Analysis of ethylene receptor genes from petunia and arabidopsis

Chapter 1 Literature Review 1.1 Biosynthesis, perception and signal transduction of ethylene The simple gaseous hormone ethylene plays an important role in many aspects of plant growth

Chapter 1 Literature Review

1.1 Biosynthesis, perception and signal transduction of ethylene

The simple gaseous hormone ethylene plays an important role in many aspects of plant growth and development It elicits diverse morphological changes of plant organs, including seed germination, leaf and flower senescence and fruit ripening It is also involved in the process of responding to stress and pathogen attack (Abeles et al., 1992) Ethylene production during plant development is tightly regulated by internal signals Higher level of ethylene production can be observed in plants that are subjected to environmental stresses such as wounding, pathogen attack, flooding or freezing The induced ethylene in turn can elicit defense responses such as accelerated senescence, abscission of infected organs, and the induction of specific defense proteins (Chang and Shockey, 1999)

The current understanding of ethylene biosynthesis as well as its perception and signal transduction pathway has been enhanced by isolation of mutants that have defective ethylene responses Many dark-grown dicotyledonous seedlings in the presence of ethylene or after treatment with the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), exhibit a phenotype collectively called “triple response” (Goeschl et al., 1966; Taylor et al., 1988; Guzman and Ecker, 1990) Triple response refers to the

Ethylene-insensitive mutants are those that fail to generate the triple response when

treated with ethylene or exhibit only a weak response These mutants include etr1 (for ethylene response), etr2, and ein-type (for ethylene insensitive) (Bleecker et al., 1988;

Sakai et al., 1998; Roman et al., 1995; Chao et al.,1997) The constitutive triple response

mutants, including ctr1 (for constitutive triple response), and eto2 (for ethylene

overproducer), exhibit the triple response even in the absence of added ethylene (Kieber et al., 1993; Vogel et al., 1998) The two classes of mutants behave differently upon treatment with inhibitors of either ethylene synthesis or perception, including silver

thiosulfate and aminoethoxyvinyl glycine (AVG) The mutants in the ctr1 category are

unaffected by the ethylene inhibitors, they constitutively exhibit the triple response

Whereas the phenotype of the eto2 category mutants will revert to normal morphology if

treated with ethylene inhibitors This demonstrates that these mutants are defective in ethylene biosynthesis regulation and this defect can be overcome by providing exogenous ethylene inhibitors

Figure 1.1 Effects of ethylene in plant development (from Johnson and Ecker, 1998)

a) Wild-type Arabidopsis seedlings treated with ethylene show “triple response”,

which refers to the phenotype including short and thick hypocotyls, short roots and exaggerated apical hooks

b) Tomato plants defective in ethylene perception are more prone to necrosis following pathogen infection

c) Root hair formed in nearly all the epidermal cells upon ethylene treatment

d) Ethylene promotes flower senescence and fruit ripening

e) Ethylene inhibits cell expansion in wild-type Arabidopsis plants

(a) (b)

(d)

1.1.1 Ethylene biosynthesis

1.1.1.1 Mechanistic overview of ethylene biosynthesis

The ethylene biosynthetic pathway has been studied extensively since the establishment of S-adenosyl methionine (SAM) and 1-aminocyclopropane-1-carboxylicacid (ACC) as the precursors of ethylene (Yang and Hoffman, 1984) Based on this knowledge, two key enzymes, ACC synthase and ACC oxidase that catalyze the final two-step reaction from SAM to ACC and ACC to ethylene were successfully cloned from zucchini and tomato, respectively (Sato and Theologis, 1989; Hamilton et al., 1991) Thereafter, cloning and characterization of genes encoding these two enzymes has been carried out in many

species such as Arabidopsis (Abel et al., 1995; Kieber et al., 1993), tomato (Terai, 1993;

English et al., 1995), carnation (Woodson et al., 1992) These studies demonstrated that these two enzymes are encoded by a multigene family and expressed tissue-specifically and differently in response to developmental, environmental and hormone factors (Kende and Zeevaal, 1997) Detailed studies of these enzymes also led to further understanding of molecular regulation of ethylene production (Kende, 1993)

SAM is the major methyl donor in plants and plays a role in methylation reactions including modifying lipids, proteins and nucleic acids It is involved in many biochemical

synthase also produces 5'-methylthioadenosine in addition to ACC, which is utilized for the synthesis of new methionine via a modified methionine cycle (Bleecker and Kende, 2000) This salvage pathway preserves the methyl group for another cycle of ethylene evolution Continuously high rates of ethylene biosynthesis can therefore be maintained without demanding large amounts of free methionine Finally, ACC is oxidized by ACC oxidase to synthesize ethylene, or it can be conjugated to a malonyl or glutamyl group to restrict its availability for ethylene production Both ACC synthase and ACC oxidase are involved in the positive feedback regulation of ethylene biosynthesis In this positive feedback, ethylene treatment leads to increased ethylene production in several plant species (Nakatsuka et al., 1998; Barry et al., 2000)

1.1.1.2 Regulation of ACC synthase and oxidase genes

The transcription of different forms of ACC synthase is induced under different environmental or physiological conditions (Theologis, 1992) The members of ACC

synthase (ACS) in Arabidopsis clearly exemplify this point that different isoforms are

differently regulated For example, ACS4 is induced in seedlings by wounding, cycloheximide and indoleacetic acid (Liang et al., 1992; Abel et al., 1995) ACS6 can be inducedspecifically by exposure to ozone in light-grownleaves and mechanical stress by touching (Vahala etal., 1998; Arteca and Arteca, 1999) Besides the regulation mentioned above, ACC synthase may also be post-translationally regulated as demonstrated by the

type Furthermore, cytokinin could increase ethylene production without induction of

ACS5 mRNA, suggesting that the regulation of ethylene synthesis by cytokinin may be

through posttranslational modification of ACC synthase The most obvious evidence of posttranslational regulation is shown in tomato ACC synthase (Spanu et al., 1994) Its activity in cell culture can be induced by fungal elicitors, while addition of the protein kinase inhibitor staurosporine can rapidly inactivate this enzyme

While control of ethylene production may be largely attributed to ACC synthase, the periods of ACC oxidase induction also correlate with some events of senescence and wounding, which are ethylene-regulated (Callahan et al., 1992; Pogson et al., 1995) ACC oxidase protein is also thought to be post-translationally modified, giving rise to different isoforms (Stearns and Glick, 2003) Several isoforms of ACC oxidase have been identified which are active under different physiological conditions (Arshad et al., 2002) The conversion of ACC to ethylene in most tissues occurs at very low levels, suggesting that ethylene synthesis is tightly controlled Expression of ACC oxidase can also be induced either by hormone or environmental stimuli except in fruits Thus, defining the rate-limiting step in ethylene biosynthesis may differ in different plants, tissues and in response

to environmental cues

1.1.2 Ethylene perception and signal transduction

Burg proposed that ethylene exerts its physiological action through a receptor, and a metal may be involved in the ethylene binding process (Burg and Burg, 1967) But attempts to isolate ethylene receptor by biochemical approaches failed Elucidation of the mechanism

of ethylene perception and signal transduction pathway began with the molecular and

genetic studies on Arabidopsis in the last decade Screening for mutants that have altered

triple response allowed identification of many loci involved in the ethylene-signaling pathway Cloning and characterization of the corresponding genes tentatively defined their order of action within the signal transduction pathway

1.1.2.1 Ethylene perception and the action of ethylene receptor(s)

As shown in Figure1.2, ethylene is perceived by plants through a family of integral membrane receptors These receptors are homologous to “two-component” regulators initially characterized in bacteria (Stock et al., 1985) A typical two-component regulator consists of two types of signal transducers, a sensor component and a response regulator component (Stock et al., 1990; Parkinson, 1993) The N-terminal region of the sensor kinase monitors an environmental stimulus, and transfers the signal to its transmitter domain at the C-terminal which is a conserved histidine protein kinase (Parkinson and Kofoid, 1992) The histidine residue in the transmitter domain is then autophosphorylated

Figure 1.2 The circuit of ethylene response from biosynthesis to perception and signal transduction to final gene induction (modified from Johnson and Ecker, 1998)

a) The final two-steps in ethylene biosynthesis include converting ACC to ethylene

by ACC synthase and ACC oxidase

b) Ethylene binds to the receptors on the membranes and inactives the downstream

negative regulator CTR1 which probably functions as a MAPKKK in a MAPK

cascade

c) Further transduction of ethylene signal requires positive regulators EIN2, EIN3,

EIN5, and EIN6

d) The immediate target of EIN3 is ethylene response element binding proteins

(EREBPs), which will induce specific subsets of ethylene response genes leading

EBF1/2

response regulator Through the output domain of the regulator, expression of downstream target genes and ultimately cell behavior will be modified

These two-component regulators are predominantly sensors and signal transducers of many environmental stimuli in the adaptive responses of numerous prokaryotic species (Parkinson and Kofoid, 1992) Eukaryotes also have two-component signal transducers

(Chang and Meyerowitz, 1994) In the yeast Saccharomyces cerevisiae, these genes were

found to be involved in osmosensing MAP kinase cascade regulation (Maeda et al., 1994;

Posas et al., 1996) In the fungus Neurospora, Nik1 plays a role in the hyphal growth process (Alex et al., 1996) Studies on the slime mold Dictyostelium showed that two genes Dok1 and Dhk1 were essential in the osmolarity sensing and development process (Schuster et al., 1996; Wang et al., 1996) A histidine kinase CKI1 was also implicated in the cytokinin signal transduction in Arabidopsis (Kakimoto, 1996)

1.1.2.1.1 The ethylene receptor family

In Arabidopsis, there exist at least five members of ethylene receptors: ethylene response gene ETR1, ETR2, ethylene insensitive 4 (EIN4), ethylene response sensor (ERS1) and

ERS2 (Chang et al., 1993; Hua et al., 1995, 1998; Sakai et al., 1998) Among these

receptors, ETR1, ETR2 and EIN4 contain a receiver domain at the C-terminal response

On the basis of sequence and structural similarities, irrespective of the presence of the receiver domain, the receptor family can be further divided into two classes The first

class, consisting of ETR1 and ERS1, features three hydrophobic membrane-spanning

subdomains at the N-terminal region and a well-conserved histidine kinase domain at the

C-terminal part of the protein The second class, which includes ETR2, EIN4 and ERS2, is

predicted to have four stretches of hydrophobic amino acids

Interestingly, features of these two classes are also identified in other plant species such as tomato, peach, muskmelon, indicating that the mechanism of ethylene perception is likely conserved among all the flowering plants (Klee, 2002; Rasori et al., 2002; Sato-Nara et al.,

1999) Besides Arabidopsis, the tomato receptor family is the most fully characterized in which the existing six members designated as LeETR1 to 6 also fall into two classes

Functional compensation of these two classes of receptor genes is suggested by both

absence of phenotype in single loss-of-functions mutant of Arabidopsis and also from the antisense suppression studies of tomato LeETR3 and LeETR4 receptor genes (Tieman et al., 2000) In the antisense LeETR4 transgenic tomato plants, reduction in LeETR4 mRNA

level leads to enhanced ethylene sensitivity and responsiveness This ethylene sensitivity

could be eliminated by crossing with lines that overexpress LeETR3, suggesting the

occurrence of functional redundancy among various ethylene receptors

The expression levels of these genes are spatially and temporally controlled throughout

development In Arabidopsis, expression pattern of ETR1 and ERS1 differs in each tissue although they are ubiquitously expressed LeETR4 and LeETR5 in tomato are mainly

expressed in reproductive organs and less in vegetative tissues They are differently regulated during flower development (Tieman and Klee, 1999) These genes also respond

differently upon ethylene treatment ERS1 and LeETR3 can be up-regulated by exogenous ethylene, whereas ETR1 and LeETR1 are not affected by ethylene treatment These

different expression patterns as well as different structures suggested that these receptors must have their own specialized functions in the plant Transgenic tomato with reduced

LeETR1 transcripts displayed shorter internodes and reduced auxin movement This shows

that the different receptors might play different roles during plant development Therefore,

it was proposed that ethylene signal transduction occurs through parallel paths that partially intersect (Whitelaw et al., 2002)

1.1.2.1.2 Physiological action of receptors

The mechanism of ethylene perception of receptors has been largely revealed by the recent

genetic and biochemical studies In Arabidopsis, ETR1 gene was the first one identified as

a strong dominant ethylene-insensitive mutant, followed by the isolation of etr2, ein4 mutants with the same effect on ethylene sensitivity Transgenic plants with ERS1 and

ERS2 carrying similar missense mutations also showed insensitivity to ethylene,

suggesting that similar NH2-terminal domains were conserved for ethylene binding and

High-affinity ethylene binding with ETR1 occurs in the membrane-localized pocket at the

N-terminal part of the protein and copper ion is required as a transition cofactor

(Rodriguez et al., 1999) RAN1 (Responsive to Antagonist 1) was supposed to deliver the copper ions to the receptors RAN1 encodes a copper-transporter which is highly homologous to copper-transporting p-type ATPase such as the yeast Ccc2p and human

Menkes/Wilson disease proteins (Hirayama et al., 1999) Epistasis analysis indicates that

RAN1 acts upstream of the ethylene perception and two weak mutant alleles exhibited the

triple response when treated with the receptor antagonist trans-cyclooctene (Hirayama et

al., 1999) Co-suppression of the RAN1 gene led to a constitutive ethylene response

phenotype, and this phenotype could be partially rescued by the addition of copper These observations support the view that a copper-delivery pathway is required for generating a functional ethylene receptor

All the Arabidopsis receptors are negative regulators of the ethylene response (Hua and

Meyerowitz, 1998; Cancel et al., 2002) This was determined by analysis of quadruple

mutants that have constitutive ethylene responses The later generated ers1; etr1

loss-of-function double mutants exhibited the same responses is consistent with this functional

model (Wang et al., 2002) The negative regulation model was also demonstrated by

down-regulation of only one of the six known receptor genes, LeETR4 in tomato, which

results in enhanced ethylene responsiveness (Tieman et al., 2000)

1.1.2.2.1 CTR1 and the MAPK-kinase cascade

Isolation and characterization of CTR1 has led to substantial insights on the nature of ethylene signal transduction CTR1 was placed downstream of the ethylene receptors by

epistasis analyses (Kieber et al., 1993) The recessive loss-of-function or

reduction-of-function nature of ctr1 mutant indicates that it is a negative regulator and inhibits downstream events of the response pathway CTR1 is homologous to the Raf family of

serine/threonine protein kinases that initiate mitogen-activated protein (MAP)-kinase

cascade (Kyriakis et al., 1992) The high similarity of CTR1 to the existing MAPKKKs

implies the involvement of the MAP-kinase cascade in the signaling process This model was recently proposed after the identification of ACC-activatedMAPKs in Medicago and

Arabidopsis (Ouaked et al., 2003) Compelling evidence implicates that CTR1 together

with the newly identified MAPKK and MAPKs may comprise a MAPK module in plants ethylene response regulation (Chang et al., 2003)

1.1.2.2.2 Signaling to the nucleus and the nuclear events

Phenotypic and epistasis analyses showed that signal propagation from CTR1 to the nucleus requires EIN2 Nearly all ein2 loss-of-function mutants are insensitive to ethylene, suggesting that EIN2 is an important positive regulator in the signaling pathway (Alonso

et al., 1999) EIN2 encodes a novel integral membrane protein with an amino-terminal domain similar to members of NRAMP family of metal-ion transporters which may serve

direct evidence for the nuclear signal regulation in the early ethylene signal transduction

pathway (Chao et al., 1997) Overexpression of EIN3 in the ein2 null mutant background results in constitutive responses, indicating that EIN3 acts downstream of EIN2 and positively regulates ethylene signaling pathway EIN3 is a nuclear protein with several domains similar to transcription factors Recent studies showed that EIN3 is involved in an

ubiquitin/proteasome pathway to mediate the downstream ethylene responses (Guo and

Ecker, 2003; Potuschak et al., 2003) EIN3 is rapidly induced by ethylene and this induction requires functional upstream genes like CTR1 and EIN2 While in the absence of

ethylene, EIN3 protein is degraded by mediation of two F box proteins, EBF1 and EBF2 These two proteins interact physically with EIN3 and other EIN3-like proteins (EILs) to

destabilize EIN3 Overexpression of either EBF1 or EBF2 results in ethylene-insensitive phenotype similar to the loss-of- function mutant ein3-1, which is consistent with EBF1/2 targeting EIN3 for degradation The ebf1; ebf2 double mutant showed constitutive ethylene responses and EIN3 is stabilized in this plant These observations suggest that the proteasome pathway targets EIN3 to negatively regulate ethylene responses

1.2 Ethylene response in other processes

1.2.1 Ethylene and plant defense responses

1.2.1.1 Ethylene in systemic acquired resistance (SAR) and induced systemic

resistance (ISR)

1.2.1.1.1 Ethylene and SAR responses

Plants have many weapons to cope with environmental stresses such as mechanical injury and attack by pathogens or herbivores (Yang et al., 1997) The early defense response to pathogen attack is the tissue necrosis at the site of infection and cell death to restrict growth of the pathogen, which is a complex process known as hypersensitive response

(HR) Expression of two ethylene receptor genes, LeETR3 and LeETR4, is induced in tomato (Lycopersicon esculentum Mill) leaves during an HR to Xanthomonas campestris Enhanced HR to Xanthomonas campestris was also found to be correlated with the reduced expression of LeETR4 (Ciardi et al., 2000) HR is often followed by systemic,

long lasting response in which the plant becomes more resistant to subsequent pathogen

by necrotizing pathogens (Ross and Williamson, 1951; De Laat and Van Loon, 1982; Mauch et al., 1984; Spanu and Boller, 1989) Different plant species behave differently

upon pathogen infection Arabidopsis etr1-1 mutant can fully mount a SAR response

(Lawton et al., 1995) On the contrary, transgenic tobacco plants expressing this mutated

form of etr1-1 gene showed reduced SAR responses, they became more liable to be

infected by bacteria that are not pathogenic to the wild type (Knoester et al., 1998) The

SAR responses also vary with the pathogen stains The Arabidopsis ein2 mutant is more

resistant to three virulent strains of bacterial pathogens, but more susceptible to a normally avirulent fungal strain (Bent et al., 1992; Thomma et al., 1999)

In Arabidopsis, one subset of pathogenesis-related (PR) genes requires a functional ethylene pathway to obtain efficient resistance against some pathogens EIN2 is essential for the systemic induction of chitinase PR-3 and a hevein-like gene PR-4 in Arabidopsis upon infection with the fungus Alternaria brassicicola (Thomma et al., 1999) Ethylene signaling was also crucial to Arabidopsis resistance to E carotovora infection since culture filtrate-mediated gene induction was severely reduced in the ein2-1 and etr1-1

mutants (Norman-Setterblad et al., 2000) That ethylene may mediate responses of SAR

was further demonstrated by the tomato ethylene insensitive Nr (for Never ripe) mutant SAR was abolished in Nr plants although it was similar to the wild-type plants following

initial infection with bacterial spots and speck disease pathogens While wild-type plants

reacted to the infection by wilting and loss of infected leaves, the Nr mutant showed

Another induced resistance was first reported in carnation and cucumber when ‘infection’

by a nonpathogenic strain, plant growth-promoting rhizobacteria (PGPR) was shown to induce systemic resistance (Van Peer et al., 1991; Wei et al., 1991) This induced protection is different from SAR in that it is independent of salicylic acid (SA) and not associated with PR proteins It was termed rhizobacteria-mediated induced systemic resistance (ISR) (Pieterse et al., 1998; Van Loon, 1997) ISR examination of the ethylene

mutants against virulent Pseudomonas syringae pv Tomato (Pst) showed that ISR was abolished in not only etr1-1, but also in ein2 through ein7 (Knoester et al., 1999) This

evidence demonstrated that expression of ISR requires the complete ethylene signal transduction pathway established so far Detailed study was also carried out on

rhizobacteria application to eir1 (for ethylene-insensitive root) and axr1 (for auxin

resistance) mutants which are selectively insensitive to ethylene in the roots (Knoester et al., 1999) No ISR was observed in these two mutants after the rhizobacteria treatment

While the eir1 mutant expressed ISR upon preinoculation of the rhizobacterium only in

the leaves but not the roots, indicating that intact ethylene signaling is required at the site

of application of the inducing rhizobacteria

1.2.1.2 Interaction of ethylene, jasmonate, SA pathway in plant defense responses

The compound SA is essential for SAR since transgenic plants expressing a salicylate hydroxylase enzyme (NahG), which will degrade SA, do not exhibit SAR (Vernooij, et al., 1994) Nevertheless, inducible defense to some pathogens requires concerted function of two hormones, ethylene and jasmonic acid (JA) (Thomma et al., 2001) The induction of a

subset of plant defense genes including PR-3, PR-4 and PDF1.2 failed in Arabidopsis ein2 and coi1, the plants with defects in ethylene perception and JA signaling, separately (Penninckx et al., 1998) The induction of PDF1.2 upon infection by Alternaria

brassicicola requires concomitant presence of both ethylene and JA signaling pathways

(Penninckx et al., 1996, 1998.) A recent study on ethylene response factor1 (ERF1),

which encodes a transcription factor regulating the expression of pathogen response genes further demonstrates the crosstalk of these two hormones (Lorenzo et al., 2003) Either

ethylene or jasmonate by itself, and the two hormones synergistically can induce ERF1 expression rapidly But activation of ERF1 requires both signaling pathways because

plants defective in either pathway will fail to induce expression of this gene by any of the two hormones or combination of them These data suggest that there exists an ethylene/jasmonate (ET/JA) dependent pathway in which ethylene and jasmonate co-regulate some plant defense genes

Although the SA- and ET/JA-mediated signaling on their own induce different sets of PR genes and lead to plant resistance to different pathogens, there appears to be considerable interactions between these pathways as indicated by several studies on SAR (Dong, 1998)

Analysis of the Arabidopsis ssi1 mutant revealed that PDF1.2, the marker gene of ET/JA

pathway (as mentioned above) is expressed in an SA-dependent manner (Shah et al., 1999) On the other hand, components of the ET/JA pathway are required for SA-

mediated resistance in the cpr5/6 mutant (Clarke et al., 2000), suggesting CPR5/6 may be one of the integrators between these pathways Study on Arabidopsis hrl1 mutant revealed

that the components of SA and ET/JA signaling pathways work synergistically to regulate

the expression of defense genes and resistance against Peronospora parasitica (Devadas

et al., 2002) Moreover, compatibility of the two pathways was demonstrated by

simultaneously activating SA-dependent SAR and ET/JA mediated ISR in Arabidopsis which led to an additive effect on induced resistance to Pseudomonas syringae (van Wees

et al., 2000)

Despite the above-mentioned synergistic interactions, there are reports on antagonistic effects among the various defense pathways While JA suppresses superoxide-driven cell death and results in lesion containment, SA promotes HR-related cell death (Rao et al.,

2000; Overmyer et al., 2000) Overexpression of ERF1 in Arabidopsis was shown to confer resistance to necrotrophic fungi such as Botrytis cinerea but reduced SA-mediated tolerance against Pseudomonas syringae pv tomato DC3000, suggesting the negative

regulation between ethylene and SA responses (Berrocal-Lobo et al., 2002)

1.2.2 Ethylene and tissue-specific responses

1.2.2.1 Ethylene responses in root and hypocotyl

The EIR1 gene may be a possible target of ethylene pathway in the roots since the eir1

mutant seedlings showed ethylene-insensitive phenotype only in the roots but normal

responses in apical hook and hypocotyl (Roman et al., 1995) Mutant analysis places EIR1 downstream of CTR1 and EIN2 (Luschnig et al., 1998) The eir1 roots are agravitropic which is not a phenotype found in other ethylene signaling mutants EIR1 may encode a putative auxin transporter and some of the phenotypes of the eir1 mutant are similar to that of auxin-sensing mutant These observations also revealed a role of EIR1 in auxin-

ethylene cross-talk

Studies on the Arabidopsis hookless mutant hookless1(hls1) not only suggested a role for

ethylene in differential cell elongation in hypocotyl, but also demonstrated the integration

of ethylene and auxin signaling pathways in the regulation of apical hook formation

(Lehman et al., 1996) HLS1 transcripts are increased by ethylene treatment and decreased

in ethylene-insensitive ein2 mutant Overexpression of HLS1 in transgenic Arabidopsis

caused constitusive hypocotyl hook curvature Inhibition of auxin transport also results in the similar phenotype Expression of two auxin-responsive genes is altered in the

transgenic Arabidopsis plants, indicating regulation of auxin activity is required by the

1.2.2.2 Ethylene and nodulation

It has been suggested that ethylene is involved in nodule development in legume symbiosis A hyper-nodulation legume mutant, sickle (skl) showed increased mature nodules (Penmetsa and Cook, 1997) At the same time, the skl plants also showed

Rhizobium-ethylene-insensitive phenotypes, such as delayed senescence of petals and leaves, as well

as insensitivity of seedlings to ACC treatment

1.2.3 Ethylene responses in animals

Some genes that respond to ethylene have been isolated from bacteria, marine sponge (invertebrate) and mammalian cell cultures (vertebrate) recently (Rodriguez et al., 1999;

Wang et al., 2002) A portion of an ethylene binding protein from Synechocystis sp has high sequence similarity to the transmembrane domain of ETR1 The sponge Suberites

domuncula provides the first example of ethylene signaling in an animal because this

organism could respond to ethylene both physiologically and at molecular levels (Krasko

et al., 1999) Ethylene could reduce the starvation-induced apoptosis and up-regulate

expression of two genes, SDERR and SDCCdPK in this sponge SDERR encodes a

potential ethylene-responsive protein and shares high homology to HEVER (Hevea ethylene responsive) of Hevea brasiliensis(rubber tree) (Sivasubramaniam et al., 1995)

concentration Moreover, mammalian cell cultures treated with ethephon, an releasing reagent, showed a dramatic increaseof cytosolic calcium influx (Perovic et al., 2001) Although the mechanisms are yet to be determined, these observations from cell cultures suggest the occurrences of some forms of ethylene perception and signal

ethylene-transduction in animals The recent identification of a putative ACS gene from human

makes this hypothesis more interesting although the corresponding product does not haveACC synthase activity to catalyze the synthesis of ACC, most likely due to the absence of two conservedresidues in the protein (Koch et al., 2001).

1.3 Application of ethylene-insensitivity-based technology to floriculture and agriculture

The senescence responses of leaves and flowers to ethylene are of significance for many commercial floricultural products and vegetable crops Genetic engineering has been applied to control postharvest longevity One strategy is to reduce ethylene synthesis This

is most effective in controlling fruit ripening, especially in climacteric plants like tomato and melon The cloned key enzymes in the ethylene biosynthesis pathway, such as ACC oxidase and S-adenosylmethionine hydrolase facilitate the inhibition of ethylene biosynthesis by generating antisense transgenic plants This has shown to be successful in inhibiting fruit ripening in tomato (Hamilton et al., 1990; Good et al., 1994; Oeller et al., 1991) and melon (Ayub et al., 1996) Transgenic carnation with an antisense ACC oxidase gene showed a longer shelf-life and less ethylene production than untransformed plants (Savin et al., 1995) Leaf senescence of tomato transformed with an antisense ACC isoform was delayed considerably (John et al., 1995) These plants also produced less ethylene during senescence Expression of an ACC-degrading enzyme from bacteria in tomato also caused significant delays in fruit ripening (Klee et al., 1991) Transgenic

tomato plants with reduction of the ethylene receptor ETR1 by introducing an inverted

receiver domain displayed a marked delay in abscission and shorter internode although these plants have a normal triple response and ethylene receptors are supposed to be negative regulators of ethylene response (Whitelaw et al., 2002)

Another approach to control the senescence process is to create ethylene-insensitive

plants This has been achieved so far by introducing the dominant Arabidopsis etr1-1 and

boers (from Brassica oleracea) mutant gene into heterologous plants (Wilkinson et al.,

1997; Shaw et al., 2002) Transgenic tomato and petunia expressing Arabidopsis etr1-1

showed significantly delayed fruit ripening, flower senescence and flower abscission

When etr1-1 was introduced into carnation plants, about half of the transgenic plants

displayed delayed flower senescence (Bovy et al., 1999), with 2 to 3-fold increase in vase life In addition, the phenotype of these flowers was different from that of the control as their petals remained firm and finally decolorized, instead of the typical ethylene-dependent petal inrolling during senescence The ability of these receptors to function in heterologous plants suggests that this pathway of hormone recognition and response is highly conserved and can be manipulated to the benefit of horticulture and agriculture

1.4 Studies on Coriander

The herbaceous plant coriander (Coriandrum sativum L.) is well kown for its culinary and

medicinal uses Its leaves and fruits are commonly used as flavoring agents A study on micronutrition and phytochemicals having antioxidant properties from 30 green leafy vegetables revealed that coriander leaves showed high values for ferrous iron chelating activity (Tarwadi et al., 2003) The antiperoxidative effect of coriander seeds was also demonstrated by rats administered high fat diet after they were fed with coriander seeds

essential oils from several plants (Elgayyar et al., 2001) It has been documented that coriander is one of the traditional plants which was used in diabetes treatment (Swanston-Flatt et al., 1990) When coriander seeds were served in the diets in a certain ratio, the development of streptozotocin diabetes in mice could be retarded The hyperglycaemia of the mice was also reduced Detailed studies showed that insulin-releasing and insulin–like activity exists in coriander (Gray and Flatt., 1999)

Genetic engineering has been used applied to modify fatty acid composition in seeds of oil

crops (Dehesh et al., 1996) Overexpression of Ch FatB2, a thioesterase cDNA from

Cuphea hookeriana in the seeds of canola, an oil seed crop that normally does not

accumulate any caprylate and caprate, resulted in a dramatic increase in the levels of these two fatty acids Similarly, transgenic cotton harboring seed-specific fatty acid desaturase genes showed improved nutritional quality (Liu et al., 2000) However, there is no reported genetic transformation of coriander

1.5 Tandem Affinity Purification (TAP) method

1.5.1 Overview of the TAP method

The TAP method is a generic and rapid tool for protein complex characterization and proteome exploration (Rigaut et al., 1999) The TAP-tag used in this method refers to a fusion cassette including calmodulin-binding peptide (CBP), a TEV cleavage site, and IgG

binding unit of protein A of Staphylococcus aureus (ProtA) The two high-affinity tags in

the cassette, CBP and ProtA allow efficient recovery of a fusion protein expressed at a low level in a complex mixture In the TAP method, this cassette is fused to the targeted protein and introduced to the host cell or organ (Figure1.3) As shown in Figure 1.3, the fusion protein and the potential interacting components in the cell extracts were recovered after an IgG matrix affinity selection Those bound materials were collected after the TEV protease digestion and then subjected to incubation with calmodulin (CAM) agarose, the eluate containing the bound materials is released under native condition with EGTA

There are several means to characterize the protein complex of interest in the final eluate Combined with mass spectrometry, this method is now used for analysis of protein complexes in specific groups of cells Proteins from heterocomplexs have been

successfully identified in various organisms such as Escherichia coli (Gully et al., 2003),

Figure 1.3 Schematic representation of the TAP-tag purification process

A two-step affinity selection is involved in the TAP-tag purification using the IgG and calmodulin beads as matrix The native complex is finally released by EGTA elution

yeast (Rigaut et al., 1999), mammalian cells (Knuesel et al., 2003) and recently in

Nicotiana benthamiana (Rohila et al., 2004) Since the purification steps are performed

under conditions that maintain native protein structure, this method allows for direct assay for the activity and structure of purified proteins The TAP method is also useful for purification of recombinant proteins that may be present at low levels in organisms like bacteria and yeast (Puig et al., 2001)

The TAP method is versatile in the selection of different tag systems and different purification protocols (Puig et al., 2001) The initial tag can be at the C-terminal of the target protein In case that the tag would affect the protein expression level or function, the tag could be put at the N-terminal Split TAP method involves adding the ProtA with the TEV cleavage site to one protein and the CBP to another protein (Puig et al., 2001) By this way, the complex containing the two given proteins as well as the target protein can

be purified together The third strategy is the subtraction method (Bouveret et al., 2000) This method is used when a common unit is shared in several complexes but only one of them is needed finally In the undesired complex, the ProtA without a TEV cleavage site is fused to a protein After the first IgG argarose affinity selection, only the desired complex

is eluted while the undesired one remains on the column The variation of the purification methods will depend on the objectives of different experiments For example, nonionic detergent can be added to help solubilize the target protein

1.5.2 Application of TAP method for plant heterocomplex study

The TAP method was first applied to tomato Cf-9 protein heteromultimeric complex characterization (Rivas et al., 2002) The TAP-tag was fused to the C-terminal of the Cf-9

protein and the resulting functional Cf-9: TAP was transiently expressed in Nicotiana

benthamiana leaves Western blot analysis showed that Cf-9 protein is a part of a 420 kD

complex with only one molecule in the complex The TAP-tag was also applied in pull down experiments to investigate the mechanism of ethylene signal transduction A TAP-

tagged CTR1 in Arabidopsis was co-purified with the receptor ETR1 provids definitive

proof that CTR1 is part of the signaling complex (Gao et al., 2003) Recently, an improved tag and purification method from plants were developed (Rohila et al., 2004) All these data suggest that the TAP-tag method is becoming a major tool for proteome exploration

in plants

Chapter 2 Materials and methods 2.1 Materials

2.1.1 Plant materials

Wild-type petunia (diploid Petunia hybrida cv Mitchell) seeds were obtained from Dr

Harry J Klee’s Laboratory, Department of Horticultural Sciences, University of Florida,

Gainesville, USA Wild-type coriander (Coriandrum sativum L) seeds were purchased from Known-you Seed Distribution, Taiwan Wild-type Nicotiana benthamiana seeds

were obtained from Dr Sanjay Swarup’s Laboratory, Department of Biological Sciences,

NUS Wild-type Arabidopsis ecotype Col-0 (LEHLE seeds, U.S.A.) were used in this

study

2.1.2 Bacterial strains and plasmids

The bacterial strain used for DNA cloning in this study was Escherichia coli DH5α, which

was grown in liquid LB medium (Sambrook et al., 1989) at 37°C except when indicated

otherwise The Agrobacterium tumefaciens strains used were GV3101 and GV3850

(Koncz and Schell, 1986) Both these strains were grown in the indicated medium at 28°C

gene fragment and the pGreen 0229 vector (Yu et al., 2004) with the TAP-tag that was

used for Nicotiana benthamiana transient expression analysis were obtained from

Professor Elliot Meyerowitz’s laboratory, California Institute of Technology, Pasadena, California , USA

2.1.3 Culture conditions and media

Wild-type petunia plants were grown on commercial potting compost (Flora Fleur Multi- purpose Substrate) in the greenhouse with temperature ranging from 28°C to 35°C Coriander seeds were surface sterilized with 10% Clorox (0.5% sodium hypochlorite) for

10 min, rinsed five times with distilled water and incubated on MS (Murashige and Skoog, 1962) basal medium supplemented with 3% sucrose and 2.5% phytagel in GA-7 vessels at

24ºC Wild-type Nicotiana benthamiana and Arabidopsis plants were grown on

commercial potting compost (Flora Fleur Multi-purpose Substrate) in the plant growth room maintained at 22°C ± 2°C

Culture media, chemicals and antibiotics used in this study are listed in Table 2.1 and Table 2.2

Table 2.1 Murashige-Skoog medium including Gamborg’s B5 vitamins

Macro elements CaCl2 332.02

KH2PO4 170.00 KNO3 1900.00 MgSO4 180.54

NH4NO3 1650.00 Micro elements CoCl2·6H2O 0.025

CuSO4·5 H2O 0.025 FeNaEDTA 36.70

H3BO3 6.20

KI 0.83 MnSO4·H2O 16.90

Na2MoO4·2H2O 0.25 ZnSO4·7H2O 8.60 Vitamins Myo-Inositol 100.00

Nicotinic acid 1.00 Pyridoxine HCl 1.00 Thiamine HCl 10.00

Table 2.2 Chemicals and antibiotics used for tissue culture

Abbreviations: BA, benzyladenine; NAA, 1-naphthaleneacetic acid; 2,4-D, 2,4-dichloro- phenoxyacetic acid

sterilized, and stored at 4ºC in the dark

sterilized, and stored at 4ºC in the dark

sterilized, and stored at 4ºC in the dark

Cefotaxime 100 mg/ml Dissolved in H2O, filter sterilized,

and stored at -20ºC in the dark Kanamycin 50 mg/ml Dissolved in H2O, filter sterilized,

and stored at -20ºC in the dark Gentamycin 40 mg/ml Dissolved in H2O, filter sterilized,

and stored at -20ºC in the dark

2.2 Molecular cloning of PERS1 and PETR2 from petunia

2.2.1 Extraction of RNA from petunia tissues

Precautions were taken for preventing RNase contamination before extracting RNA from petunia tissues All the glassware, mortars and pestles were baked at 180°C overnight or autoclaved for 1 h

2.2.2 PCR with degenerate primers

After RNA extraction, the quality of RNA was checked by agarose gel electrophoresis Reverse transcription (RT) was performed using AMV (avian myeloblastosis virus) reverse transcriptase (AMV-RT, Promega) Briefly, about 2 µg RNA was mixed with oligodT primer, incubated at 70ºC for 3 min, then the tube was cooled on ice for 2 min After a short spin, the following were added to the mixture: 2 µl 5X reaction buffer, 2 µl dNTP mix (10 mM each), 30 units AMV RT, 40 units rRNasin® ribonuclease inhibitor and nuclease-free water to a final volume of 20 µl The tube was incubated at 42ºC in an oven for 1 h This RT reaction was terminated by heat treatment at 75ºC for 10 min The tube was cooled on ice and centrifuged to collect the cDNA products to be used for the

AGG-3’ and ETR1r, 5’-TAS AMC CCT TSC CRA SAC CWT CAC T-3’ to isolate ETR1

homolog, ETHK1, 5’-GAA GGA CCC CAA TGG AGG TYK TCT YAC-3’ and ETHK2,

5’-CCR TCY AAA TCA GCA GGT GGA GAT CCA-3’to isolate the ETR2 homolog

(Mixed bases nomenclature: R, A/G; M, C/A; N, A/T/G/C; Y, T/C; K, G/T; S, G/C) The PCR condition was one cycle at 94ºC for 5 min, 35 cycles of denaturation at 94 ºC for 30

s, annealing at 56ºC for 1 min 30 s and extension at 72ºC for 1 min, and final extension at 72ºC for 7 min The PCR products were fractionated by 1% agarose gel electrophoresis and visualized under ultraviolet light after staining with ethidium bromide

2.2.3 Cloning PCR bands of the expected sizes

Bands of the expected size were excised from the gel using a razor blade, and the DNA was recovered with QIAEX®II Gel Extraction System (QIAGEN, Germany) Purified products were quantified and cloned into pGEM-T vector (Promega, USA) by TA cloning Ligation was set up following the instructions of the manufacturer using T4 ligase (Promega, USA) After several hours at room temperature and then overnight ligation at

4ºC, the total ligation mix of 10 µl was used to transform chemically competent E.coli

DH5α cells by heat shock transformation as described below

2.2.4 Preparation of E coli competent cells for heat-shock transformation

E coli competent cells were prepared as described by Inoue et al (1990) with some

modifications Frozen stock E coli cells were thawed, streaked on an LB agar plate, and

cultured overnight at 37ºC A single colony was inoculated into 1.5 ml SOB medium (Tryptone 20 g/l, yeast extract 5 g/l, NaCl 0.58 g/l, KCl 0.19 g/l, MgCl2·6H2O 2.03 g/l, MgSO4·7H2O 2.46 g/l) in a 15 ml culture tube, and grown for 12 h with vigorous shaking (200 rpm) at 37ºC 500 µl of the above culture was then inoculated to 100 ml SOB medium in a 1-liter flask, and grown to an A600 of 0.6 at about 20ºC with vigorous shaking (225 rpm) The culture was transferred to 2 ice-cold Falcon tubes, and placed on ice for 10 min before centrifugation at 3000 rpm for 5 min at 4 ºC The pellet was gently resuspended in 20 ml of freshly prepared ice-cold TB (10 mM Pipes, 55 mM MnCl2, 15

mM CaCl2, 250 mM KCl, pH 6.7), incubated on ice for 10 min, and centrifuged as above The cell pellet was gently resuspended in 4 ml of TB, and DMSO was added with gentle swirling to a final concentration of 7% After incubating on ice for 10 min, the cell suspension was aliquoted (100-200 µl) into pre-chilled 0.6 ml microfuge tubes, immediately frozen by immersion in liquid nitrogen, and stored at -80ºC

2.2.5 Transformation of E.coli competent cells

A tube with 100 µl frozen competent cells was thawed by holding in the palm Just as the cells thaw, the tube was put on ice The ligation mixture was added to each tube and mixed well with the cells by gently tapping, and the cells were incubated on ice for 30 min The cells were heat shocked for 90 s at 42ºC in a heat block and transferred to ice, incubated for 3-5 min After 1ml SOB was added, the cells were allowed to recover at 37ºC for 1 h with shaking at 200 rpm The cultured cells were centrifuged in an eppendorf centrifuge (5417C) at 9000 rpm for 1 min at room temperature, and 800 µl of SOB medium from the top was discarded The cells in the rest of the medium were resuspended and spread onto LB agar plates containing the appropriate antibiotic selection for the plasmid The plates were incubated overnight at 37ºC

2.2.6 Screening of the clones containing the expected fragments

Colony-direct PCR was carried out to screen the white colonies following Paton et al (1993) with some modifications Sterilized white tip was lightly touched on the surface of the test white colony on the agar medium to collect the bacteria The collected sample was then resuspended in 6-7 µl of sterile water in a sterile 0.6 ml microfuge tube 2 µl of this suspension was used as a template for the PCR reaction with SP6 and T7 primers, which

2.2.7 DNA sequencing and alignment analysis

Plasmid DNA was isolated using Miniprep kit (Promega, USA) according to the manufacturer’s recommendations The cloned cDNAs for both strands were sequenced by the dideoxy method (Sanger et al., 1977) using ABI PRISM BigDyeTM Terminator Sequencing Ready Reaction Kit (Perkin-Elmer Applied Biosystem, USA) The cycle sequencing reaction was prepared by starting with a mixture of 200 ng double-stranded DNA, 1.6 pmol appropriate primers, 4 µl Terminator Ready Reaction Mix with deionised water to a final volume of 10 µl Then PCR was performed by 25 cycles of denaturation at 96ºC for 10 s, annealing at 50ºC for 5 s and extension at 60ºC for 4 min The amplified products were precipitated for 15 min in 80 µl 75% isopropanol at room temperature and centrifuged for 20 min at 14 000 rpm The pellet was washed with 250µl 75% isopropanol, air dried and dissolved in 4 µl loading buffer consisting of formamide, 25

mM EDTA (pH 8.0) The sample was heat-denatured for 2 min at 95ºC and cooled on ice before loading Sequencing was performed using ABI PRISMTM 377 DNA sequencer (Perkin-Elmer Applied Biosystem, USA at the DNA sequencing lab of the Department of Biological Sciences) The sequencing data obtained were used to identify the cDNAs using BLAST nucleotide homology search at the National Centre for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/BLAST/)

2.2.8 Rapid Amplification of 5’- and 3’- cDNA Ends of PERS1, PETR1,

PETR2 genes

The SMARTTM RACE cDNA Amplification Kit (Clontech Laboratories) was used to

identify the 5’- and 3’- cDNA (5’/3’-RACE) end sequences of PERS1 and PETR2 genes

The RNA quality isolated from petunia flower was checked by agarose gel as described earlier before cDNA synthesis using the 5’/3’-RACE kit The 5’ (3’)-RACE ready cDNA were generated according to the manufacturer’s instructions using the BD PowerSript reverse transcriptase (a type of MMLV reverse transcriptase) and primers provided in the kit In the reverse transcription process, the BD PowerSript reverse transcriptase exhibits a terminal transferase activity, adding 3-5 residues (predominantly dC) to the 3’ end of the first-strand cDNA when this transcriptase reached the end of the RNA template The terminal stretch of dG residues of the SMART oligo (provided in the kit) therefore annealed to dC-rich cDNA tail and served as an extended template for the reverse transcription The BD reverse transcriptase thus switched the template from the mRNA molecule to the SMART oligo, followed by generation of a complete cDNA copy of the original RNA with the additional SMART sequence at the end Because the dC-tailing activity of reverse transcriptase was most efficient if the enzyme reached the end of the RNA template, the SMART sequence was typically added only to complete first-strand cDNAs If high quality RNA is used as the template, this process will guarantee the formation of a set of cDNAs with longer 5’ sequences

For the first strand synthesis of the 5’-RACE cDNA, the 5 µl reaction mixture consisted of

1 µg total RNA, 1µl 5’-CDS primer (the modified oligo (dT) primer) and RNase-free H2O After incubation at 72ºC for 2 min and then on ice for another 2 min, 2 µl 5X first-strand buffer was added followed by, 1 µl DTT(20 mM), 1 µl dNTP mix (10 mM each), and 1 µl

BD RT (200 U /µl ), and incubated at 42ºC for 1.5 h After heat treatment at 72ºC for 7 min, the product (5’-RACE ready cDNA) was diluted with Tricine-EDTA buffer before storage for later PCR amplification

For the first strand synthesis of the 3’-RACE cDNA, the 3’-CDS primer which contains 30 dTs and an adapter at the 5’-end of the primer was used The reaction conditions were the same as that for 5’-RACE cDNA synthesis except that the 5’-CDS and SMART II oligo were replaced by the 3’-CDS primer The following PCR reaction was prepared in a 50 µl reaction mix containing 2.5 µl 5’ (3’)-RACE ready cDNA, 5 µl 10 X Advantage 2 PCR buffer, 1 µl dNTP mix (10 mM each), 5 µl UPM, 1 µl Advantage 2 polymerase, 34.5 µl sterile distilled water and 1 µl gene-specific primer (10µM) PCR was performed for 35 cycles of 10 s denaturation at 94ºC, 20 s annealing at 68ºC and 3 min extension at 72ºC The PCR products were fractionated by gel electrophoresis The appropriate bands were purified and cloned into the pGEM-T vector for sequence analysis The gene-specific

primers used for RACE of PERS1, PETR2, and PETR1 gene are given below:

ERS1race-5, 5’-CATGGGCTCGCATGGACTCCTCCAGAAT -3’,

ERS1race-3, 5’-TGGAGGAGTCCATGCGAGCCCATGATCA-3’,

HKrace-5, 5’-TTCCTCCACCTC CCTACTGCAACGTCTTT -3’,