CIRCULARES INFORMATIVAS Y UNA

hypothetical linear pathway is based on both genetic (epistasis) analysis, ectopic gene expression studies, and biochemical interactions. Progressing from left to right,

ethylene is thought to regulate negatively a family of membrane-associated receptors that are related to the bacterial two-component superfamily of catalytic receptors. The histidine-kinase transmitter domains of members of the receptor family interact with the regulatory domain of the Raf-like kinase CTR 1 . This receptor/CTR 1 complex negatively regulates a membrane protein (EIN2) related to a superfamily of metal transporters. The cytoplasmic C-terminal domain of EIN2 positively signals

downstream to the E I N3 family of transcription factors located in the nucleus. A target of the E I N3 transcription factors is the promoter of the ERF1 gene, a member of a second family of transcription factors_ ERF1 is rapidly induced in response to ethylene and is capable of activating a subset of ethylene responses when ectopically

expressed. This figu re was reproduced from Hende and Bleecker (2000) with permission.

Chapter 1, Introduction

type, and also had reduced ethylene binding capacity. The gene encoding ETR1 was subsequently cloned (Chang et aI., 1993), and showed sequence homology to a superfamily of catalytic receptors in bacteria known as two-component regulators (Wurgler-Murphy and Saito, 1 997).

1 6

Two-component regulators are typically composed of a sensor protein with an input domain that receives signals and a catalytic transmitter domain that autophosphorylates on an internal histidine residue. The second component is a response regulator protein, which is composed of a receiver domain that receives phosphate from the transmitter on an aspartate residue and an output domain that mediates responses depending on the phosphorylation state of the receiver. The transmitter domain of ETR1 has all the conserved residues essential for histidine kinase activity and is capable of

autophosphorylating on the conserved histidine when expressed in yeast (Gamble et aI., 1998).

The sensor component of these two-component regulators contains an input domain that interacts directly with a signalling ligand. The N-terminal hydrophobic domain of ETR1 showed no homology to any other functional domain in protein databases. The

importance of this novel domain for ethylene signalling was suggested because all point mutations that caused insensitivity to ethylene in planta were located to this domain (Chang et aI., 1993). Schaller and Bleecker (1995) demonstrated high affinity binding of ethylene to ETR1 when expressed in yeast. Further, Rodriguez et al. (1 999) showed that the 128 residues of the hydrophobic domain of ETR1 were necessary and sufficient for ethylene binding activity. Both the ability to bind ethylene, and the homology to the bacterial two-component regulators suggests that the ETR1 protein is in fact an ethylene receptor.

Four other genes have been isolated from Arabidopsis that are related to ETR1 (Hua et aI., 1995; Hua et aI., 1998; Sakai et aI., 1998). They are split into two groups based on structural similarities. The ETR1-like family includes ETRl and ERSl and is

characterised by similar hydrophobic N-terminal domains and a conserved histidine kinase domain. The ETR2-like subfamily, which includes ETR2, ERS2 and E1N4, has

Chapter 1, Introduction

an additional hydrophobic structure compared to the ETR1-like subfamily, and lacks some of the elements considered necessary for catalytic activity in the histidine kinase domain (Bleecker and Kende, 2000).

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The next enzyme thought to be involved in ethylene signalling is CTRl. CTRl was cloned by Keiber et al. (1993) and sequence analysis showed that the gene encodes a protein related to mammalian Raf kinases that initiate mitogen-�ctivated Qrotein kinase (MAPK) cascades. The homology of CTRl with MAPKKKs suggests that ethylene signalling may operate through a MAP-kinase cascade. Although genes homologous to MAPKKs and MAPKs exist in Arabidopsis, none are known to be associated with ethylene signalling. Further, biochemical and yeast two-hybrid experiments indicate that CTRl interacts directly with the transmitter domains of ethylene receptors (Clarke et aI., 1998). However, ethylene treatment caused enhanced MAPK-like activity and

phosphorylated MAPK-like protein accumulation in Arabidop sis (Novikova et aI.,

2000).

Loss of function mutations in CTRl and/or mUltiple receptor genes leads to constitutive activation of the ethylene-response pathway, suggesting that the receptors form a complex with CTR1, which negatively regulates responses in the absence of ethylene. Ethylene binding to receptors would then reduce the activity of the ETRfCTRl complex and result in a derepression of the pathway (Bleecker and Kende, 2000). Ethylene signalling downstream of CTRl is not very well understood. The protein encoded by EIN2 is related to the eukaryotic Nramp family of metal-ion transporters (Alonso et aI., 1999) and is required for ethylene signalling, acting somewhere between CTRl and the EIN3 family of transcriptional regulators (Chao et aI., 1997). A loss of function

mutation in EIN3 caused a reduced responsiveness to ethylene, and EIN3 was shown to encode a nuclear protein (Chao et aI., 1997). Over-expression of EIN3 and related genes, EIL I and EIL2, caused constitutive activation of response pathways (Figure 1.2).

Sensing of ethylene causes up-regulation of a number of transcripts that are responsible for eliciting the ethylene response, such as chitinases (Ishage et aI., 1991), cellulase (Ferrarese et aI., 1995) and cysteine protease (Jones et aI., 1995). Promoter elements for

Chapter 1. Introduction _{1 8}

all these ethylene-induced genes contain a conserved 1 1 base pair sequence

T AAGAGCCGGCC, known as the GCC box. These �thylene response �lements (EREs) are crucial for ethylene signalling, as deletion of these elements causes loss of ethylene response (Shinshi et aI., 1995), and addition of these elements to a minimal cauliflower mosaic virus (CaMV) 35S promoter conferred ethylene responsiveness (Ohme-Takagi and Shinshi, 1 995). The �thylene response factor (ERFl ) gene was identified by Solano et al. ( 1 998) and was found to have a target promoter for the EIN3 family of proteins (Figure 1 .2). ERFl is a member of a group of plant-specific

transcription factors referred to as �thylene-response-�lement-hinding-Qroteins (EREBPs). EREBPs were originally identified as trans-acting DNA binding proteins that bound to specific elements in ethylene-inducible genes (Ohme-Takagi and Shinshi,

1 995). ERF 1 expression was rapidly induced by treatment with ethylene in Arabidopsis

(Solano et aI., 1 998) and heterodimers of EIN3 were shown to interact in vitro with a

promoter element of ERF l . Further, when ERF 1 is constitutively expressed in an EIN3

mutant background, ethylene reponsiveness is restored. These results suggest that ERFl is in the primary transduction chain downstream of the previously identified

components and clearly indicate that a transcriptional cascade is operating in ethylene signalling (Bleeker and Kende, 2000).

Not only the ability to sense ethylene, but also changes in sensitivity to ethylene appear to be important. For example, a particular developmental state is required for response to ethylene for both ripening tomato fruits (Grierson and Kaber, 1986) as well as senescing leaves (Bleecker and Patterson, 1 997).

The concentration of ethylene also plays an important role. Transgenic tomato fruit with lowered levels of ethylene production ripen more slowly (Klee, 1 993) and in some cases fail to ripen altogether (Oeller et aI., 1 99 1 ). Furthermore, transgenic tomato plants unable to produce high levels of ethylene in response to flooding stress, when compared with wild-type plants, displayed reduced epinastic curvature of the petioles (English et aI., 1 995). These findings suggest that ethylene production is also an important aspect of how this hormone regulates a diverse array of responses in plants. This, together with the fact that the two dedicated ethylene biosynthetic enzymes, ACC synthase and ACC

Chapter 1, Introduction

oxidase, are encoded by multi gene families in plants that are differentially expressed, may provide another tier of regulation for this hormone.