Role of ethylene receptors during senescence and ripening in horticultural crops

Abstract

The past two decades have been rewarding in terms of deciphering the ethylene signal transduction and functional validation of the ethylene receptor and downstream genes involved in the cascade. Our knowledge of ethylene receptors and its signal transduction pathway provides us a robust platform where we can think of manipulating and regulating ethylene sensitivity by the use of genetic engineering and making transgenic. This review focuses on ethylene perception, receptor mediated regulation of ethylene biosynthesis, role of ethylene receptors in flower senescence, fruit ripening and other effects induced by ethylene. The expression behavior of the receptor and downstream molecules in climacteric and non climacteric crops is also elaborated upon. Possible strategies and recent advances in altering the ethylene sensitivity of plants using ethylene receptor genes in an attempt to modulate the regulation and sensitivity to ethylene have also been discussed. Not only will these transgenic plants be a boon to post-harvest physiology and crop improvement but, it will also help us in discovering the mechanism of regulation of ethylene sensitivity.

Introduction

Sessile organisms like plants cannot move and are dependent on themselves for their requirements and to combat environmental assaults. Where after they are equipped by nature with the simplest known yet powerful phytohormone “ethylene”, a gaseous plant hormone, which is known to produce myriad effects on plant physiology and its growth and development.¹ Ethylene is known to produce marked phenotypic changes in etiolated seedlings. It is globally characterized by the so called “triple” response reported for the first time in pea demonstrating the inhibition of epicotyl and root elongation, radial swelling of epicotyls and root cells, diagravitropical (horizontal) growth pattern and apical hook formation.² Ethylene is perceived by a family of ethylene receptors in the presence of a copper cofactor.³ Ethylene receptors and the immediate follower of receptor elements, Constitutive Triple Response (CTR1) element are the negative regulators of the cascade. The ethylene receptors are classified into 2 subfamilies. Subfamily I (ETR1, ethylene receptor1/ethylene resistant1 and ERS1, ethylene response sensor1) receptors have three membrane spanning domains at the N-terminus of the hydrophobic sensor domain where as subfamily II (ETR2, ERS2, EIN4, ethylene insensitive 4) have four domains, 3 of which are the membrane spanning domain and one is a putative signal peptide. Subfamily I receptors show histidine kinase (HK) activity^4,5 while, subfamily II receptors possess serine/threonine kinase activity.⁵ ERS1 is known to possess both the activities. Reversion to ethylene sensitivity (RTE1), co localized on endoplasmic reticulum (ER) with ETR1 acts as a positive regulator of ETR1 and is specific to ETR1, barring other receptors. It regulates ethylene signaling by physically interacting with ETR1.⁶ ETR1 is found to be both essential and sufficient for nutations among the ethylene receptors. However, it was concluded that ETR1 HK activity and phosphotransfer through the receiver domain are not needed to rescue nutations.⁷ Of the five Arabidopsis ethylene receptor, ETR1 is the best and most exhaustively studied. Almost every possible information about it has been deciphered Whether, it is ethylene binding activity,⁸ need of a copper co factor for better affinity,³ HK activity,⁴ localization in ER,⁹ interaction with CTR1¹⁰ or its disulfide linked dimer formation.⁸ The other companion of this receptor in subfamily I i.e.ERS1 has also been shown to depict ethylene binding activity.¹¹ Binder (2008)¹² has elaborated on the models for receptor function and output. Epistatic studies have revealed that ethylene receptors namely ETR1, ETR2, ERS2, ERS1and EIN4 act upstream of the CTR1, whereas the EIN2, EIN3, EIN5, EIN6 and EIN7 act downstream of CTR1.^13,14 EIN3 and related EIN3-LIKE (EIL) are known to function as transcription regulators of ethylene primary response genes.¹⁵ CTR1 activates Ethylene Insensitive 2 (EIN2), cytoplasmic protein which is related to Nramp family of metal transporters.¹⁶ Ethylene Insensitive 3 (EIN3) has a DNA binding ability and is located in nucleus. It acts upon Ethylene response Factor1 (ERF1) gene which encodes for an ethylene response element binding protein (EREBP) now known as ERF (ethylene- responsive element binding factor) that binds a GCC-box, a cis element of many ethylene response genes.¹⁵

A short post-harvest longevity has been a detrimental factor for many crops. Separation from plant leads quickly to ripening of fruits and senescence of flowers. In many species ripening and senescence are ethylene regulated. Thus, attempts have been made to retard the post-harvest processes by applying chemicals that inhibit ethylene synthesis like 1-MCP (1-Methylcyclopropene) marketed as antisenescence agent EthylBloc, silver thiosulfate, AVG (amino ethoxyvinylglycine hydrochloride) marketed as Retain. 1-alkyl-cyclopropenes and AgNO₃ are the most prominently used ethylene antagonists.^17-22 Another class of strained alkenes, cyclobutenes and trans cyclooctene have recently been brought into picture because of their greater convenience in handling and administration.²³ Kazemi et al. (2011)²⁴ reviewed the effect of various chemicals at different concentrations on Gebrera cut flowers. However, the long-term solution will probably be based on genetically modified plants with genes that either suppress synthesis or reduce sensitivity to ethylene. A few mutants that impart ethylene insensitivity are known e.g., tomato Nr and dominant mutants of Arabidopsis ethylene receptor sensor genes ETR and ERS. Genetically modified crops with etr1–1 allele have shown delayed fruit ripening, flower senescence and flower abscission of tomato and petunia. Mutants of ETR1 and ERS1 receptor from Zea mays has shown to confer ethylene insensitivity in Arabidopsis but in the presence of subfamily I Arabidopsis ethylene receptors.²⁵ This is an example of functional conservation of ethylene receptors across the genus. ERS1 not only represses ethylene responses but also promotes ethylene responses in ETR1 dependent manner implying collaboration among ethylene receptors and regulation of receptor mediated signaling.²⁶ Recent finding by Liu and Wen (2012)²⁶ showed a difference in ethylene responses when dominant ethylene-insensitive receptor was used in the background of wild type ethylene receptor. It was opined from this observation that different combination of ethylene receptors can facilitate differential receptor signal output thus regulating the ethylene activity. Collaboration among the receptors was further substantiated by the finding that they form protein complexes to relay signal as per the specific cellular environment and responses.²⁵ Characterization of ethylene receptor genes provide clues to understand how plants regulate their ethylene sensitivity. Therefore, an alteration of ethylene action is a valuable target for the genetic engineering of crops. Manipulation of ethylene biosynthesis or perception allows us to modulate these processes and thereby create plants with more robust and desirable traits, giving us a glimpse into the role of ethylene in the plant. Having known about the ethylene biosynthetic pathway and signaling molecules, the approach has been to manipulate the perception of hormone not directly involving the biosynthesis.

Modification in the levels of plant hormones and their signaling have augmented the life and yield of many agriculturally important crops as reviewed by Csukasi et al. (2009).²⁷ Ethylene is not just a ripening hormone in addition, the phytohormone plays role in physiological processes throughout the life cycle of the plant.^1,28 Its impact on nodulation in legumes mediated by manipulated ethylene biosynthesis is reviewed in detail by Shaharoona et al. (2011).²⁹ Its involvement in such agronomically important processes such as senescence, abscission and fruit ripening has made ethylene a target for manipulation by genetic engineering methodologies.¹⁶ The role of ethylene in controlling abscission has been reviewed by Binder and Patterson (2009).³⁰ Key aspects of research on regulatory networks controlling ethylene synthesis, its role in flower development and fruit ripening integrating knowledge from the model plant Arabidopsis and other plant species has been focused on by Lin et al. (2009).³¹ This review aims at findings involving regulation of ethylene perception and its hormonal levels mediated mainly by the receptors and the downstream molecules involved in the signaling cascade. Expression studies have also been covered to get a better insight into the role of these molecules in regulating senescence and ripening in a crop specific manner.

Targeting Ethylene Perception, and Not Ethylene Biosynthesis

There have been many prolific attempts to procrastinate senescence by exploiting ethylene biosynthetic pathway. Little et al. (2009)³² have elaborated on how an altered ethylene manufacturing modified reception by its receptors or an intervened signaling keeps a check on senescence, stress and disease, at the same time regulating ripening and flowering. Stearns and Glick (2003);³³ Czarny et al. (2006)³⁴ have documented how the biosynthetic pathway rate limiting enzyme ACC synthase, ACC oxidase along with other enzymes on being suppressed using antisense approach or overexpressed have fetched with the desired outcome. The approach has been put to use in different crops like tomato, tobacco, potato, carnation, begonia, torenia, cantaloupe, broccoli, canola, melon, etc. Reverse genetics approach using TILLING platform was exploited in melon to identify novel alleles involved in ripening by Mardas et al.,³⁵ Blocking ethylene production by targeting enzymes has been an approach to combat senescence to good extent but with contraindicated repercussions. Another approach that has gathered attention in recent years is reducing the sensitivity of plant for ethylene without altering the biosynthetic pathway.

A few points are enumerated that could have led to the paradigm shift to modulate the plant’s sensitivity to ethylene instead of manipulating the biosynthetic pathway.

(1) ACC synthase, the rate limiting enzyme and ACC oxidase enzyme of the pathway are coded by a multigene family and undergo posttranslational modifications to give rise to different isoforms which are active under different physiological conditions.^1,36,37-39 Different isoforms of these enzymes limits the success of antisense suppression of the genes coding for these enzymes in other crops, e.g., Oeller et al. (1991)⁴⁰ observed limited success while using antisense ACC synthase to lower plant ethylene levels in tomato. In addition, ACC oxidase is present in meagre amounts in plant tissues.⁴¹

(2) Antisense suppression of carnation ACC oxidase resulted in noticeable decline in ethylene production during flower senescence and enhanced longevity of flowers. This could only check the endogenous production of ethylene⁴² and application of exogenous ethylene which would re-establish the impact of ethylene. However, the same idea did not work in case of begonia.⁴³

(3) S-adenosylmethionine (SAM), initiator of ethylene biosynthetic pathway acts a universal methylating agent for proteins, nucleic acids, lipids, carbohydrates and other biomolecules. In an attempt to reduce the availability of SAM for ethylene production by overexpression of SAM hydrolase⁴⁴ or sense and antisense expression of SAM decarboxylase (this enzyme produces decarboxylated SAM, used for polyamine biosynthesis) in potato leads to unusual phenotype.⁴⁵

(4) There are certain events in flower development like flower opening, ovary development or pedicel elongation which are elicited by ethylene even in the flowers where petal senescence is virtually irresponsive to ethylene. So, it would not be a good idea to disturb the biosynthetic pathway thus hampering the normal development of the flower.²¹

(5) Pollination induces increased ethylene production leading to enhanced senescence, which is described as a mechanism to terminate the life of flower after successful pollination to benefit the survival of species.⁴⁶ Decreased ethylene sensitivity by using mutated ethylene receptors would retard senescence.⁴⁷

(6) Czarny et al. (2006)³⁴ and Hoffman et al. (1999)⁴⁸ have reviewed that blocking the ethylene biosynthetic pathway can put a check on scattering effect of damage caused due to an infection or stress in plant. But it seems unreckonable to check the upsurge of second ethylene peak which leads to more profound and collateral damage. The second peak of ethylene is originated due to faster transcription of ACC synthase genes and antisense suppression of such a large pool of ACC synthase genes does not seem feasible.

(7) Numerous studies have also revealed that antisense expression of ethylene synthesis genes varied according to organ,⁴⁹ developmental stage, age of organ⁵⁰ and enzyme induction factors.⁴⁹ Transgenic expression will further vary according to the promoter employed, transformation method, species and other variable factors e.g., transgenic petunia were developed using antisense broccoli ACC synthase (BoACS1) and ACC oxidase (BoACO1) genes. BoACO1 was able to reduce ethylene biosynthesis and delay flower senescence of Petunia hybrida more efficiently than the other gene.⁵¹

(8) Dandekar et al. (2004)⁵² used antisense versions of apple ACC synthase and ACC oxidase to transform the apple with the aim of studying the role of ethylene in apple ripening. Ethylene production was drastically hampered and fruits ripened very slowly but with a side effect of decreased concentration of flavor ester forming enzymes.

(9) Transgene flow through pollen dispersal is one of the major concerns because it could lead to introduction of undesirable traits in other plants and weeds. Transgenics raised by targeting ethylene biosynthetic pathway did not offer any solution to the problem. However, using mutated ethylene receptor, the issue was sorted out.

Role of Ethylene in Climacteric and Non-Climacteric Plants

Flowers can be categorized as being ethylene sensitive or ethylene insensitive. In climacteric or ethylene sensitive flowers such as carnations and orchids, senescence is accompanied by a sudden transient increase in ethylene production and respiration, while treatment of non-senescent flowers with ethylene rapidly induces petal senescence. In non climacteric flowers such as Gladiolus, tulip, and iris, generally, no increases in ethylene production and respiration are apparent during flower senescence, and exogenous ethylene application has little or no effect on petal senescence.^21,53 Several studies to date suggest that abscission and senescence of flowers may be triggered by the perception of endogenous ethylene by ethylene receptors. Abscission is a typical ethylene response induced through ethylene receptors and is influenced by mutations in ethylene receptors.⁵⁴ Therefore, investigations of ethylene receptors and associated signal transduction pathways are essential for understanding ethylene perception in flowers. However, these changes are not understood in best capacity when it comes to non climacteric fruits. The molecular mechanism of fruit ripening is a complex phenomenon, as the dynanism and degradation of ethylene receptor has a significant part to play. The major difference between climacteric and non-climacteric plants is marked by the presence or absence of autocatalytic ethylene production. There are several aspects which are focused on to modulate ripening and senescence in plants and authors have reviewed these different perspectives. For example, Palma et al. (2011)⁵⁵ has focused on the total proteome involvement in the molecular physiology of fruit ripening in important climacteric and non climacteric crops. Bapat et al. (2010)⁵⁶ has enlisted the cases where ripening of fleshy fruits is arrested by targeting the ripening related genes. Here, we have listed all the horticultural crops which are either genetically modified using ethylene receptor gene for altered perception and senescence (Table 1) or in which the expression of these receptors and transcription factors involved in signaling cascade has been studied.

Table 1. Transgenic crops with altered phenotype, senescence and ripening due to overexpression and suppression of ethylene receptor and downstream signaling molecules.

Crop	Gene	Function of the Gene	Expression	Effect/Phenotype	Conclusion	References
Tomato	NR (Receptor gene) Semi Dominant	Functionally redundant ethylene insensitive in all tissues.	Antisense suppression	Normal ripening as in wild type fruit. No effect on ethylene sensitivity, increased levels of LeETR4 mRNA. No effect on ethylene sensitivity of seedlings	Not essential for ripening. Ethylene perception and signaling via the NR receptor is consistent with the receptor inhibition model. Functional compensation of LeETR4 for reduced NR expression	Hackett et al., 2000172 Lanahan et al., 1994163 Tieman et al., 2000167
		NR (Never Ripe) Semi Dominant	Functionally redundant ethylene insensitive in all tissues.	Overexpression in LeETR4 suppressed lines	Eliminates ethylene sensitivity, lack of epinasty, flower senescence and normal fruit set, rescued the ethylene response phenotype.	Proves the functional redundancy of the receptors. Negative Regulators of ethylene response. Only associated with ripening.	Tieman et al., 2000167
		Nr (Mutant NR gene)	Unable to bind ethylene, delay senescence, imparts ethylene insensitivity.	Antisense suppression	Activated ethylene responses and onset of normal ripening, plant with two homozygous copies of transgene produced wild type fruit. Single gene in a homozygous state gave intermediate phenotype.	Threshold level of mutant receptor is needed to suppress the ethylene response pathway. Support receptor inhibition model.	Hackett et al., 2000172 Lanahan et al., 1994163
		LeEIL(1-3) Transcription Factor	Functionally redundant, positive regulator of ethylene signaling.	Antisense suppression	Single LeEIL suppression results no change. Multiple LeEIL gene suppression delays leaf epinasty, flower abscission, flower senescence.	Normal levels of expression of the other two LeEILs can compensate for the loss of single gene.	Tieman et al., 200123
		LeEIL1 in tomato Nr mutant	Functionally redundant, positive regulators of multiple ethylene receptors.	Overexpresession in ethylene insensitive non ripening Nr mutant of tomato	Partial restoration of ripening. Seedling triple response, upregulation of all ripening related genes were not restored.	LeEIL1 may not have expressed at sufficiently high levels to restore these functions,LeEIL2, LeEIL3 are also required.	Chen et al., 2004175
		LeEIN2	Positive mediator of ethylene signaling and fruit development	Suppression (VGIS)	Delayed fruit development and ripening with fewer seeds and locules, suppressed ethylene inducible and ripening related genes, partially blocked ripening with large green parthenocarpic sector on fruit	Probable involvement of LeEIN2 in a crosstalk between ethylene and auxin during fruit development	Zhu et al., 2006174
		LeETR4	Imparts ethylene in sensitivity, delay the onset of ripening until the seeds have matured.	Suppression	Increased ethylene sensitivity, fruits ripen prematurely flower senesce early without an increase in ethylene level. Severe epinastic curvature of petioles, no change in the levels of NR.	Negative regulator of the ethylene signaling pathway, lowering the receptor levels increases sensitivity to ethylene. An important regulator of ripening	Tieman et al., 2000167
		LeETR4 using immature fruit specific Tfm7 promoter	Delays onset of ripening in immature fruits	RNAi mediated suppression	Early ripening, total yield, fruit size and flavour related chemical composition had no negative effect. Leaves and other tissues were unaffected.	Tissue specific approach to hasten fruit development without unwanted effects and enhanced ethylene response by ethylene receptor depletion.	Kevany et al., 2008171
		LeETR1	Functionally redundant	Antisense expression of receiver domain and the 3′ UTR of LeETR1	Delayed abscission and shorter internodes with no effect on ethylene sensitivity, normal triple response, and reduced auxin movement. No effect on NR transcripts.	Receiver domain of LeETR1 and LeETR2 has a role to play in signaling and can not be compensated by NR receptor	Whitelaw et al., 2002176
		etr1-1 (mutated ethylene receptors from Arabidopsis)	Imparts dominant ethylene insensitivity	Overexpression	Long thin hypocotyls, normal root growth, more open and expended apical regions. Fruits with delayed ripening and senescence similar to Nr mutants	Confers ethylene insensitivity in heterologous plants	Wilkinson et al., 1997112
	LeETR1 and LeETR2	Non redundant, plays role in ethylene perception	Antisense suppression	Shortened, thickened hypocotyls, enhanced abscission of petiole explants, faster senescence of flowers both in presence and absence of flowers.	LeETR1 might have a more specific role to play in signaling, each member has a specific role to play and are not totaly redundant.	Wang et al., 2003170
Carnation	etr1-1 using CaMV 35S promoter	Imparts global ethylene insensitivity	overexpression	Diminished ethylene insensitivity and diseased resistance with delayed senescence. No petal inrolling, firm petals.	Heterologous expression imparts insensitivity in carnation	Bovy et al., 199979
	etr1-1 using flower specific fbp1 promoter	Imparts global ethylene insensitivity	overexpression	Delayed flower senescence with increased disease resistance. Ethylene sensitivity increased in other parts of flower apart from petals, better seed germination and reduced leaf abscission.	Flower specific promoter fixes the compromised disease resistance status	Bovy et al., 199979
Petunia	etr1-1/Nr	Imparts global ethylene insensitivity	overexpression	Ethylene insensitivity with delayed flower senescence. Turgid corollas of flower, delayed abscission.	Cross species transfer	Wilkinson et al., 1997112
	etr1-1 using flower binding protein from Petunia (fbp1) or apetala 3 promoter from Arabidopsis	Imparts global ethylene insensitivity	overexpression	Diminished disease resistance and reduced adventitious root hair formation, increased ethylene insensitivity with delayed senescence, seedling root hair formation observed. 73% of fbp1 and 32% of ap3 plants showed more than 100% increase in flower life.	Flower specific promoter enhances disease resistance in cross species transfer	Cobb et al., 2002114
	boers (mutated form of BOERS from Brassica oleracea)	Imparts ethylene insensitivity	overexpression	Plants retained turgidity and pigmentation longer, larger flowers, extended flower longevity, diminished disease resistance with higher mortality, increased ethylene evolution and reduced ethylene sensitivity, self incompatible.	boers is a dominant allele imparting insensitivity in the heterozygous transformed plants	Shaw et al., 2002177
	PhEIN2 (downstream signaling protein)	Involved in regulation of accumulation of EIN3 and EIL1 proteins. Positive regulators of ethylene cascade	Suppression by expressing sense RNA and RNAi mediated silencing	Transgenic lines using sense RNA expression showed moderate delay in flower senescence and fruit ripening with no premature death reported. RNAi construct suppressed lines showed great delay in flower senescence and fruit ripening with great deal of premature death. Corollas were turgid even after the ovaries were turgid.	Reduced or inhibited adventitious root formation (sense RNA expression) Reduced ethylene sensitivity (RNAi suppression)	Shibuya et al. 2004119
Coriander	AtERS1 (mutated dominant receptor)	Confers ethylene insensitivity	overexpression	Delayed leaf senescence with prolonged total chlorophyll content in transgenic leaves, delayed flower senescence.	Delayed senescence is not due to drastic reduction in ethylene biosynthesis rather it is due to the effect of dominant negative ERS1 transgene. Distantly related crop species can have extended shelf life exploiting heterologus gene transfer.	Wang and Kumar 200491
Campanula	etr1-1 with fbp1	Imparts global ethylene insensitivity	Overexpressed	Enhanced tolerance to exogenous ethylene and reduced ethylene sensitivity, stably passed to next generation of plants, weak expression of etr1-1 in leaves, intact fertility and no compromise on disease resistance.	Sensitivity to ethylene in plants is independent of the copy number of the T-DNA.	Srikandarajah et al., 200757
Nemesia	CmETR1/H69A, missensed mutated ethylene receptor from Cucumis melo	Imparts reduced ethylene sensitivity	Overexpression	Delayed flower senescence by 1--3 days and lesser root hair formation compared to non transgenic wild type.	Artificially mutated ethylene receptor gene conferred reduced ethylene sensitivity in heterologus plants	Cui et al., 2004109
Lettuce	CmERS1/H70A	Imparts stable sterlity, reduced fertility along with ethylene insensitivity	Overexpressed	Apart from reduced ethylene sensitivity, induced sterility in vegetatively propagated plants and pollen propagated plants	This mutated receptor gene checks the pollen dispersal from transgenic plants and prohibit unwanted development of traits in other plants and weeds, can induce sterility in heterologus transgenic plants.	Takada et al., 2007140
Chrysanthemum	mDG-ERS1 (etr1-4), mutated nucleotide substitution corresponding to etr1-1.	Imparts ethylene insensitivity	Overexpressed	Delayed leaf yellowing on exposure to exogenous ethylene and leaves on lower part of stem remain attached to it without yellowing, reduced ethylene sensitivity, detached shoots in darkness without ethylene treatment showed reduced senescence.	Heterologus gene transformation remained unsuccessful; the mutated receptor could work for compositae family members.	Narumi et al., 2005b85 Satoh et al., 2006, 2007, 200887,88,89
	Cm-ETR1/H69A (missensed mutated ethylene receptor from Cucumis melo)	Imparts ethylene insensitivity, male sterility, prevents transgene flow via pollen	Overexpression	Drastic reduction in number of pollen grains at temp 20--35 °C but observed at 10-15 C. Due to suppression of Cm-ETR1/H69A expression at low temperature and optimal growth temp for plant at 15--20 °C. Female fertility was also reduced.	Optimal growth temperature for Chrysanthemum collides with its vegetative, reproductive growth resulting in mature pollens to flourish	Shinoyama et al., 201290
Lotus	Atetr1-1	Mutated dominant ethylene insensitive ethylene receptor	Overexpression	Delayed abscission and senescence of petals, ethylene insensitive, twisted coiled hypocotyls, no triple response, fewer lateral roots, 7-fold increased nodulation in highly ethylene insensitive transformants, 1.7-fold increases in bacteroid infection.	Multiple roles of ethylene in nodule initiation by influencing root cell interactions and radial positioning, independent of autoregulation and nitrate inhibition of nodulation	Lohar et al., 2009105
	Cm-ERS1/H70A	Inhibits ethylene sensitivity, promotes root nodulation.	Overexpression	Confers ethylene insensitivity and fixes the transgene in T3 generation, reduced ethylene sensitivity due to 1-ACC resistance, increased flowering	Transgenic alteration of ethylene perception alters the rhizobial infection and nodulation phenotype.	Nukui et al., 2004104
Cucumber	At-etr1-1 with CaMV 35S, AP3, CRC promoter	Imparts dominant ethylene insensitivity	Overexpression	Prevents carpel development resulting in male flowers with CaMV35S promoter. Increased femaleness and conversion of bisexual to female buds with CRC promoter. Exclusive male flower production with AP3 promoter.	Heterologus ethylene insensitivity. Ethylene perception is required to promote femaleness in melon. Dual role of ethylene in sex expression and carpel maturation. Site for ethylene perception lies in stamen primordia and not in carpel primordial.	Little et al., 2007145

Genetically Modified Ornamental Horticultural Crops and Role of Ethylene Receptors, Downstream Molecules in Regulating their Senescence and Ripening

Campanula

Campanula carpatica is an ethylene-sensitive ornamental horticultural crop, which wilts in three days on ethylene exposure. The plant was transformed exploiting etr1-1 gene from Arabidopsis under the influence of a flower specific fbp1 promoter from Petunia. Enhanced tolerance to exogenous ethylene was observed in transgenic lines and this reduced sensitivity to ethylene was stably passed on to the next generation. The better part of the result was that there was no compromised disease resistance, no interference with root forming ability and fertility of the plant. On crossing, a two T-DNA containing transgenic plant with an ethylene sensitive cultivar, the T1 progeny obtained plants were with high tolerance to ethylene. The degree of ethylene tolerance in plants with a single or double copy number did not vary at all showing that the sensitivity is independent of the copy number of the transgene.⁵⁷

Carnation

During natural and pollination-induced senescence of the flowers, ethylene is first produced in the gynoecium (pistil) and the evolved ethylene acts on petals and induces the expression of genes for ethylene biosynthetic enzymes and cysteine proteinase resulting in the autocatalytic ethylene production from the petals and wilting of the petals.^58-60 Also, exogenous ethylene applied to carnation flowers, which have not yet started ethylene production, induces autocatalytic ethylene production in petals, resulting in wilting of the petals.^61,62 Thus, carnation (Dianthus caryophyllus L.) flowers are considered to be an excellent model system for the study of ethylene perception and signaling. Three ethylene receptor genes have been isolated in carnation namely DC-ERS1,⁶³ DC-ERS2⁶⁴ and partial sequence of DC-ETR1.⁶⁵ The expression analysis revealed that only DC-ERS2 and DC-ETR1 are involved in flower opening but in a tissue specific manner as the mRNAs of both the genes are present in abundance in petals at the fully opened stage and level of both the mRNAs, DC-ERS2 in particular declined as the senescence progresses. However, the level of DC-ERS2 increased slightly in ovaries and that of DC-ETR1 remained constant whereas, expression was observed to be constant during opening and senescence in the style.⁶⁶ Flowers treated with 1, 1-dimetly-4-(phenylsulfonyl)-semicarbazide (DPSS) are known to be blocked in ethylene production⁶⁷ and result in decreased expression of the genes in petals independent of ethylene production. In presence of exogenous ethylene, the expression stayed the same as it was under the influence of increase in endogenous ethylene production. The decrease in DC-ERS2 mRNA in petals with the progress of senescence goes in accordance with the negative regulation model of ethylene signaling but further work needs to be done at the protein level to confirm this.⁶⁶ An EIN3-like protein (DC-EIL1), downstream to receptors was identified in carnation by Waki et al. (2001).⁶⁸ DC-EIL1 transcript levels decreased in petals during natural senescence, marginal increase in DC-EIL1 expression has been reported in ovaries as the senescence approaches, which otherwise was almost constant in styles and ovaries even during senescence. Exogenous ethylene and ABA treatment showed increased expression initially followed by a decline as the senescence progresses. However, small amount of RNA accumulated on ABA and ethylene treatment. Since the exogenous application of ethylene or ABA leads to declined DC-EIL1 mRNA level and increased ethylene production during natural senescence demonstrated the same results. Therefore, it can be interpreted that EIN3 level decreased with the increase in ethylene production, contradicting the findings of Chao et al. (1997)⁶⁹ and Kosugi and Ohashi (2000)⁷⁰ which states that EIN3 and TEIL mRNA levels in Arabidopsis and tobacco respectively are not affected by ethylene. This indicates that there could be other senescence factors apart from ethylene resulting in decline of DC-EIL1 expression. It is known that EIN3 and its homologs act as positive regulator of ethylene response, the DC-EIL1 transcripts were therefore expected to increase or remain unchanged in the presence of ethylene. However, the declined DC-EIL1 mRNA levels indicate the decrease in ethylene response of petal tissues, thus mitigating the ethylene response in senescing carnation petals in response to exogenous and natural ethylene produced. This contradicts the predicted enhanced ethylene response in the tissues due to decreased DC-ERS2 and DC-ETR1 expression in carnation.⁶⁶

Overexpression of several EIN3/EIL members confers constitutive ethylene responses without significant changes in transcript levels suggesting that EIN3 may be regulated at the protein level⁷¹ as shown by Guo and Ecker (2003);⁷² Potuschak et al. (2003)⁷³ and Yanagisawa et al. (2003).⁷⁴ Apart from DC-EIL1 three more EIN3-like (EIL) genes DC-EIL1/2, DC-EIL3, DC-EIL4 were isolated, cloned and analyzed for their expression in vegetative and flower tissues (petals, ovaries and styles) during growth and development under natural and ethylene induced senescence in carnation (Dianthus caryophyllus) by Iordachescu and Verlinden (2005)⁷⁵ to get a better insight into ethylene responsiveness during flower development and senescence leading to ethylene climacteric. DC-EIL3 mRNA levels in flower petals and style increased both under normal flower development and exogenous ethylene application. The EIN3 like proteins are also known to interact with ethylene responsive elements (EREs).¹⁵ These observations suggest that DC-EIL3 may be playing a role in regulating gene expression, therefore regulating senescence. However, dip in DC-EIL3 expression in wounded plant suggests the suppression of onset of senescence in leaves either by decreasing the tissue sensitivity to ethylene or activation of some defense mechanisms as observed in ovaries during senescence with a temporary decline in DC-EIL3 mRNA status when higher ethylene production is reported in the flower (negative correlation of ethylene production and DC-EIL3 mRNA accumulation). Thus, showing differential regulation of DC-EIL3 in different parts of the flower. Another evidence of dynamism of DC-EIL3 was supported by the unexpected maintained increase in its mRNA level in sucrose treated ovaries of the flower. Sucrose and other sugars are known to extend the vase life of flowers,⁷⁶ however sucrose induced the decline of ethylene responsiveness in carnation petals,⁷⁷ raising the expected possibility of delayed increase in DC-EIL3 mRNA. Also the elevated levels of DC-EIL1/2 and DC-EIL4 mRNA in flowers treated with sucrose were in contradiction with depleted ethylene responsiveness, prompting tissue dependent and complex ethylene signaling.

Exploiting the fact that ethylene production shoots up post pollination in carnation flowers at different intervals of time,⁷⁸ an attempt was made to interpretate the regulation of DC-EINs with the ethylene production in flower. NBD (an ethylene inhibitor) treated pollinated flowers showed higher accumulation of DC-EIL1/2 and DC-EIL4 transcripts (at 12 and 48 h after pollination) which was expected to show lower DC-EILs mRNA levels. DC-EIL3 transcript levels showed subtle differences between NBD non treated flowers and only pollinated flowers. It was therefore concluded that EIN3 like genes and DC-EIL3 gene in particular is involved in significant regulation by ethylene in initiating and sustaining of the senescence process in Carnation petals.⁷⁵

Transgenic carnation plants were obtained by using etr1-1 allele driven by constitutive CaMV 35S or flower specific petunia fbp1 promoter,⁷⁹ CMB2 (Carnation MADS box) containing promoter.⁸⁰ Lower transformation efficiency along with diminished disease resistance and lower ethylene sensitivity^48,81was observed using constitutive promoter compared with flower specific promoters, which took care of both the above stated drawbacks.^79,82 Symptoms like petal in rolling phenotype, hallmark of ethylene-dependent carnation flower senescence was not observed in transgenic carnation flowers. Instead, the petals remained firm throughout the senescence process and gradually became brown on the edges (loosing color) and died from rotting.⁷⁹ Transgenic carnation cut flowers had three times the vase life of non transformed flowers and lasted longer than flowers treated with inhibitors of ethylene biosynthesis or ethylene antagonists.^79,80

Catharanthus

The ETR1 gene deciphered in Catharanthus roseus showed highest transcript levels in petals and ovaries. The expression level was almost unperturbed by ethefon treatment, under any abiotic stress or by other hormonal actions. However, the amount of monoterpene indole alkaloid Ajmalicine in the cells subjected to exogenous ethylene was reduced on being treated with inhibitors of HK. This prompted the involvement of this receptor protein in ethylene related enhancement of alkaloid biosynthesis in Catharanthus.⁸³

Chrysanthemum

Chrysanthemum is an ornamental plant known to be insensitive to ethylene. However, some cultivars of Chrysanthemum are ethylene sensitive. Narumi et al. (2005a)⁸⁴ observed that in an ethylene insensitive cultivar of Chrysanthemum the DG-ERS1 transcript level was highest and declined consistently thereafter. The same was not observed in an ethylene-insensitive cultivar, though. The observed difference between the two cultivars is probably related to the variation in sensitivity to ethylene between them and this suggests that the DG-ERS1 gene and its resultant DG-ERS1 protein may be involved in the perception of ethylene signal in flower and leaf tissues of the cut Chrysanthemum.

DG-ERS1 gene was subjected to one nucleotide substitutions corresponding to Arabidopsis etr1-1, etr1-2, etr1-3, etr1-4 and tomato Nr. These mutated DG-ERS1 receptors were transformed into Chrysanthemum (ethylene-sensitive cultivar) to attain reduced ethylene sensitivity. Failure with heterologous transgene expression in Chrysanthemum, led to this idea of mutating the Chrysanthemum ethylene receptor for imparting reduced ethylene sensitivity. Out of the five DG-ERS1 transgenes tested, DG-ERS1 (etr1-4) emerged the best resulting in 37% reduced ethylene sensitive transgenic Chrysanthemum leaves.⁸⁵ Sumitomo et al. (2008)⁸⁶ used the transgenic Chrysanthemum expressing a mutated ethylene receptor genes, DG-ERS1 (etr1-4) and DG-ERS1(Nr) with reduced ethylene sensitivity to assess the involvement of ethylene response pathway in temperature induced dormancy of Chrysanthemum. Satoh et al. (2008)⁸⁷ evaluated ethylene sensitivity of the transgenic grown in soil. They observed delayed leaf yellowing in shoots after being detached from soil grown plants on ethylene treatment under continuous light. Also, these shoots when kept in darkness without ethylene treatment resulted in reduced senescence. Natural senescence in shoots was also analyzed from soil grown plants by Satoh et al. (2006, 2007).^88,89 All the observations and failure of heterologus transformation in Chrysanthemum suggests that the mutated ethylene receptor DG-ERS1 (etr1-4) could be used to combat senescence in composite leafy vegetables plants, which cannot be stored for a long time at cool temperatures because of cold injury to plants.

Transgene flow from transgenic plants to wild types (horizontal gene transfer) has always been a concern. To tackle the problem male sterility needs to be introduced to prevent transgene flow via pollen. Shinoyama et al. (2012)⁹⁰ overexpressed the mutated ethylene receptor gene from melon (CmETR1/H69A) into Chrysanthemum which resulted in delayed tapetum degradation of anther sac and reduced pollen grain formation. However, the concurrence of healthy growth pattern and flourished pollen formation at a temperature of 15–20°C was a hindrance to put a check on pollens from genetically modified chrysanthemum to mingle with its wild type counterparts.

Coriander

Delaying senescence in coriander leaf and flower using a dominant negative mutant ERS1 of Arabidopsis is yet another attempt of heterologous expression. Expression analysis of ACO and ERS1 homologs in both transgenic and wild­type plants revealed subtle downregulation of ACO in transgenic plants compared with wild type. The marginal downregulation of ACO was attributed to the failure of feedback control mechanism of ethylene synthesis by heterologous dominant negative receptor. Thus, the delay in senescence in transgenic plant resulted due to the effect of dominant negative ERS1 and not due to reduction of ethylene biosynthesis. Also ERS1 homolog in coriander was not altered significantly indicating no co-suppression by the transgene to confer its function.⁹¹

Delphinium

Delphinium flowers show a climacteric-like rise in ethylene production before abscission of sepals. These are abscised by exposure to exogenous ethylene.⁹² Two ethylene receptor genes in Delphinium have been isolated namely Dl-ERS1 type1 and Dl-ERS1 type 2 differing by three amino acids.⁹³ Two more cDNA encoding ethylene receptors Dl-ERS1–3 and Dl-ERS2 were isolated from Delphinium flowers by Tanase and Ichimura, (2006).⁹⁴ However, the most common ethylene receptor ETR1 seems to be missing from Delphinium. The flower parts showed higher Dl-ERS1-3 transcript levels than leaves and stems whereas, Dl-ERS2 transcript levels in leaves and flowers were found to be equal but greater than that of stem. These observations suggest differential regulation of receptor genes (Dl-ERS1-3 and Dl-ERS2) and their involvement in ethylene signaling of Delphinium flower senescence.

Considering the fact that ethylene receptors are not specifically expressed in the abscission zone,⁹⁵ it was concluded by Tanase and Ichimura, (2006)⁹⁴ that Dl-ERS1-3 and Dl-ERS2 are not expected to determine the ethylene response of the abscission zone. Therefore, ethylene induced sepal abscission is controlled both by ethylene signaling components and Dl-ERS1-3 and Dl-ERS2 proteins.

Kuroda et al. (2003)⁹³ also concluded that Dl-ESR1 expression is more or less proportional to the endogenous ethylene production implying that ethylene evolved by the florets is perceived by elevated levels of Dl-ERS1 leading to flower senescence.

Dendrobium

Dendrobium “Pompadour”, one of the most important commercial orchids in Thailand is an ethylene sensitive variety and has relatively short vase life. A subfamily one ethylene receptor, ERS1, lacking a receiver domain was found in Dendrobium with a single copy in the genome by Thongkum et al. (2009).⁹⁶ However, the possibility of presence of anther ethylene receptor gene cannot be ruled out.

Transcript analysis of Den-ERS1 was also performed in various vegetative tissues like psuedobulb, young and mature leaves, root, peduncle, bud and pen floret. The transcript expression remains unperturbed with the development of leaves. During the flower development the expression of Den-ERS1 declined as the senescence progressed and the ethylene production was enhanced suggesting the receptors to be negative regulators of ethylene signaling during natural course of senescence.⁹⁶

Geranium

Two ethylene receptors, PhETR1 and PhETR2 are deduced till date, though there is scope for more receptors to be decoded.⁹⁷ They have also been characterized for their distribution, temporal and spatial regulation in Geranium (Pelargonium x hortorum L.H. Bailey). The rapid nature of ethylene response was figured out by Clark et al. (1997)⁹⁸ by demonstrating that during the receptive stage of pollination, Geranium florets were observed to undergo complete abscission even in the turgid petals when treated with very low concentrations of exogenous ethylene.⁹⁸ Self pollination of Geranium flower causes rapid burst of ethylene production by the gynoecium within 1 h, which leads to petal abscission in 2–4 h. Enhanced sensitivity of Geranium florets to exogenous ethylene at the pollination receptive stage was also reported by Evensen et al. (1991).⁹⁹

PhETR expression analysis revealed that it was expressed least in roots and maximum in pistils. Considering these observations, hypothesis put forth by Dervinis et al. (2000)⁹⁷ cannot be ruled out, which states that the control of ethylene sensitivity in Geranium florets may not be determined by the amount of receptor protein present justifying the repression of ethylene signal transduction pathway by the receptors unless ethylene binds the receptors. Abundant expression of ethylene receptor prior to ethylene production might be a factor responsible for allowing non-autocatalytic plants like Geranium to respond quickly to the ethylene generated after pollination.⁹⁷ These observations raise a possibility of overlapping functions and genetic redundancy adopted by PhETR genes.

Gladiolus

Gladiolus is an ethylene-insensitive flower as exogenous ethylene and ethylene inhibitors have no effect on the petal senescence.⁵³ Two ethylene receptor paralogous genes, GgERS1a and GgERS1b, have been isolated from Gladiolus grandiflora hort, an ethylene-insensitive flower.¹⁰⁰ The cDNA sequence of the two genes is almost identical except that GgERS1 b lacks 636 nucleotides, encoding first and second in the HK motifs present in GgERS1a. The analysis of the genomic DNA sequences (4,776-bp nucleotide designated as GgERS1 long DNA and 3,956-bp nucleotide designated as GgERS1 short DNA) revealed that both sequences were identical except that GgERS1 short DNA was devoid of an 820 bp long nucleotide segment in the first intron of GgERS1 long DNA. These data suggested that each of the GgERS1 genes was generated by duplication and splicing from different genomic DNA. The GgERS1b mRNA level decreased in petals during flower development, whereas the expression of GgERS1a mRNA was constitutive, however, with a high accumulation level, suggesting that high expression level of GgERS1a conferred the ethylene insensitive nature in petals of Gladiolus. On the other hand, the sensitivity to ethylene might be regulated by GgERS1b expression.

Kalanchoe

Kalanchoe blossfeldiana was transformed with the mutant ethylene receptor gene etr1-1 from Arabidopsis under the influence of the flower specific fbp1 promoter from Petunia. The transgenic flowers were reported with reduced ethylene sensitivity and increased flower longevity. However, one of the transgenic lines was found to be infertile and only slightly less ethylene sensitive. Out of the three transgenic lines containing etr1-1, one ethylene resistant, fertile line was crossed with ethylene sensitive cultivar to create T1 progeny. The ratio of the ethylene resistant to ethylene sensitive plants was 3:1. The etr1-1 gene was found to be expressed only in petals and stamens but not in carpels and sepals, as expected from fbp1 promoter.¹⁰¹

Lotus

Lotus japonicus B-129 “Gifu”¹⁰² was transformed with Cucumis melo ethylene receptor gene ERS1 (Cm-ERS1) point mutated at 70th amino acid (histidine to alanine) designated as Cm-ERS1/H70A¹⁰³ under Cauliflower mosaic virus 35S (CaMV-35S) constitutive promoter and the transgenic lines designated as Lj-H70A showed inhibitory ethylene mechanism and nodulation along with reduced ethylene sensitivity.¹⁰⁴ Parallel work in Lotus using a well characterized two component histidine kinase type Atetr1-1 allele was done by Lohar et al. (2009).¹⁰⁵ A gradient of ethylene insensitivity was observed in transgenic Lotus plants from hyper insensitive to hypo insensitive. The transgenic plants were insensitive to both endogenous and exogenous ethylene and formed more nodule loci compared with wild type plants. Nodulation varied in transgenic lines as per the insensitivity gradient. The more ethylene insensitive the line was, the more the nodulation had occurred. Some prominent work involving the effect of ethylene perception on nodulation has been contributed by other groups as well.^106-108

Nemesia

Nemesia strumosa, an ethylene-sensitive ornamental plant belongs to Scrophalariaceae, the figwort family. On the lines of mutated Arabidopsis etr1-1 gene, the missense mutation His-69 to Ala (H69A) was introduced into Cm-ETR1 to create the mutant gene Cm-ETRI/H69A. Reduced ethylene sensitivity and flower longevity in transgenic plants of Nemesia was demonstrated by introducing this mutated melon ethylene receptor gene Cm-ETR1/H69A.^109,110 The Cm-ETR1/H69A expression resulted in longer vase life by 1–3 days due to inhibited ethylene response. The root hair formation in transgenic lines was also reduced to a good extent as compared to wild­type non transgenic plants.

Oncidium

The controversy of Oncidium being ethylene sensitive or insensitive was put to rest by Huang, (1998)¹¹¹ and it is now known to be a climacteric crop. Having known the problem, Huang et al. (2007)⁵¹ reported an ethylene receptor in Oncidium, OgERS1 and examined its expression during the flowering process in response to exogenous ethylene and pollinia cap dislodgment. OgERS1 mRNA levels were piled up in flowers at bud stage and at lower levels with concomitant increase in ethylene production in fully bloomed flowers. However, the expression was always on the rise in the roots signifying, a differential sensitivity acquired by roots for its development. Comparative expression analysis in dislodged and un-dislodged pollinia caps revealed that the expression of OgERS1 was on decline throughout the course of senescence in dislodged flowers compared with intact capped flowers. Exogenous ethylene application had even more profound effect within an hour and it lasted for 36 h. They also concluded that petal senescence in Oncidium is anchored to OgERS1 expression. On being exposed to exogenous ethylene, the OgERS1 expression upsurge with early advent of senescence.

Petunia

Tomato and Petunia plants transformed with the Arabidopsis mutated dominant ethylene insensitive etr1-1 cDNA showed that cross species transfer of an ethylene insensitivity phenotype is possible.¹¹² The creation of ethylene-insensitive tomato and Petunia plants via the introduction of dominant Arabidopsis ethylene receptor alleles depicts the functional conservation for this component of ethylene signaling.¹¹³ This strategy of developing plants without ethylene perception however, does not lead to a normal development of plant. Therefore, in an attempt to improvise the previous work Cobb et al. (2002)¹¹⁴ transformed Petunia with etr1-1 under the influence of floral specific promoters FBP1 (floral binding protein) and AP3 (involved in floral organ development) resulted in increased vase life of transgenic flowers up to 5 times compared with control flowers.

Mutated version of BOERS, an Ethylene Response Sensor (ERS) gene of broccoli, Brassica oleracea, boers¹¹⁵ conferred ethylene insensitivity (because of non functional sensor domain, not able to bind ethylene) when transformed into petunia x hybrida Hort. Vilm.-Andr. Transformed petunia flowers retained pigmentation longer and showed longer life span irrespective of either its storage is in water or exposed to exogenous ethylene. These observations were consistent with those of Bovy et al. (1999)⁷⁹ and Wilkinson et al. (1997),¹¹² depicting successful cross species transfer of ethylene receptor gene. The delay in senescence observed in transgenic etr1-1 flowers was longer than in flowers pretreated with chemicals that inhibit either ethylene biosynthesis (amino-oxyacetic acid) or the ethylene response (silver thiosulfate) and blocked ethylene biosynthesis by ACO1 gene co-suppression.⁷⁹ The possible reason for this difference might be that ACO1 co-suppressed flowers are still sensitive to basal levels of ethylene, either produced endogenously or by flowers and fruits in the vicinity, whereas etr1-1 transformed flowers are not. This observation is also of great importance to non-climacteric flowers and fruits that can be damaged by ethylene produced in the surrounding. However, the disease resistance was compromised as the plant became more susceptible to fungal disease. Earlier, Clark et al. (1999);¹¹⁶ Gubrium et al. (2000)¹¹⁷ and Clevenger et al. (2004)¹¹⁸ reported reduced adventitious root formation, enhanced ethylene production in pollinated flowers, slightly faster flowering along with delayed senescence in ethylene-insensitive transgenic petunia.

Shibuya et al. (2004)¹¹⁹ figured out the central role of signaling molecule EIN2 in petunia plant development. Transgenic petunia plants with reduced PhEIN2 expression were produced using co suppression (expressing PhEIN2 sense RNA) and RNA-interference (PhEIN2-RNAi) approach. Out of 68 lines produced, two homozygous lines, EIN2s-182 and EIN2r-12, exhibiting the greatest flower longevity after ethylene treatment and pollination in T1 generation were picked from PhEIN-sense and PhEIN2-RNAi lines, respectively. RNAi mediated gene silencing was more efficient than co suppressed lines. These transgenic plants were compared with wild-type Petunia x hybrida cv Mitchell Diploid (MD) and two ethylene insensitive petunia plants transformed with Atetr1 namely etr-44568, (Wilkinson et al., 1997)¹¹² and etr-56 (Clevenger, 2004).¹¹⁸ EIN2s-182 and etr-56 showed moderate delay in fruit ripening and flower senescence with no premature death of flowers and fruits compared with EIN2r-12 and etr-44568. Langston et al. (2005)¹²⁰ showed that there was delayed induction of senescence specific nuclease and DNA fragmentation ethylene insensitive 35S::etr1–1 transgenic petunia.

Rose

Rosa hybrida should have shown decreased expression of ethylene receptors and CTR1 undergoing senescence in compliance with the negative regulation model of ethylene signaling. However, study on rose tells a different story, CTR1, CTR2 (CTR1 homolog) were constitutively expressed during senescence and increased in response to exogenous ethylene. Also, RhETR3 (one of the four ethylene receptors in rose) expression increased in senescing flowers. RhEIN3, a positive regulator of ethylene signaling was also constitutively expressed throughout the process under the influence of both exogenous ethylene and ABA.^121,122 Constitutive expression of Rh-EIN3-1 and Rh-EIN3-2 in cut rose is consistent with previous reports in Arabidopsis,⁶⁹ tomato,¹²³ or miniature potted roses,¹²⁴ where the expression of EIN3 remain unchanged even on being exposed to ethylene. A step further, Muller et al. (2000a)¹²⁵ demonstrated the cultivar difference in ethylene receptor levels during flower development. The group also demonstrated the ethylene treatment mediated flower sensitivity to ethylene by RhETR1 regulation. Though the exact mechanism by which ethylene sensitivity is regulated by ethylene receptor dynamism is still not understood fully. An effort has been made to unravel the mechanism to an extent by receptor expression analysis in rose. Higher expression of RhETR1 was observed in cultivar “Bronze” (having shorter flower life) as compared with “Vanilla” (longer vase life). Maximum expression of RhETR1 has been observed in the bud and young flower stage both in bronze and vanilla cultivar respectively. As far as the RhETR3 expression levels were concerned, in “Bronze” the transcript level increased as the senescence approached however, in “Vanilla” flowers it was constitutively expressed at very low levels. Constitutive expression of RhETR2, was observed during senescence although the transcript level was different for the two cultivars. Early expression of RhETR1 i.e., before the ethylene production, along with the increase in RhETR3 expression in senescencing flowers of “Bronze” prompts that ethylene response system in rose is mediated via overlapped expression of the multiple receptors. This also explains the reason for the multiplicity of ethylene receptors to some extent. Thus, it can be concluded that RhETR1 and RhETR3 are rate limiting for ethylene perception and determinant for flower longevity. These observations also justify Lashbrook et al. (1998)¹²⁶ theory which states that increase in ethylene sensitivity is manifested by increase in receptor abundance.

Ma and coworkers (2003)¹²⁷ explained ethylene’s regulatory role in flower opening, effect of ethylene on flower pre-pollination opening and identified key regulatory components in ethylene biosynthesis and signaling pathways in cut roses. Exogenous ethylene promoted ethylene production in petals, but 1-MCP did not inhibit ethylene biosynthesis. Moreover decrease in ethylene production was also not observed. This confirmed that ethylene regulates flower opening of roses through signaling pathway and not through biosynthetic pathway. It was also established that continuous ethylene perception is required for flowers to open without the feedback regulation of ethylene biosynthesis, in accordance with Wang and Woodson’s (1989)⁶² theory of flower opening.

Xue et al. (2008)¹²⁸ showed that ethylene enhanced both ethylene production and the expression of Rh-ACS2 or Rh-ACS3 in gynoecia, petals, and receptacles, with gynoecia showing the earliest ethylene enhancement. However, 1-MCP did not suppress ethylene production and the expression of the ethylene biosynthetic genes in these three tissues. In stamens, no obvious changes were found in ethylene biosynthesis either by ethylene or 1-MCP treatment. This suggests that gynoecia are the most sensitive tissue to ethylene treatment, and none of the five floral tissues exhibits positive feedback regulation of ethylene biosynthesis. Among the five floral tissues studied, ethylene-induced expression of Rh-ETR genes occurred first in gynoecia, and only the expression of the Rh-ETR3 gene was regulated in a positive feedback manner by ethylene and 1-MCP. These results suggest that transcriptional regulation of the Rh-ETR3 gene in gynoecia may play an important role in ethylene-enhanced flower opening. These results differ from the findings in carnation, geranium, and Delphinium.

Out of seven signaling component genes studied in rose, including three ethylene receptors (Rh-ETR1, Rh-ETR3 and Rh-ETR5), two CTRs (Rh-CTR1 and Rh-CTR2), and two transcription factors (Rh-EIN3–1 and Rh-EIN3-2), transcripts of Rh-ETR5, Rh-EIN3–1 and Rh-EIN3-2 were accumulated in a constitutive manner and had no or little response to ethylene or 1-MCP, while transcript levels of Rh-ETR1 and Rh-CTR1 were substantially elevated by ethylene and those of Rh-ETR3 and Rh-CTR2 were greatly enhanced by ethylene. 1-MCP reduced all the four genes to levels much less than those in control flowers. These results show that ethylene triggers physiological responses related to flower opening in cut rose cv Samantha, and that continued ethylene perception results in flower opening. Ethylene may regulate flower opening mainly through expression of two ethylene receptor genes (Rh-ETR1 and Rh-ETR3) and two CTR (Rh-CTR1 and Rh-CTR2) genes.¹²⁷

Ethylene Perception and Receptor Expression in Fruit Crops

Apple

In apple (Malus domestica), the expression of ethylene has been observed during fruitlet abscission and early development.¹²⁹ The expression of MdETR1 and MdERS1 is found to be tissue specific. Although the expression of MdETR1 is at steady-state but maximum expression has been observed in the peduncle, abscission zone and in seed, than in the cortex of early developing fruits. A possible role of MdERS1 receptor in abscission is suggested. The expression of gene encoding for specific cell wall hydrolases is regulated by increase in ethylene evolution at abscission zone which results in abscission zone cell separation and fruitlet shedding. Wiersma et al. (2007)¹³⁰ has identified two new ethylene receptors ETR2 and ETR5 along with ethylene control element in Summer Apple, “Sunrise” (SR) and late season “Golden Delicious” (GD). For analyzing quantitative expression of four ethylene receptors (ETR1, ETR2, ETR5 and ERS1) and CTR1, EIN2 only minor changes in expression were observed in two apple cultivars. The expression of ERS1was in accordance with feedback inhibition model of ripening fruit in GD fruit, however in SR fruit such expressions was not observed. With a difference in both the cultivars a very small expression of CTR1 was observed at maturity .A decline in expression of CTR1 in GD was due to increase in ethylene sensitivity in mature tissues. During various stages very little variation was observed in Apple EIN2.¹³¹

Avocado

In avocado (Persea americana) the activity of PA-ERS1 mRNA increased gradually until the climacteric peak where, it is hyper induced. The stimulated induction of PA-ERS1 at climacteric peak may be due to suppression by 1-MCP; this may be mechanism adapted by avocado fruit to dissipate high levels of autocatalyic ethylene being produced by the plant.¹³²

Citrus

Although Citrus (Citrus sinensis) fruit is known to be nonclimacteric, exogenous ethylene is able to affect its ripening in. It accelerates respiration, biosynthesis of carotenoids, degradation of chlorophyll and induction of pigment change of peel in citrus. The expression of CsERS1 has been detected immediately after harvest in young Valencia fruitlets which is induced further in subsequent days.¹³³ The expression of CsERS1 has been found to be ethylene independent in young fruitlets. In mature fruit, 1-MCP or propylene treatments has no affect on expression of both CsERS1 and CsETR1 genes. CsERS1expression modulates the differential sensitivity to ethylene in mature and young fruitlets.

Cucumber

Three ethylene receptor genes CSETR1, CSETR2 and CSERS were isolated from Cucumis sativus and the expression of these genes was analyzed to decipher the role of ethylene in inducing femaleness in cucumber. A correlation between ethylene production and ethylene receptor genes was also established. The expression of CSETR2 and CSERS was more profound in gynoecious cucumber as compared to monoecious one. This expression deepened even further in presence of ethrel and declined in presence of AVG (ethylene biosynthesis inhibitor). This observation was complimented by higher ethylene production and better expression of ethylene biosynthetic gene CSACS2 in gynoecious cucumber than in monoecious.¹³⁴

Cucurbita pepo

To study the mechanism by which ethylene metamorphoses the sex expression and development, two CTR1 homologs were identified and the transcript levels of the same were also studied during the male and female flower development and when exposed to external ethylene.¹³⁵ During development of male and female flowers, the expression of both the CTR1 homologs increased along with ethylene production in flowers indicating that both the genes are upregulated by ethylene in flowers but not in seedlings and leaves. Exogenous ethylene also did not have any impact on the expression of CTR1s in seedlings and leaves but, upregulated the expression in flowers. During earlier days of male flower development higher expression of both the CTR1s was observed when lesser ethylene was produced within the flowers, indicating lower ethylene sensitivity in male flowers as compared with female flowers. Role of ethylene in imparting femaleness in Cucurbitaceae is well known. This member of the family also abides by the same principle. Manzano et al. (2010)¹³⁵ showed that a particular inbred line of Cucurbita pepo, vegetable spaghetti, exhibited extreme male phenotype which can be attributed to either mutation in a receptor or response of gene for ethylene which resulted in weaker sensitivity to ethylene, evident from weaker triple response. The more sensitive F2 plants showed increased femaleness.

Grapes

Although grapes (Vitis vinifera) are regarded as a non-climacteric fruit 1-MCP has shown to alter the berry ripening process. There are four ethylene receptor genes known in grapes namely VvETR1, VvETR1, VvERS1 and VvEIN4. Other orthologs of ethylene signaling cascade identified in grapes are VvRTE1, VvCTR1 and VvEIN3. Exogenous ethylene application resulted in increased transcript accumulation of VvETR2 and VvCTR1. Ethylene antagonist 1-MCP application on the other hand resulted in increased transcript levels of VvEIN3 after 9 weeks and VvRTE1 after 7 weeks. However, no significant change was observed in VvEIN4 transcript accumulation. These responses by cascade molecules against ethylene and its antagonist in grapes also existed in climacteric crop like tomato and in Arabidopsis. Taken together, these observations prompt the prevalence of a common regulatory pathway in climacteric and non-climacteric fruit.¹³⁶

Kiwifruit

Kiwifruit (Actinidia deliciosa) is a typical climacteric fruit, extremely sensitive to ethylene. Yin et al. (2008)¹³⁷ showed the expression profile of eight signaling molecules in Actinidia, including five ethylene receptors namely AdERS1a, AdERS1b, AdETR1, AdETR2, AdETR3, two CTR1 like genes AdCTR1, AdCTR2 and an EIN3 like gene AdEIL1. None of the gene was found to be fruit specific and all were differentially expressed among various kiwifruit tissues. Like in tomato, no ERS2-type receptor is reported in kiwifruit. Except, AdERS1a all the receptor genes were expressed in good amounts in roots, stems and fruits implying that AdERS1a has a precise role to play in flower development. Strong expression of AdERS1a and AdETR3 in the early developmental stage, when substantial cell division happens is suggestive of ethylene involvement at this growth stage. However, AdERS1b and AdETR2 were expressed after full bloom prompting the association of these genes with ethylene involvement in cell expansion and development. AdERS1b exhibited no ethylene response but its expression was explicitly associated with fruit ripening. AdERS1a, AdETR2 and AdETR3 showed the most promising expression during fruit ripening on exposure to ethylene. Authors have speculated AdETR2 to play a role in senescence process rather than its involvement in early ripening event. In the light of the negative regulation model of ethylene receptors, the AdETR1 downregulation during ripening and on exposure to ethylene was justified as making the signaling pathway active and more responsive or it may be because of the degradation of receptor protein on exposure to ethylene. This suggests, less receptor protein cause de-repression of the pathway. Considering the fact that upregulated receptor proteins have better ethylene affinity for ripening to proceed, the upregulated receptors AdERS1a, AdETR3 are perhaps important in activating signaling pathway on ethylene binding and AdETR1 may act as a principal suppressor of the pathway. AdCTR1 and AdCTR2 showed low levels of transcriptional response to ethylene and AdEIL1 showed no response to ethylene.

Lettuce

After the success of Cm-ERS1/H70A missensed mutated melon receptor gene in inducing stable sterility in transgenic tobacco plants.^110,138,139 Takada et al. (2007)¹⁴⁰ exploited this gene to make transgenic lettuce (Lactuca sativa). This heterologus transformation effectively induced stable sterility like in tobacco. However, the only pleiotropic phenotype observed in lettuce was reduced pollen production. Reduced ethylene sensitivity imparted by this gene has already been reported in Nemesia strumosa, Lotus japonicus and Nicotiana tabacum. Cm-ERS1/H70A acts as an effective tool in the armoury of a genetic engineer to impart sterility in vegetatively propagated plants and if used with an anther/pollen specific promoter can serve the purpose in seed propagated plants.¹³⁸ This system demonstrated how a mutated ethylene receptor could contribute in checking the pollen dispersal from transgenic plants.

Mango

Mango (Mangifera indica) is an important tropical fruit crop. There are two ethylene receptor genes known in mango namely MiETR1¹⁴¹ and recently discovered MiERS1.¹⁴² The MiETR1 expression increased during ripening and wounding. However, in the abscission zone, the MiETR1 transcript levels remained unchanged and MiERS1 expression increased. During fruit ripening, MiERS1 expression was almost negligible compared with MiETR1 which increased both in mesocarp and seed tissue.¹⁴² The comparative expression profiling of the two genes suggest that the ERS1 counterpart might have a role to play in regulation of abscission and ETR1 in fruit ripening with a possibility of more members to be added in the family to have a better picture.

Melon

Due to distinct anatomical, reproductive and developmental features, the melon fruit developmental stages are categorized into ethylene sensitive and ethylene insensitive stage with the developing fruit having lower affinity to ethylene than the ripening fruit.^143,144 During fruit ripening in muskmelon, Cm-ERS1 transcript level increased slightly in the pericarp of fruit. Also there was a concomitant increase in Cm-ETR1 transcripts along with climacteric ethylene production. Elevated levels of Cm-ERS1 transcript compared with Cm-ETR1 mRNA at low ethylene levels and ethylene production suggests Cm-ERS1 to be more sensitive to even lower levels of ethylene, whereas Cm-ETR1 is thought to be responsive to high level of ethylene.¹⁰³ Studies performed to examine the temporal and spatial expression pattern of Cm-ERS1 protein, during fruit development, revealed that a posttranscriptional regulation of Cm-ERS1 expression affects stage and tissue-specific accumulation of the protein.¹⁴⁴ The melon subfamily II ethylene receptor, Cm-ETR2 mRNA, exhibits earlier accumulation compared with Cm-ETR1 during ripening, and its transcript accumulation increased during melon ripening, and declined in parallel with a reduction in ethylene production.

Sex determination is another repercussion of altered ethylene perception in melon as reported by Little et al. (2007).¹⁴⁵ The transgenic melon with At-etr1-1 gene resulting in reduced ethylene sensitivity showed change in sex expression. The expression of etr1-1 was studied under the influence of a constitutive promoter, CaMV35S and tissue specific inflorescence targeted Apetela3 (AP3) and Crab’s Claw (CRC) promoters. The 35s::etr1-1 melons showed inhibition of carpel bearing and bud production suggesting involvement of ethylene perception in determination of sex. However, AP3 promoter resulted in increased maleness, poor carpel development and hardly any production of bisexual flowers. On the other hand with CRC promoter, predominant femaleness with increased number of carpel bearing buds was observed. This suggested that ethylene perception by stamen has a significant role to play during sex determination period. However, the hormone perception by carpel is important for maturation of carpel-bearing flowers to anthesis.

Significance of endogenous ethylene production for pistillate flower development has been demonstrated in melon by Papadopoulou et al. (2005).¹⁴⁶ Constitutive overexpression of ethylene biosynthetic gene, ACS resulted in enhanced endogenous ethylene accelerating bisexual flower initiation, development of mature bisexual flowers and fruit set in melon.

Passion fruit

During ripening in Passiflora edulis no significant change was observed in the expression of two ethylene receptors, PeETR1 and PeERS1. However, high levels of PeETR1 and PeERS1 mRNA were detected in arils than in seeds. PeERS2 transcript levels were elevated in general but a heightened level it was noticed in particular in the arils of ripened fruit. PeERS2 mRNA is speculated to be involved in repressing ethylene response; this can be inferred when mature green fruits are exposed to ethylene.¹⁴⁷

Peach

The expression of Pp-ETR1 peach (Prunus persica) appeared to be upregulated by propylene treatment. In peach its expression appeared to be constitutive and ethylene independent during fruit development and ripening.¹⁴⁸ It was seen that application of 1-MCP delays fruit ripening, evolution of ethylene and also downregulates the activity of Pp-ERS1. Its activity was abolished on keeping the fruits in air and rapid activity of Pp-ERS1 mRNAs stimulated ethylene evolution.

Pear

A gradual increase in ethylene production and expression of ethylene receptor mRNA was observed during cold treatment of late season pear (Pyruscommunis cv Passe-Crassane) by (El-Sharkawy et al., 2003).¹⁴⁹ Cold treatment upregulated the expression of Pc-ETR1 mRNA during ripening, whereas the expression of Pc-ETR1 and Pc-ETR5 were found to be less affected by cold treatment. During ripening high accumulation of Pc-ETR1 and Pc-ERS1 mRNA was observed in the early-season pear cultivar as compared to the gradual increase seen in late-season pear cultivar, Passe-Crassane. A stark difference in accumulation of Pc-ETR5 transcript was observed both during early-season cultivar and late season cultivar PC for example: in early season pear fruit the level of Pc-ETR5transcript reduced sharply before and during the ethylene climacteric whereas it sharply increased in late-season cultivar. Increased ethylene sensitivity during the early ripening phase of early fruit development can be due to decrease in expression of negative regulator.

Persimmon

The expression of three ethylene receptor genes DkERS1, DkETR1 and DkETR2 has been determined during the ripening of persimmon (Diospyros kaki) fruit.¹⁵⁰ A correlation was found between the levels of DkERS1, DkETR1 mRNA and ethylene production during fruit development and ripening. The expression of DkETR1 mRNA was found to be ethylene independent and is expressed during all the stages of fruit ripening. Decreased level of DkERS1 protein prior to fruit maturation suggests its involvement in ethylene perception during fruit ripening.

Plum

Japanese plum (Prunus salicina L.) falls in the category of climacteric fruit. Unlike other climacteric fruits, stone fruit plum, exhibit a double sigmoid growth pattern during fruit development stages (S1–S4). Two Japanese plum cultivars “Early Golden” (“EG”) with normal climacteric pattern and “Shiro” (“SH”) with suppressed climacteric pattern were chosen by El-Sharkawy et al. (2007, 2009)^151,152 for isolation and molecular characterization of 11 ethylene signaling elements, which includes two receptors (ETR1 like proteins, Ps-ETR1, Ps-ERS1), one CTR1 like protein (Ps-CTR1), an ethylene responsive element binding factor (Ps-ERF). Ps-ETR1 and Ps-ERF1 were absent in the late cultivar ‘SH’. Ps-ERS1 lacks the C-terminal receiver domain, which was present in Ps-ETR1. The predicted Ps-CTR1 protein contained both ATP binding site and Ser/Thr kinase activity. The receptors’ expression was upregulated post fertilization and then subsided. During S1 and S2 stages all the four molecules were downregulated in an ethylene independent manner. However, Ps-ETR1 and Ps-CTR1 transcripts were accumulated during S3 stage in an ethylene independent manner. The threshold level requirement of ethylene in late cultivar like “Shiro” (“SH”) and once this requirement is fulfilled, the developmental changes are accelerated. The transcript levels of Ps-ETR1 and Ps-CTR1 increased sharply during climacteric, S4 stage. But it was higher in ‘EG’ cultivar as compared with “SH”. Ps-ETR1 was constitutively expressed during development with further increased expression during fruit ripening. Ps-ERS1 and Ps-ERF1 transcript level increased sharply with the climacteric peak of “EG” fruit in an ethylene dependent manner. However, in “SH” cultivar the expression was constitutive and low. A positive correlation was established between the Ps-ERS1 and Ps-ERF1 expression.¹⁵¹ As many as seven Ps-ERFs were characterized during fruit development and ripening.

Strawberry

When compared with climacteric fruits, there is no other growth regulator corresponding to the role played by ethylene in nonclimacteric fruits. Dearth of literature regarding the role of ethylene receptors in nonclimacteric fruits and an observation that they synthesize ethylene adding to postharvest pernicious effects, suggests a need to establish a relationship between ethylene and ripening of fruits. Three ethylene receptors, FaEtr1, FaErs1 and FaEtr2, have been isolated and their expressions were determined in strawberry (Fragaria ananassa) fruits. Ethylene receptors were suspected to be involved in ripening of nonclimateric strawberries,¹⁵³ as suggested by the expression of FaErs1 gene in responses to ethylene at red stage, FaEtr1 and FaEtr2 genes in white fruits. The expression of FaEtr1 mRNA was found to be low in flowers but subsequently increased in small green fruits and then decreased in large green fruits followed by steep increment throughout ripening phase. The expression of FaETR2 mRNA showed 3-fold increase to reach maximum in white fruits whereas, FaErs1 mRNA expression was very high in flowers and decreased to minimum in large green fruits.

Ethylene Perception and Role of Ethylene Receptors in Tomato

Tomato is not only an important vegetable crop but has also emerged as a model crop like Arabidopsis and tobacco. Simpler transformation and genetic manipulation with rapid life cycle and distinct anatomical features has made it a crop of choice for genetic studies. Also there is a detailed information about its genomics.^154,155 Transcriptomic, metabolomic and proteomic profiling of Nr mutant has revealed several aspects of regulation and metabolism during ripening and development.¹⁵⁶ Klee (2004)¹⁵⁷ has elaborated on the reasons for tomato to be a model system for study and has also postulated on the dynamic behavior of ethylene receptor expression and function. Nr is a semi dominant ethylene receptor mutant, has a mutation in ethylene binding domain of the NR receptor, unable to bind ethylene and the first to be identified in tomato.¹⁵⁸ In tomato, there are six ethylene receptors LeETR1,^126,159 LeETR2,^126,160 LeETR4, LeETR5,¹⁶¹ LeETR6¹⁶² and NR.¹⁵⁸ Nr, mutant of NR.^163,164 Three receptors have a potential extra N-terminal membrane-spanning domain. Only one receptor, NR, lacks the receiver domain. Three receptors (LeETR4-6) are missing one or more conserved HK domains, thus resembling the Subfamily II Arabidopsis receptors. NR and LeETR4 expression increases during fruit ripening^126,161,165 whereas LeETR1 and LeETR2 are constitutively expressed in all tissues throughout development,^126,159 with LeETR1 expressed at about 5-fold higher level than LeETR2. In contrast, expression patterns of the other four genes are highly regulated. For example, during fruit development,ovaries express high levels of NR mRNA at anthesis.^126,158,165 The level then drops approximately 10-fold until the onset of ripening whereupon, it rises approximately 20-fold. LeETR5 expression was high in reproductive and lower in vegetative tissues.¹⁶¹

Ethylene Receptor Regulation in Tomato is Consistent with Receptor Inhibition Model

As per the Arabidopsis negative regulation model less receptor level increases ethylene sensitivity while more receptor reduces ethylene sensitivity i.e., more receptor requires more ethylene to turn on an ethylene response. Receptor inhibition model states that receptors are active and repress ethylene response in the absence of hormone and vice-versa. The first evidence in favor of the negative regulation model of ethylene receptors was put forth by Ciardi et al. (2000).¹⁶⁶ Overexpressed NR receptor in plant reduces the ethylene sensitivity quantitatively when there is a several fold increase in NR protein.

Considering the fact that there are no examples of decreased ethylene receptor gene expression during ethylene response in tomato there is a need to justify the observation of enhanced LeETR4, LeETR5, LeETR6¹⁶⁷ and NR gene expression in ovaries and ripened fruit with the ripening process and thus with ethylene.^{126, 158} This ripening associated rise is an example of developmentally dependent ethylene inducible i.e., the gene is ethylene inducible in ripening but not in mature fruit.¹⁵⁸ Gallie (2010)¹⁶⁸ demonstrated the ability to control ethylene responses temporally and in amount through the control of mutant receptor (etr1-1) expression.

One justification could be that at the onset of fruit ripening ethylene production becomes autocatalytic, resulting in rapid increase in ethylene levels. To minimize the effect of ethylene, ethylene sensitivity is reduced by increased expression of receptors. Another justification could be in accordance with Schaller and Bleecker (1995)⁸ findings that the dissociation time of ethylene saturated ETR1 receptor is 12 h and under such circumstances the only way a plant can opt for a turn off of an ethylene response is by synthesis of new receptors. Substantiating the negative regulation even further, Tieman et al. (2000)¹⁶⁷ developed transgenic plants with reduced LeETR4 gene expression resulting in enhanced ethylene sensitivity (severely epinastic and almost all flowers senesced before anthesis). This observation is in accordance with the negative regulation model. However, it does defy the Arabidopsis loss of function mutation findings because in tomato only a single gene reduced expression leads to severe ethylene triple response, whereas in Arabidopsis multiple receptor inactivations exhibited such severe responses. When the same strategy (antisense suppression) was used with NR, negligible phenotypic alterations were observed. Now, having known that the expression of LeETR4 and NR under normal circumstances is more or less the same, the question that needs to be answered is why the loss of expression of LeETR4 leads to severe responses and the same event in NR did not lead to any severe responses? The answer lies in the functional compensation exhibited by the receptors in a multigene family. This is revealed by expression analysis of LeETR4 in Nr suppressed lines. Even the modest reductions in NR results in elevated levels of LeETR4 by almost 3–4 times. However, the other receptor levels did not differ significantly. On the contrary, in LeETR4 suppressed lines there was no significant increase in other receptor gene. Thus, LeETR4 takes care of ethylene sensitivity and compensates for the reduction in expression of other receptors, which is not reported in Arabidopsis. Kevany et al. (2007)¹⁶⁹ deciphered the role of LeETR4 and LeETR6 in modulating ethylene responses including fruit maturation. Depleted levels of either of these receptors by antisense suppression or ethylene mediated protein degradation resulted in early fruit ripening. Also, the NR protein levels were parallely depleted on ethylene exposure. In contrast to very high rise in mRNA expression of NR, LeETR4 and LeETR6 at the onset of ripening, the protein levels of these receptors showed a significant decline and remained low on exposure to ethylene. However, the same proteins were expressed at their maximum in immature fruits. Thus, it was demonstrated that the reduction in the levels of either of the two family members, LeETR4 or LeETR6, causes an early ripening. The authors hypothesized that the ligand induced conformational change in the receptors could be a probable reason for receptors’ subsequent degradation via 26S proteasome apparatus. Unlike the Arabidopsis model of knockouts where only triple and quadruple mutants depicted profound ethylene response, here the LeETR4 and LeETR6 suppressed plants showed phenotypes with exaggerated ethylene response including epinastic growth, premature flower senescence and an early fruit ripening. The results are in contradiction with Wang et al. (2003)¹⁷⁰ theory which says subfamily I receptors are more responsive to ethylene effect than the subfamily II receptors.

The success of antisense suppressed ethylene receptors, LeETR4 and LeETR6, leading to desired premature ripening^167,169 was incomplete as it was accompanied by many unwanted effects on growth of plant. It was hypothesized that the fruit specific suppression would result in early ripening without undesirable issues. Kevany et al. (2008)¹⁷¹ deciphered the role of ethylene during immature fruit development. The depleted LeETR4 levels resulted in early ripening without compromising on agronomic features, fruit size, yield and flavour related chemical composition were also not penalized. However, this observation of single ethylene receptor suppression resulting in altered phenotype is in contrast with Arabidopsis model, where only multiple loss of function receptor mutants resulted in obvious ethylene effects. It validates Kevany et al. theory (2007)¹⁶⁹ that if receptors are not replaced after ethylene mediated degradation, the fruit becomes more sensitive to ethylene and ripens faster.

Hackett et al. (2000)¹⁷² used antisense inhibition of a mutant tomato ethylene receptor, Nr gene to show that the mutant tomato restored the normal ripening suggesting that the mutant Nr receptor is unable to bind ethylene and prevents its inactivation, therefore suppressing the ethylene responses. The transgenic plants developed using antisense Nr produced three different phenotypes depending upon the transgene dosage in the progeny gave intermediate fruit type if they were hemizygous for both copies, in case of single transgene inheritance in a homozygous state fetched fruits with intermediate (between Nr and wild type) phenotype, whereas hemizygotes produced Nr-type fruit. This finding suggests that a threshold level of mutant receptor is needed to suppress the ethylene response pathway and prevent normal ripening. However, antisense inhibition of the wild type NR gene showed no effect on ripening, raising a question about normal function of this gene. Probable reason of the functional NR gene might be to achieve wild type levels of LeETR4 expression. The Nr mutants are known to affect ethylene responses in tissues other than ripe fruit (Lanahan et al., 1994),¹⁶³ including seedlings. Nr seedlings were devoid of ethylene triple response (shortened, swollen hypocotyls, exaggerated apical hook and shortened root) when germinated in the dark on media with ethylene precursor ACC. Similar results were observed in case of homozygous NR antisense transgenic and Nr seedlings when given similar treatment. These data suggests that although downregulation of the mutant Nr gene was sufficient to alleviate its effect on fruit ripening in the transgenic raised, insensitivity of seedlings to ethylene was not altered. Results indicated that wild type NR gene product is not required for normal ripening and antisense inhibition of the mutant Nr gene products restores normal ripening. This provides enough evidence in support of receptor inhibition model and negative regulation of ethylene signaling in tomato.

Under pathogen challenge, the LeETR4 expression is induced because of hypersensitive response triggered by infection. In antisense lines with severely reduced LeETR4 expression there is a heightened hypersensitive response to pathogen infection manifested by increased ethylene synthesis and pathogenesis related gene expression as a consequence of enhanced defense response. Had the receptor levels not increased in response to infection, the tissue would have undergone a hypersensitive ethylene response state. This observation suggests that plant takes measures to prevent itself from the collateral damage that could be caused as a result of subsequent ethylene response after pathogen infection.¹⁷³

Conclusion and Future Prospects

The varied expression pattern of ethylene receptor in agriculturally important crops gives an insight regarding regulation of ethylene perception in different species of plants. Though, the expression of receptors varies with crops and their developmental stages and environmental condition, the negative regulation theory is never violated. This information helped in designing plants with altered perception to ethylene with delayed senescence and ripening of fruit, vegetables and flowers. Being a multigene family, the expression analysis of each receptor tells about redundancy, compensatory and regulatory role of each member of the family. Structural understanding of the receptor has led to the development of ethylene receptors mutated in the ethylene binding domain leading global ethylene insensitivity in crops even across the species. Different expression of ethylene receptors at various developmental stages in non-climacteric crops, where only basal level of ethylene is produced, for example in Gladiolus, is an indicator of receptor mediated regulation of senescence and a possible reason of imparting insensitivity to ethylene. It would be interesting to see if this can be validated by using ethylene receptor genes in its natural form from such ethylene-insensitive systems and used in different crops and measure the effect on transformed plants.

The central idea of this review is to present every possible detail about the role of ethylene receptor expression in flower senescence and fruit ripening along with the transgenics developed using mutated ethylene receptors and transcription factors involved in the cascade. To date, only the receiver domain of an ethylene receptor protein is crystallized. If structure of other domains of the receptor protein can be modeled then, this would help in more precise targeting of ethylene signaling at the receptor level itself thus, regulating the relay of signals from ethylene binding domain via HK domain and receiver domain to the downstream CTR1 molecule.

With the advent of next generation sequencing, genomes and transcriptomes of several crops are available leading to rapid discovery of genes and transcription factors governing the ethylene signaling and biosynthesis. These transcription factors can be exploited to control the expression of desired genes, thus regulating the flux of the proteins formed, affecting ripening and senescence. Also, understanding the role of micro RNAs in fruit ripening and development, and flower senescence will help in precisely manipulating the complex process of regulation of ripening and senescence.