Differential expression of two novel members of the tomato ethylene-receptor family

Abstract

The phytohormone ethylene regulates many aspects of plant growth, development, and environmental responses. Much of the developmental regulation of ethylene responses in tomato (Lycopersicon esculentum) occurs at the level of hormone sensitivity. In an effort to understand the regulation of ethylene responses, we isolated and characterized tomato genes with sequence similarity to the Arabidopsis ETR1 (ethylene response 1) ethylene receptor. Previously, we isolated three genes that exhibit high similarity to ETR1 and to each other. Here we report the isolation of two additional genes, LeETR4 and LeETR5, that are only 42% and 40% identical to ETR1, respectively. Although the amino acids known to be involved in ethylene binding are conserved, LeETR5 lacks the histidine within the kinase domain that is predicted to be phosphorylated. This suggests that histidine kinase activity is not necessary for an ethylene response, because mutated forms of both LeETR4 and LeETR5 confer dominant ethylene insensitivity in transgenic Arabidopsis plants. Expression analysis indicates that LeETR4 accounts for most of the putative ethylene-receptor mRNA present in reproductive tissues, but, like LeETR5, it is less abundant in vegetative tissues. Taken together, ethylene perception in tomato is potentially quite complex, with at least five structurally divergent, putative receptor family members exhibiting significant variation in expression levels throughout development.

Figure 1

Schematic representation of the structure of the tomato LeETR proteins. By analogy to the Arabidopsis ETR1 protein, the three domains of the proteins include the sensor domain, with three or four hydrophobic regions (represented by black boxes) capable of spanning a membrane (amino acids 1–325 of ETR1); the signaling domain (amino acids 326–609 of ETR1), with the conserved domains of bacterial His kinases represented by black boxes and the conserved His represented by a star; and the response-regulator domain (amino acids 610–738 of ETR1), with the conserved domain containing an Asp that is capable of receiving the phosphate from the His kinase represented by a black box (Chang et al., 1993). The percentage of amino acid identity and similarity between the Arabidopsis ETR1 protein and each of the tomato ETR1 homologs within each of the three domains is shown below the schematic of each protein.

Figure 2

Amino acid sequence alignments of the five tomato ETR1 homologs and Arabidopsis ETR1 and ETR2. Tomato sequences are designated LeETR1, LeETR2 (Lashbrook et al., 1998), LeNR (Wilkinson et al., 1995), LeETR4, and LeETR5 (this paper). Arabidopsis ETR1 and ETR2 are designated AtETR1 and AtETR2, respectively (Chang et al.,1993; Sakai et al., 1998). Shaded areas represent putative membrane-spanning domains. Cons, Consensus sequence; ⇓ , Cys residues involved in dimerization (Schaller et al., 1995); ↓, amino acids in which mutations result in dominant ethylene insensitivity in Arabidopsis (Ala-31 to Val, Ile-62 to Phe, Cys-65 to Tyr, and Ala-102 to Thr) or tomato (Pro-36 to Leu in NR) (Wilkinson et al., 1995); * at nucleotide 391, the autophosphorylated His (Gamble et al., 1998); * at nucleotide 718, Asp suggested to act as a receiver of the phosphate; boxed areas are conserved regions in bacterial His kinases and response regulators (Parkinson and Kofoid, 1992).

Figure 3

Phylogenetic analysis of the Arabidopsis and tomato ETR1 homologs aligned by the Clustal program. Tomato sequences are designated LeETR1, LeETR2 (Lashbrook et al., 1998), LeNR (Wilkinson et al., 1995), LeETR4, and LeETR5 (this paper). Arabidopsis sequences are designated AtETR1 (Chang et al., 1993), AtERS1 (Hua et al., 1995), AtEIN4 (Hua et al., 1998), AtETR2 (Sakai et al., 1998), and AtERS2 (Hua et al., 1998). Numbers represent the percentages of amino acids that differ between two proteins (Saitou and Nei, 1987).

Figure 4

Phenotype of etiolated Arabidopsis seedlings transformed with the mutated or wild-type tomato putative ethylene-receptor genes, LeETR4 or LeETR5, under the control of a constitutive promoter grown in the presence of ACC, as described in Methods. A, Wild-type Columbia; B, ethylene-insensitive mutant ein3-1; C, transgenic Arabidopsis expressing the mutated LeETR4 gene; D, transgenic Arabidopsis expressing the wild-type LeETR4 gene; E, transgenic Arabidopsis expressing the mutated LeETR5 gene; and F, transgenic Arabidopsis expressing the wild-type LeETR5 gene.

Figure 5

LeETR4 and LeETR5 mRNA expression in fruit, flowers, and vegetative tissues of cv Pearson tomato plants. Fruit at 30, 40, 50, and 58 d after anthesis are approximately immature, mature green, turning, and red ripe, respectively. RNase protection assays were performed using 5 and 10 μg of total RNA for LeETR4 and LeETR5, respectively.