Fig. 1. Alignment of NaHD20 amino acid sequences with HD-Zip I proteins from different plant species. Multiple alignment of the NaHD20 amino acid sequence.

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Fig. 1. Alignment of NaHD20 amino acid sequences with HD-Zip I proteins from different plant species. Multiple alignment of the NaHD20 amino acid sequence with its closest homologues from other plant species. ATHB7, A. thaliana homeobox 7 (At2g46680); ATHB12, A. thaliana homeobox 12 (At3g61890); SlHD, S. lycopersicum homeodomain (AK323400); RcHD, Ricinus communis homeodomain (XM_002529914); PtHD, Populus trichocarpa homeodomain (XM_002302623); HD, homeodomain; Zip, leucine-zipper domain. From: Nicotiana attenuata NaHD20 plays a role in leaf ABA accumulation during water stress, benzylacetone emission from flowers, and the timing of bolting and flower transitions J Exp Bot. 2010;62(1):155-166. doi:10.1093/jxb/erq252 J Exp Bot | © 2010 The Author(s).This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

Fig. 2. Analysis of NaHD20 transcript levels in N Fig. 2. Analysis of NaHD20 transcript levels in N. attenuata leaves and roots after different stress treatments. Total RNA was extracted from leaves or roots of rosette stage wild-type (WT) or transgenic N. attenuata plants after different times and treatments, reverse transcribed, and NaHD20 mRNA levels were quantified by qPCR. (A) Leaf tissue from WT plants treated with exogenous JA (100 μM), SA (300 μM), or solvent (SV). (B) Leaf tissue from wounded WT plants with the addition of either solvent (W+SV) or 18:3-Glu (W+FAC). (C) Leaf tissue from wounded WT, ir-lox3, ir-coi1, and ir-jar4/6 silenced plants. (D) Leaf tissue from WT plants subjected to water stress (by withholding soil irrigation) or control conditions for several days. (E) Roots of WT plants subjected to water stress (by air drying) or control conditions for up to 24 h. *Student t-test, P <0.05 [control versus treatment (A, D, E); wounding versus FAC elicitation (B); WT versus RNA interference-silenced plants (C)]; (n=3, bars denote ±SE). From: Nicotiana attenuata NaHD20 plays a role in leaf ABA accumulation during water stress, benzylacetone emission from flowers, and the timing of bolting and flower transitions J Exp Bot. 2010;62(1):155-166. doi:10.1093/jxb/erq252 J Exp Bot | © 2010 The Author(s).This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

Fig. 3. Tissue-specific expression of NaHD20 mRNA Fig. 3. Tissue-specific expression of NaHD20 mRNA. Total RNA was extracted from different tissues of rosette stage and mature N. attenuata wild-type (WT) plants, reverse transcribed, and used for quantification of NaHD20 mRNA levels by qPCR (n=3, bars denote ±SE). Different letters indicate significant differences (univariate ANOVA, F<sub>8,18</sub>=228.8, P <0.001, followed by a Scheffé post-hoc test, P <0.05). From: Nicotiana attenuata NaHD20 plays a role in leaf ABA accumulation during water stress, benzylacetone emission from flowers, and the timing of bolting and flower transitions J Exp Bot. 2010;62(1):155-166. doi:10.1093/jxb/erq252 J Exp Bot | © 2010 The Author(s).This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

Fig. 4. Analysis of ABA accumulation and gene expression during water stress conditions. (A) Rosette stage EV and NaHD20-silenced plants were water stressed for several days and leaves (one leaf per plant for each time point) were harvested at different times, extracted, and ABA levels quantified by LC-MS/MS. The arrow marks the day of re-watering (day 18 after the beginning of the experiment). *One-way ANOVA with Fisher's PLDS, P <0.05 (n=5). (B) Analysis of NaNCED1, NaOSM11, and NaLTP1 expression levels in EV and NaHD20-silenced plants during conditions of water deficit. Total RNA was extracted from leaves at different times, reverse transcribed, and used for quantification of NaNCED1, NaOSM1, and NaLTP1 mRNA levels by qPCR. *Student t-test, P <0.05 (EV versus NaHD20-silenced plants) (n=3, bars denote ±SE). From: Nicotiana attenuata NaHD20 plays a role in leaf ABA accumulation during water stress, benzylacetone emission from flowers, and the timing of bolting and flower transitions J Exp Bot. 2010;62(1):155-166. doi:10.1093/jxb/erq252 J Exp Bot | © 2010 The Author(s).This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

Fig. 5. Analysis of benzylacetone (BA) emitted by night flowers Fig. 5. Analysis of benzylacetone (BA) emitted by night flowers. Volatiles emitted by night flowers of EV and NaHD20-silenced plants were trapped from 14:00 h to 9:00 h and analysed by GC-MS. BA levels correspond to 10 biological replicates (n=10) for EV and NaHD20-silenced plants and they are expressed as the normalized peak area to the internal standard. *Mann–Whitney U-test; P <0.05; NaHD20-silenced plants versus EV plants. From: Nicotiana attenuata NaHD20 plays a role in leaf ABA accumulation during water stress, benzylacetone emission from flowers, and the timing of bolting and flower transitions J Exp Bot. 2010;62(1):155-166. doi:10.1093/jxb/erq252 J Exp Bot | © 2010 The Author(s).This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

Fig. 6. Analysis of reproductive phase change and flower transitions in NaHD20-silenced plants grown under control conditions. EV and NaHD20-silenced plants were grown under control conditions in ambient growth chambers. For all experiments, day 0 (D0) was defined as the time in which the first EV plants bolted and time therefore refers to days after D0. (A) Bolting time expressed as the percentage of bolted plants relative to D0 [two-sample Kolmogorov–Smirnov (K–S) test; P <10<sup>−4</sup>]. (B) Rate of stalk elongation. (C) Percentage of plants that had at least one opened bud (OB; two-sample K–S test; P <10<sup>−4</sup>). (D) Number of closed buds per plant (buds per plant). (E) Number of opened buds (OBs) per plant. (F) Number of opened corolla flowers (OCFs) per plant. *Mann–Whitney U-test; P <0.05; NaHD20-silenced plants versus EV plants. Each data point corresponds to data collected for 15 plants per plant type. (This figure is available in colour at JXB online.) From: Nicotiana attenuata NaHD20 plays a role in leaf ABA accumulation during water stress, benzylacetone emission from flowers, and the timing of bolting and flower transitions J Exp Bot. 2010;62(1):155-166. doi:10.1093/jxb/erq252 J Exp Bot | © 2010 The Author(s).This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

Fig. 7. Analysis of reproductive phase change and flower transitions in NaHD20-silenced plants during water stress and after recovery. Rosette stage EV and NaHD20-silenced plants were subjected to conditions of water deficit for 18 d and afterwards re-watered (black arrows: day 18). Day 0 (D0) was defined as the time in which the first EV plants bolted and time therefore refers to days after D0. The data corresponding to stalk length and number of buds for EV plants under control growth conditions (Fig. 6) are also presented (grey dotted lines) to help with the comparisons between treatments. (A) Rate of stalk elongation. (B) Number of closed buds per plant. (C) Number of opened buds (OBs) per plant. (D) Number of opened corolla flowers (OCFs) per plant. *Mann–Whitney U-test; P <0.05; NaHD20-silenced plants versus EV plants. Each data point corresponds to data collected for 15 plants per plant type. (This figure is available in colour at JXB online.) From: Nicotiana attenuata NaHD20 plays a role in leaf ABA accumulation during water stress, benzylacetone emission from flowers, and the timing of bolting and flower transitions J Exp Bot. 2010;62(1):155-166. doi:10.1093/jxb/erq252 J Exp Bot | © 2010 The Author(s).This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)