes is dynamic, i.e., they are involved in cell wall expansion leading to opening of flowers and growth as well as in abscission of flowers and floral parts.
Genes encoding cysteine proteases and ubiquitin
Genes encoding cysteine proteases
Possible biological functions
Remobilization of nutrients from the petals to the developing ovary
Jones et al. (1995)
Hydrolysis of soluble proteins (indicating Petal PCD)
Valpuesta et al. (1995)
Dehydration responsive and postharvest protein degradation
Guerrero et al. (1998)
Developmental markers of senescence
Noh and Amasino (1999)
BnSAG12-1 and BnSAG12-2
Eason et al. (2002)
Proteolysis and remobilization during later stages of senescence
Hunter et al. (2002)
Wagstaff et al. (2002)
9 genes (PhCP2–PhCP10)
Protein degradation and remobilization
Jones et al. (2005)
In15 and In21
Yamada et al. (2007)
Protein degradation and petal abscission
Tripathi et al. (2009)
Genes encoding ubiquitin ligases
Possible biological functions
Development-induced PCD and ethylene synthesis/signaling
Kosslak et al. (1997)
A part of the brassinosteroid response/pathogen response
Molnar et al. (2002)
Hu et al. (2002)
Serrano and Guzman (2004)
Not clearly demonstrated
Nodzon et al. (2004)
Drought resistance and homeostasis of various plant hormones
Ko et al. (2006)
XB3 (XA21 binding protein 3)
Pathogen-induced type of programmed cell death
Wang et al. (2006)
Coordination of the senescence program
Xu et al. (2007b)
Thus, there has been upregulation of both cysteine proteases and 26S proteasome-mediated ubiquitin pathway during flower senescence suggesting the two alternative pathways of protein degradation (proteasomal as well as non-proteasomal). However, some flowers show upregulation of both cysteine endopeptidases as well as ubiquitin ligases during flower senescence (e.g., carnation, Alstroemeria) while others show only upregulation of ubiquitin ligases (e.g., M. jalapa). The role of cysteine proteases has been implicated in major protein degradation and remobilization besides abscission of flowers or floral parts as the ubiquitin genes have been found to only fluctuate during senescence. Of the various RING Zinc-finger ankyrin proteins (ubiquitin ligases), only XBAT32 has been found to be involved in ethylene synthesis/signaling while MjXB3 (isolated from M. jalapa) has no known function but thought to be involved in coordination of the senescence program.
Genes involved in nucleic acid degradation
Genes involved in nucleic acid degradation
Panavas et al. (1999)
DEAD/DEAH box helicases
Breeze et al. (2004)
Langston et al. (2005)
Narumi et al. (2006)
Rose cultivars (‘Black magic’ and ‘Maroussia’)
RhCG1 and RhCG2
Hajizadeh et al. (2011a)
Farage-Barhom et al. (2008)
In conclusion, degradation of nucleic acids by specific nucleases during flower senescence has been demonstrated in various flower systems. A number of cDNAs encoding such nucleases have been isolated and found to be expressed well before the initiation of senescence suggesting their role in programmed execution of flower senescence. PhNUC1 has been found to be a cobalt-dependent senescence specific nuclease and both PhNUC1 and DcNUC1 have been found to be ethylene-responsive nucleases. The BFN1 nuclease has been well characterized and its intracellular localization has also been investigated. The evidences so far have suggested that nuclear degradation by the nucleases occur well before the senescence symptoms become apparent and that they might play an important role in developmental PCD-related processes as well as in progress of senescence. The involvement of nucleases is also indicative of the fact that flower senescence involves PCD.
Genes encoding various transcription factors
Genes encoding transcription factors
Possible biological functions
A homeodomain protein
Waki et al. (2001)
A MYB-like DNA-binding protein, a MYC protein, a MADS-box factor
Regulation of senescence but the exact role is unclear
Hoeberichts et al. (2007)
MADS box transcription factor
Delays petal senescence and abscission
Fang and Fernandez (2002)
MADS Box gene
van Doorn et al. (2003)
Guo and Gan (2006)
CCA1 and F935 (Myb transcription factors)
Photoperiodic control, flower opening and maturation
Xu et al. (2007a)
b-Zip and HD-Zip protein
Regulates osmotic and water relations of the opening and senescing flowers
CEBP (Carnation ethylene-responsive element binding protein)
Ethylene signaling in carnation flower development and senescence
Iordachescu et al. (2009)
Stress and senescence regulation
Balazadeh et al. (2010)
Myb, Lim, Hap5B and MADS box transcription factors
Stress and flower senescence regulation
Wagstaff et al. (2010)
In conclusion, MADS-box transcription factors, MYB-like DNA-binding proteins, MYC protein and CEBP have been identified as downregulated genes whose transcript abundance peaked during initial stages of flower development (up to flower opening). The possible role of these genes in flower senescence is still unclear; however, they have been implicated to be involved in the initial steps of senescence process as there are evidences that overexpression of these transcription factors delays senescence. The upregulated transcription factors consist of HD-Zip proteins, B-Zip proteins and Zinc-finger proteins. NAC-domain transcription factor has been found to show differential expression in various tissue systems. It has been identified as downregulated gene in carnation petals, but found to be expressed in senescent leaves of Arabidopsis. Further SEPALLATA3 (a MADS-box transcription factor) has been found to function as upstream regulator of NAC gene. The expression of these transcription factors is either controlled by developmental signals (CCA1) or induced in response to changing osmotic and water relations of the opening and senescing flowers (HD-Zip proteins).
Genes involved in ethylene synthesis (ACC synthase and ACC oxidase genes)
In ethylene-sensitive flower systems, ACC synthase and ACC oxidase are the key enzymes involved in ethylene biosynthesis. Several genes encoding 1-amino cyclopropane-1-caboxylate (ACC) synthase and ACC oxidase have been found to be upregulated during petal wilting in senescing carnation flowers. Initially, CARACC3 has been cloned by Park et al. (1992), and found to be abundantly expressed in petals during natural and ethylene-induced flower senescence. Later, Henskens et al. (1994) isolated two cDNA clones encoding carnation ACC synthase. One of the clones has been found to be identical to CARACC3 while the other clone (CARAS1) has been found to share only 66 % sequence similarity to CARACC3 (in the amino acid sequence). CARAS1 has been found to be more abundantly expressed in the styles rather than in the petals, thereby confirming the petal-specific nature of CARACC3. In the deduced amino acid sequence of the cDNA clone CARAS1, the amino acid residue tyr-215 (conserved residue among many known aminotransferases and all known ACC synthetases) is replaced by Phe (Henskens et al. 1994; Zarembinsky and Theologis 1994). The residue has been thought to be involved in the binding of essential co-factor, Pyridoxal phosphate (Mehta et al. 1989). Henskens et al. (1994) has suggested that the gene might encode a non-functional enzyme; however, the positive correlation between CARAS1 abundance and stylar ethylene production during aging is indicative of the fact that CARAS1 does encode a functional enzyme (Have and Woltering, 1997). Ma et al. (2005) while studying the differential induction features of three ACS genes in roses found that Rh-ACS2 is strongly induced by senescence. The tissue specificity of Rh-ACS2 has been found to be quickly induced by ethylene in gynoecia (Xue et al. 2008). Similar observations have also been reported in carnation, where CARAS1 (also named as DCASC2) showed a quicker and stronger response to ethylene treatment in gynoecia than in petals (Have and Woltering 1997). All these observations suggest that Rh-ACS2, a senescence-associated gene in rose petals, might play an important role in the induction of ethylene biosynthesis in gynoecia and in promoting the flower opening process. The genomic DNA structure of DcACS1 has been successfully revealed in senescing carnation petals (Dianthus caryophyllus and D. superbus). The gene has been found to express in two different isoforms (DcACS1a and DcACS1b). Genomic PCR analysis of 32 carnation cultivars has shown that most cultivars have only DcACS1a while some have both DcACS1a and DcACS1b. Both the genes were found to have five-exon and four-intron structure. Nucleotide sequences of exons 1–3 in DcACS1a have been found to be completely identical to those in DcACS1b. However, substitution of several nucleotides has been found in exon4 and 5. Exon5 of DcACS1b has been found to be 18 nucleotides shorter than that of DcACS1a, causing shorter stretch of threonine residues characteristic to DcACS1 gene. Introns 1–3 varied from 56 to 70 %, while the nucleotide sequence of Intron 4 has been shown to be completely identical in the two genes. Nucleotide sequence of 5′-UTR has been found to be conserved in DcACS1a and DcACS1b, but that of 3′-UTR was not. Moreover, DcACS1 orthologous genes have been isolated D. superbus var. longicalycinus, designated as DsuACS1a and DsuACS1b. Exogenously applied ethylene has been found to induce autocatalytic ethylene production in petals of D. superbus var. longicalycinus with simultaneous accumulation of transcripts of DsuACS1 (Harada et al. 2011a). ACC oxidase gene has been isolated from carnation (Wang and Woodson 1991). Constitutive expression of this gene has been reported in the styles but not in other floral organs (Woodson et al. 1992). Spanu et al. (1994) suggested that the post-translational regulation of ACC synthetase protein is achieved through phosphorylation and dephosphorylation of associated proteins.
To sum up, it can be concluded that ACC synthase and ACC oxidase genes (involved in ethylene biosynthesis) have been successfully isolated and characterized in various flower systems (carnation and rose) and their differential expression in different tissue systems has also been revealed. CARACC3 gene from carnation has been found to have petal-specific expression, whereas CARAS1 from carnation and Rh-AS1 from rose have been found to express in gynoecia. The genomic DNA structure of DcACS1 (both isoforms: DcACS1a and DcACS1b) has also been revealed and its orthologous genes have also been identified. The only ACC oxidase gene identified in carnation has been reported to be constitutively expressed in the styles and not in other floral organs.
Genes encoding ethylene receptors
Three N-terminal hydrophobic domains capable of reversibly binding to ethylene.
Phytochrome-related T2L and R2L domains (homologous domains with the chromophore attachment domains of phytochrome photoreceptors).
A GAF domain (homologous domain found in photo-transducing proteins).
Two domains homologous to a histidine kinase.
A receiver of the bacterial two-component histidine kinase system.
Genes encoding ethylene receptors
ETR1and ETR1-like genes (ETR2, EIN4, ERS1 and ERS2)
NR gene, LeETR1, LeETR2, LeETR4 and LeETR5
Vriezen et al. (1997)
Cm-ETR1 and Cm-ERS1
PE-ETR1 and PE-ERS1
Mita et al. (1998)
RhETR1, RhETR2, RhETR3 and RhETR5
DcERS1, DcERS2 and DcETR1
Shibuya et al. (2002)
DlERS1type1 and DlERS1type2, Dl-ERS1-3 and Dl-ERS2
Narumi et al. (2005)
Huang et al. (2007)
Zhou et al. (2010)
The expression pattern of various ETR1 genes from different flower systems has revealed a differential expression as is evidenced by the higher expression of RhETR1 in long-lasting miniature rose cultivar and that of RhETR3 in short-lasting one, the constitutive expression of Rh-ETR5 (from cut roses) and RhETR2 (from miniature roses) throughout flower development (Müller et al. 2000a, b, 2001; Ma et al. 2006; Tan et al. 2006) and by the higher expression of Dg-ERS1 in petals of ethylene-sensitive chrysanthemum flowers (Narumi et al. 2005). Moreover, the ETR genes have been reported to show temporal regulation. A classical example of temporal regulation is provided by the carnation ETR genes (Dc-ERS1, Dc-ERS2 and Dc-ETR1) of which Dc-ERS2 has been found to be expressed at pre-opening stage while Dc-ETR1 exhibited constitutive expression during senescence and that Dc-ERS1 has not been detected throughout senescence (Shibuya et al. 2002). Similarly, the cDNAs (Dl-ERS1-3 and Dl-ERS2) from Delphinium flowers have been found to exhibit constitutive levels during flower senescence while as the Dl-ERS1 genes [Dl-ERS1 (Delphinium little-strain ERS1) type-1 and Dl-ERS1 type-2] have been shown to have increased expression prior to flower senescence following a decline thereafter (Kuroda et al. 2003; Tanase and Ichimura 2006). Likewise, the cDNA (OgERS1; phylogenetically related to ETRs from monocots) from Oncidium has been found to be abundantly expressed in roots and flower buds and to a lesser extent in pseudobulbs, leaves, and fully opened flowers (Huang et al. 2007). Similar studies in tree peony have also revealed the constitutive expression of Ps-ETR1-1 as it has been found to remain at a constant level throughout different opening stages (Zhou et al. 2010). As far as the effect of exogenous ethylene on the expression levels of ethylene receptor genes is concerned, it has been found to induce the expression of RhETR1, RhETR2, RhETR3, DlERS1-3 and DlERS2 on one hand and substantially inhibit the levels of Ps-ETR1-1 mRNA on the other hand. Moreover, the expression levels of Rh-ETR5, Dc-ERS2 and Dc-ETR1 have been found to be ethylene independent. It has been speculated that the exogenous ethylene-independent expression pattern might result from the lower amount of ethylene produced during natural flower senescence than that of exogenous ethylene and that abscission of florets in Delphinium is caused by the elevated levels of ethylene receptor (ERS1), influenced by exogenous ethylene. Moreover in tree peony, there exists an inverse relationship between the level of ETRs and the sensitivity to ethylene, and the reduction in the amount of ethylene receptor proteins have been found to increase ethylene sensitivity of plant tissues. Therefore, the decrease in the level of Ps-ETR1-1 mRNA in petals of ethylene-treated flowers has been suggested to increase the sensitivity of the petals to ethylene and hence accelerate their senescence (Shibuya et al. 2002; Kuroda et al. 2003, 2004; Ma et al. 2006; Tan et al. 2006).
In conclusion, it can be stated that the perception of ethylene during flower senescence is mediated by ethylene receptor genes (ETRs) which have been found to encode transmembrane ER proteins with similarity to bacterial two-component histidine kinase. A number of ETR genes have been identified and characterized from various flower systems. The ETR1 gene from Arabidopsis has been reported to have hydrophobic regions capable of reversibly binding to ethylene with the involvement of copper ions associated with the domain. It has been postulated that RAN1 delivers copper ion to ETR1. The differential expression of various ETRs have been found to be associated with varying longevity in miniature potted roses with the long-lasting cultivar expressing ETR1. From the available data, it may be speculated that there exists an inverse relationship between the level of ETRs and the sensitivity to ethylene in tree peony, but in Delphinium, it has been reported that abscission of florets is caused by elevated levels of ethylene receptor (ERS1) and that too influenced by exogenous ethylene.
Genes involved in ethylene signaling
Genes involved in ethylene signaling
Kieber et al. (1993)
Nakano et al. (2006)
Lin et al. (1998)
Tieman et al. (2001)
Kuroda et al. (2004)
Shibuya et al. (2004)
Liu et al. (2011)
In29 and In42
Yamada et al. (2007)
Manzano et al. (2008)
Yang et al. (2008)
Zhou et al. (2010)
RhCTR1 and RhCTR2
Hajizadeh et al. (2011b)
Fu et al. (2011a)
Fu et al. (2011b)
Hoeberichts et al. (2003)
Yang et al. (2011)
DlHD2, DlERF1 and DlERF2
Kuang et al. (2012)
Based on double-mutant analysis, it is proposed that CTR1 acts at or downstream from ETR1, ERS1 and EIN4, and that EIN2, EIN3, EIN5, EIN6 and EIN7 act after CTR1 (Hua et al. 1995; Roman et al. 1995). Chen et al. (2005) has reviewed that CTR1 passes the signal to EIN2 (an integral membrane protein that acts as a positive regulator of ethylene pathway) through a series of MAPK cascades, and then to EIN3/EILs (transcriptional factors) that trigger the expression of downstream target genes such as ERFs. However, it has been recently demonstrated that CTR1 interacts and directly phosphorylates the cytosolic C-terminal domain of EIN2 in Arabidopsis (Li and Guo 2007; Ju et al. 2012). Although overexpression of the C-terminus of EIN2 (an ER-localized membrane protein) has been reported to result in constitutive induction of a subset of ethylene responses and genes, it has been found to be inefficient in restoring ethylene sensitivity to an ein2 null mutant (Alonso et al. 1999; Bisson et al. 2009). The regulation of EIN2 has been found to be brought about by two F-box proteins—ETP1 and ETP2 (EIN2-TARGETING PROTEIN) that negatively regulate ethylene signaling as the presence of ethylene downregulates both ETP1 and ETP2 (which otherwise degrade EIN2 in presence of ethylene) leading to accumulation of EIN2 and consequently an ethylene response (Qiao et al. 2009). About 700 F-box genes have been reported in Arabidopsis which are known to mediate proteolysis via ubiquitin-mediated proteasomal degradation, e.g., ORE9 required for initiation of Arabidopsis leaf senescence (Woo et al. 2001; Vierstra 2003). Recent studies in senescing carnation flowers have led to the identification of ethylene-dependent DCEIN2 (3,828 bp ORF encoding 139.5 kDa protein of 1275 amino acids) encoding protein-containing 12 putative transmembrane domains close to the N-terminus similar to the Arabidopsis EIN2, Petunia PhEIN2, and tomato SIEIN2 protein (Alonso et al. 1999; Fu et al. 2011a). Ethylene signaling downstream of EIN2 has been found to be mediated by EIN3 or EIN3-like EIL proteins (plant-specific transcription factors: Chao et al. 1997; Solano et al. 1998) that are regulated by two F-box proteins EBF1 and EBF2 (EIN3-binding F-box proteins) in a similar manner as EIN2 regulation by ETP1 and ETP2 (Guo and Ecker 2003, 2004; Bishopp et al. 2006). The expression of EBF2 has been found to be transcriptionally induced by EIN3 that directly binds to the promoter of EBF2, thereby allowing a negative feedback regulation to desensitize ethylene signaling and that EIN5, a 5′–3′ exoribonuclease, is most likely involved in moderating EBF1 and EBF2 transcripts (Gagne et al. 2004; Olmendo et al. 2006; Konishi and Yanagisawa 2008). Recently, a carnation cDNA (DCEBF1; 1,878 bp) encoding EBF-like protein has been isolated whose expression has been reported to be enhanced by endogenous/exogenous ethylene, and inhibited by STS in petals and ovaries (Fu et al. 2011b). EIN3 or EIN3-like proteins (EIL1, EIL2, EIL3, EIL4 and EIL5; nuclear-localized transcription factors) have been found to be upregulated during senescence (Waki et al. 2001; Alonso et al. 2003; Hoeberichts et al. 2003; Yanagisawa et al. 2003; Shibuya et al. 2004; Iordachescu and Verlinden 2005; Zhou et al. 2010). As far as structural aspect of ArabidopsisEIN3 protein is concerned, it has been found to harbor a highly acidic domain at N-terminus, five small clusters of basic amino acids throughout the EIN3 polypeptide, a proline rich domain, and an asparagine-rich domain at the C-terminus (Chao et al. 1997). Moreover, the Ps-EIN3-1 (from tree peony) has been reported to be strongly inhibited by ethylene and that decrease has been attributed to the activation of some defense mechanisms, thereby decreasing the tissue sensitivity to ethylene (Lorenzo et al. 2003; Zhou et al. 2010). In carnation, the upregulation of ACC synthase and ACC oxidase genes has been linked to the upregulation of EIL genes and it has been suggested that a master-switch controlling the coordinated upregulation of numerous ethylene responsive genes is involved in the senescence of carnation flowers, of which Dc-EIL3 might be part of. It has also been speculated that endogenous levels of soluble sugars in carnation act as a regulator of flower senescence by influencing Dc-EIL3 gene expression (Hoeberichts et al. 2007). Tieman et al. (2001) have demonstrated that reduced EIL expression in tomato (LeEIL1) affects ethylene responses, including leaf epinasty, flower abscission, flower senescence and fruit ripening. However, the expression of EIN3-like gene (EIL1) in Nicotiana sylvestris plants has not been found to consistently alter the progression of senescence (Yang et al. 2008). Genetic analysis revealed that EIL1 and EIN3 cooperatively but differentially regulate a wide array of ethylene responses, with EIL mainly inhibiting leaf expansion and stem elongation in adult plants and EIN3 largely regulating a multitude of ethylene responses in seedlings. When EBF1 and EBF2 are disrupted, EIL and EIN3 constitutively accumulate in the nucleus and remain unresponsive to exogenous ethylene application. Recently, it has been reported that EIN2 is indispensable for mediating ethylene-induced EIN3/EIL1 accumulation and EBF1/2 degradation (An et al. 2010).
EIN3 or EILs have been found to induce the expression of other transcription factors, including the ERFs (ethylene responsive factors formerly known as ethylene-responsive element binding protein; EREBP) and EDFs (ethylene-responsive DNA binding factors) (Ohme-Takagi and Shinshi 1995; Suzuki et al. 1998; Li and Guo 2007), which is evident by the presence of ethylene responsive elements (EREs) in some senescence-related genes (SR5, SR9 and SR12) and related transcription factors (Hunter and Reid 2001; Verlinden et al. 2002). ERFs are plant-specific AP2/EREBP-type transcription factors, characterized by the presence of a highly conserved DNA-binding domain (the ERF domain consisting of 58 or 59 amino acids) and regulate gene expression by binding specifically to the 11 bp GCC box of the ethylene responsive element of senescence-related genes (Ohme-Takagi and Shinshi 1995; Hao et al. 1998; Riechmann and Meyerowitz 1998; Yang et al. 2011). A number of ERF genes have been identified (as listed in Table 7) and classified into small groups on the basis of structural similarities, e.g., 12 groups (group I-X, VI-L and Xb-L) in Arabidopsis (Nakano et al. 2006). Similarly, Liu et al. (2011) has also characterized and classified the 13 ERFs from Petunia (PhERF1-PhERF13). Of the 13 ERFs, PhERF2 and PhERF3 have been shown to be associated with flower senescence. Yang et al. (2011) has identified a novel transcription factor (Cit ERF) in ERF family which has been suggested to play a variety of roles in some biological processes particularly fruit ripening and in enhancing different stress tolerances. It has been suggested that histone deacetylation plays an important role in epigenetic control of gene expression, e.g., HD2, a plant-specific histone deacetylase is able to mediate transcriptional repression in many biological processes. In longan fruit senescence, one histone deacetylase 2-like gene, DlHD2, and two ethylene-responsive factor-like genes, DlERF1 and DlERF2, have been cloned and characterized. The application of nitric oxide has been found to delay fruit senescence (by enhancing the expression of DlHD2 and suppressing the expression of DlERF1 and DlERF2) indicating that a possible interaction between DlHD2 and DlERFs in regulating longan fruit senescence. The direct interaction between DlHD2 and DlERF1 suggests that DlHD2 might act with DlERF1 to regulate gene expression involved in longan fruit senescence (Kuang et al. 2012).
The molecular and genomic revolutions have undoubtedly led to a revolution in the research being conducted in the field of plant senescence in general and flower senescence in particular. It is through molecular, mutational or transcriptomic approach that we have isolated and characterized numerous senescence-associated genes (genes coding for proteases, nucleases, transcription factors, ethylene biosynthesis and signaling, etc.). Moreover, the use of microarray technology, comprehensive transcriptomic sequencing projects and transgenic approaches will be of great help in bringing valuable information about the putative genes involved in flower senescence. This will provide us with a range of genes putatively involved in the implicated pathways leading to flower aging that may be blocked or induced to modify the progression of senescence. Although most of the genes or their corresponding proteins have been elucidated in detail; however, we are still far from developing an integrated picture of the executive mechanisms that control various aspects of senescence at molecular level that hinders our progress in addressing many open challenges regarding it. Thus, the major challenge for the researchers is to efficiently integrate the available information scattered in various flower systems into a flower senescence database (FSD) as has been developed for leaf senescence using bioinformatics approach. Moreover, the information gathered so far is based on studies conducted in a few model species like Arabidopsis, Petunia,Mirabilis, Rosa, Alstroemeria, etc.; therefore, another challenge in understanding the complex senescence regulation pathways is to extend this understanding to other species particularly the commercial ones (ornamentals) so that their vase life could be extended by exploiting the control points regulating flower senescence.
The authors thank Head of the department, Prof. I. A. Nawchoo and Ex-head Prof. Z. A. Reshi for cordial support.