Volume 8, Issue 1, Pages 153-162 (January 2015) Divergence in the Enzymatic Activities of a Tomato and Solanum pennellii Alcohol Acyltransferase Impacts Fruit Volatile Ester Composition  Charles Goulet, Yusuke Kamiyoshihara, Nghi B. Lam, Théo Richard, Mark G. Taylor, Denise M. Tieman, Harry J. Klee  Molecular Plant  Volume 8, Issue 1, Pages 153-162 (January 2015) DOI: 10.1016/j.molp.2014.11.007 Copyright © 2015 The Author Terms and Conditions

Figure 1 Phylogenetic Analysis of the Protein Sequences of the Five Tomato AATs and Representative AATs from Additional Species. The trees were inferred by using the maximum likelihood method. The percentage of trees in which the associated taxa are clustered together is shown next to the branches. The trees are drawn to scale, with branch lengths measured in the number of substitutions per site. GenBank accession numbers are as follows: AcAT16 (HO772640), AeAT9 (HO772637), BanAAT (CAC09063), BPBT (AAU06226), CbBEAT (AF043464), CbBEBT (AAN09796), CmAAT1 (CAA94432), CmAAT2 (AAL77060), CmAAT3 (AAW51125), CmAAT4 (AAW51126), FaAAT2 (JN089766), MdAAT2 (AAS79797), MpAAT1 (AY707098), RhAAT1 (AAW31948), SAAT (AAG13130), VAAT (AX025504), and VpAAT1 (FJ548611). Molecular Plant 2015 8, 153-162DOI: (10.1016/j.molp.2014.11.007) Copyright © 2015 The Author Terms and Conditions

Figure 2 Alcohol Acyltransferases Expression in S. lycopersicum cv. M82 Fruit. (A) Transcript abundance of the five SlAAT in ripe fruit (±SE, n = 4). (B) Transcript levels of SlAAT1 in four stages of fruit development (immature green, breaker, turning, ripe) (±SE, n = 4). Molecular Plant 2015 8, 153-162DOI: (10.1016/j.molp.2014.11.007) Copyright © 2015 The Author Terms and Conditions

Figure 3 Silencing SlAAT1 in Tomato Decreases the Emission of Esters. (A) Transcript levels of SlAAT1 in ripe fruits of three silenced transgenic lines relative to their control, Flora-Dade (±SE, n = 6). (B) Emission rate of ester volatiles from the cut tomato fruits of transgenic plants and their control (±SE, n = 6) (**P < 0.01). Molecular Plant 2015 8, 153-162DOI: (10.1016/j.molp.2014.11.007) Copyright © 2015 The Author Terms and Conditions

Figure 4 Emission Rate of Acetate Ester Volatiles from the Cut Tomato Fruits of M82 (Control) and the Introgression Line 8-1-5 (±SE, n = 6). All esters detected are significantly different between M82 and IL 8-1-5 (P < 0.01). Molecular Plant 2015 8, 153-162DOI: (10.1016/j.molp.2014.11.007) Copyright © 2015 The Author Terms and Conditions

Figure 5 Specific Activity of SlAAT1 and SpAAT1 for Different Alcohols Found in Tomato (±SE, n = 3). (A) The transferase activity was measured with 5 mM of each alcohol and 0.01 mM of acetyl-CoA. The differences in activity between the S. lycopersicum and the S. pennellii enzyme are significant for all the substrates (P < 0.01) except sec-butanol. (B) Molecular structure of the alcohols used in the assay with the corresponding numbers used in (A). Molecular Plant 2015 8, 153-162DOI: (10.1016/j.molp.2014.11.007) Copyright © 2015 The Author Terms and Conditions

Figure 6 Specific Activity of SlAAT1 and SpAAT1 for Different Acyl-CoAs (±SE, n = 3). The assay was performed with 20 mM of 2-methyl-1-butanol and 0.01 mM of each acyl-CoA (C2, C3, C4, and C6). The differences in activity between the S. lycopersicum and the S. pennellii enzyme are significant for all the substrates (P < 0.01) except acetyl-CoA. Molecular Plant 2015 8, 153-162DOI: (10.1016/j.molp.2014.11.007) Copyright © 2015 The Author Terms and Conditions

Figure 7 Balance between Ester Synthesis and Degradation in the Fruits of Tomato and S. pennellii. S. pennellii accumulates a large amount of ester volatiles, while the content remains low in tomato.SpAAT1 is more efficient overall in ester synthesis and prefers different alcohols than SlAAT1. SlCXE1 is highly expressed in tomato resulting in a rapid conversion back to the alcohol pool. The low expression rate of SpCXE1, illustrated by a dashed line, prevents significant degradation of the esters in S. pennellii. Molecular Plant 2015 8, 153-162DOI: (10.1016/j.molp.2014.11.007) Copyright © 2015 The Author Terms and Conditions