OCL 2021, 28, 42DOI: 10.1051/ocl/2021029© T. Berton et al., Published by EDP Sciences, 2021

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Data paper

Leaf metabolomic data of eight sunflower lines and their sixteen hybrids under water deficit☆

Données métabolomiques foliaires de huit lignées de tournesol et de leurs seize hybrides sous déficit hydrique

Thierry Berton¹,2a, Stéphane Bernillon¹,2a, Olivier Fernandez¹b, Harold Duruflé³c, Amélie Flandin¹,2, Cédric Cassan¹,2, Daniel Jacob¹,2, Nicolas B. Langlade³, Yves Gibon¹,2 and Annick Moing¹,2*

¹ INRAE, Univ. Bordeaux, Biologie du fruit et pathologie, UMR 1332, Centre INRAE de Nouvelle Aquitaine–Bordeaux, 33140 Villenave d’Ornon, France
² Bordeaux Metabolome, MetaboHUB **, PHENOME, IBVM, Centre INRAE de Nouvelle Aquitaine–Bordeaux, 33140 Villenave-d’Ornon, France
³ LIPME, Université de Toulouse, INRAE, CNRS, Castanet-Tolosan, France

* Correspondence: annick.moing@inrae.fr

Received: 20 May 2021 Accepted: 13 July 2021 Published online: 16 August 2021

Abstract

This article describes how metabolomic data were produced on sunflower plants subjected to water deficit. Twenty-four sunflower (Helianthus annuus L.) genotypes were selected to represent genetic diversity within cultivated sunflower and included both inbred lines and their hybrids. Drought stress was applied at the vegetative stage to plants cultivated in pots using the high-throughput phenotyping facility Heliaphen. Here, we provide untargeted and targeted metabolomic data of sunflower leaves. These compositional data differentiate both plant water status and different genotype groups. They constitute a valuable resource for the community to study the adaptation of crops to drought and the metabolic bases of heterosis.

Résumé

Cet article décrit comment les données métabolomiques ont été produites sur des plants de tournesol soumis à un déficit hydrique. Vingt-quatre génotypes de tournesol (Helianthus annuus L.) ont été sélectionnés pour représenter la diversité génétique du tournesol cultivé et comprennent à la fois des lignées consanguines et leurs hybrides. Une limitation hydrique a été appliquée au stade végétatif aux plantes cultivées en pots à l’aide de la plateforme de phénotypage à haut débit Heliaphen. Ici, nous mettons à disposition des données métabolomiques non ciblées et ciblées de feuilles de tournesol. Ces données de composition permettent de différencier l’état hydrique des plantes et différents groupes de génotypes. Elles constituent une ressource précieuse pour la communauté afin d’étudier l’adaptation des cultures à la sécheresse et les bases métaboliques de l’hétérosis.

Key words: Helianthus / abiotic stress / drought stress / LC-MS / metabolomic profiling

Mots clés : Helianthus / stress abiotique / stress hydrique / LC-MS / profils métabolomiques

☆

Contribution to the Topical Issue “Sunflower / Tournesol”.

a

Co-first authors.

b

Present address: SFR Condorcet CNRS 3417, Université de Reims Champagne-Ardenne, Unité résistance induite et bioprotection des plantes, EA4707, Reims, France.

c

Present address: INRAE, ONF, BioForA, UMR 0588, 45075 Orléans, France.

**

https://doi.org/10.15454/1.5572412770331912E12.

Highlights

Leaf metabolomic data were produced on sunflower plants of inbred lines and their hybrids subjected to water deficit at the vegetative stage.

They differentiate both plant water status and different genotype groups.

They constitute a valuable resource to be combined with other omics data and study the adaptation to drought and the bases of heterosis.

1 Specifications table

Subject area
Biology

More specific subject area
Metabolomic data

Type of data
LC-MS: LC-MS acquisition files, R command text file for spectra processing, LC-MS/MS acquisition files, Word file for LC-MS annotation table, tab file for calculated data table
Targeted analyses: tab file for calculated data table

How data was acquired
The Heliaphen robot and targeted robotized analyses of major compounds or LC-MS analyses of polar extracts

Data format
Targeted-analyses processed data: txt
LC-MS data, metadata, raw and processed data: tab, mzML, docx, tab, txt

Experimental factors
24 genotypes of Helianthus annuus in two environmental conditions (irrigated or not) with three replicates

Experimental features
Absolute contents of major compounds of sunflower leaf
Relative contents of LC-MS based metabolite signatures of sunflower leaf

Data source location
The outdoor Heliaphen phenotyping platform at INRAE station, Auzeville-Tolosane, France (43°31’41.8”N, 1°29’58.6”E)
Bordeaux Metabolome Facility, https://doi.org/10.15454/1.5572412770331912E12

Data accessibility
The LC-MS data are publicly available in Data INRAE repository (https://data.inrae.fr/dataverse/sunflodry, https://doi.org/10.15454/2KOXOH) under license etalab-2.0
The targeted analyses data are publicly available in Data INRAE repository (https://data.inrae.fr/dataverse/sunflodry, https://doi.org/10.15454/STJH47) under license etalab-2.0

Related research article
(Blanchet et al., 2018; Gody et al., 2020; Balliau et al., 2021)

2 Value of the data

Drought stress is a crucial issue for crop adaptation to climate change and sunflower is particularly impacted as it is mostly cultivated in marginal lands (Debaeke et al., 2017). In the present experiment, plants were subjected to two treatments (Well-Watered or Water-Deficit) during the vegetative stage. This experiment was performed in the outdoor high-throughput, semi-automated phenotyping facility Heliaphen (https://www6.inrae.fr/phenotoul_eng/WHO-we-are/PhenoToul/HeliaPhen).

Heterosis is an outstanding phenomenon involved in natural selection and used in crop breeding to adapt plants to environmental constraints. Twenty-four genotypes of cultivated sunflower consisting in four maintainer lines, four restorer lines and their 16 corresponding hybrids are included in this experiment which allows studying heterosis effect on metabolism.

This dataset provides metabolomic data of sunflower leaves of lines and hybrids under control and water deficit conditions.

These data consist in unique untargeted and targeted metabolomic profiles of sunflower responses to drought based on a large genetic variability.

3 Data

Climate change is affecting plant biodiversity, and crop choice and yields. A better knowledge of plant adaptation mechanisms to this recent phenomenon is, therefore, of major interest for crop science, agriculture and for feed and food security (Porter et al., 2019). Helianthus annuus L., the domesticated sunflower, is the fourth most important oilseed crop in the world (USDA, 2019). It seems promising for agriculture adaptation to global change because it can maintain stable yields across a range of environmental conditions, especially during stress induced by water limitation (Debaeke et al., 2017). It can be considered as an archetypical systems biology model with large drought stress response which involves many molecular pathways (Moschen et al., 2017) and subsequent metabolic and physiological processes.

In this data article, we are sharing the metabolomic data of 24 sunflower genotypes grown in two environmental conditions in an outdoor phenotyping facility. This dataset is part of a larger project that integrates other omics data (Blanchet et al., 2018; Gody et al., 2020; Balliau et al., 2021).

The LC-MS data and metadata associated with this article were deposited in the Data INRAE repository. The targeted analyses data were deposited in the Data INRAE repository.

4 Experimental design, plant material and growth conditions

The experiment was performed from May to July 2013 on the outdoor Heliaphen phenotyping facility at the Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE) station, Auzeville, France (43°31’41.8”N, 1°29’58.6”E) as previously described (Blanchet et al., 2018; Gosseau et al., 2019). Briefly, germinated plantlets were transplanted into individual pots filled with 15-l potting soil and covered with a 3-mm-thick polystyrene sheet to prevent soil water evaporation. Plants were fertilized with Peters Professional fertilizer (17-07-27, 500 mL, 0.6 g/L) and an oligo-element mixture solution (Hortilon, 0.46 g/L) at 17 days after germination (DAG), and treated with Polyaxe (5 mg/L applied on foliage) against thrips at 21 DAG.

In total, 144 plants, corresponding to 24 genotypes, four maintainer (SF009, SF092, SF109 and SF193) and four restorer (SF279, SF317, SF326 and SF342) lines and their corresponding hybrids obtained by crossing, were grown in two conditions: well-watered (WW) and water-deficit (WD) with three biological replicates (Blanchet et al., 2018; Gody et al., 2020). Before the beginning of the water deficit application at 35 DAG, pots were saturated with water and excessive water was drained. Pots were weighed to obtain the full soil water retention mass. At 38 DAG, irrigation was stopped (approximately 20-leaf stage) for WD plants as described previously (Gosseau et al., 2019). Plants were weighed by the Heliaphen robot to estimate transpiration (Gosseau et al., 2019). WW plants were re-watered at each weighing to reach soil water full retention capacity. Pairs of WD and WW plants were harvested when the fraction of transpirable soil water of the stressed plant reached 0.1 (occurring between 42 and 47 DAG). Two out of the three SF342 line plants died under the WW condition. The corresponding plant samples could not be harvested and data could not be obtained.

At harvest, leaves for metabolome analyses were cut without their petiole and immediately frozen in liquid nitrogen from 11 a.m. to 1 p.m. The harvested leaf was the leaf above the leaf that had reached its maximum size the most recently, as for the proteomic and transcriptomic studies (Blanchet et al., 2018; Gody et al., 2020; Balliau et al., 2021).

5 Metabolite analyses

5.1 Metabolite extraction

Leaf sample grinding was performed using a ZM200 grinder (Retsch, Haan, Germany) as described for transcriptome analysis (Gody et al., 2020). Fresh-frozen powdered samples were then lyophilized. Aliquots of about 10 ± 2 mg of dry powder were weighed in 1.1 mL MicronicTM tubes (Lelystad, The Netherlands) and extracted with a robotized Star/Starlet platform (Hamilton, Villebon-sur-Yvette, France) using ethanol/water (80:20, v/v) added with 0.1% formic acid as solvent at room temperature. Methyl vanillate was used as internal standard to check for the quality of injection for LC-MS. Two successive extractions consisting in 1 min vigorous shaking followed by 15 min ultra-sonication were performed with 300 μL of extraction solvent. The two supernatants were combined and filtered with 0.22 μm hydrophilic Durapore filtering microplates (Merck Millipore, Carrightwohill, Ireland). Nine blank extracts from the same procedure, but without sample powder, were also prepared. A QC sample was produced for LC-MS by pooling 10 μL of each sample extract.

5.2 Targeted analyses of major compounds

The targeted analyses of major compound in all samples were performed as done previously for the parents only (Fernandez et al., 2019) and as previously described (Biais et al., 2014) using enzymatic analyses and colorimetric assays performed using a robotic Star/Starlet platform (Hamilton, Villebon sur Yvette, France) and spectrophotometers. Glucose, fructose and sucrose were determined in the ethanolic supernatant obtained as described above (Stitt et al., 1989) and expressed in μmol per g dry weight (DW). Total free amino acids were determined in the supernatant with a fluorescamine-based assay (Bantan-Polak et al., 2001) and expressed as glutamate equivalents. Protein content was determined (Bradford, 1976) on the pellet re-suspended in 100 mM NaOH and heated at 95 °C for 20 min and expressed as mg bovine serum albumine equivalents per g DW. After neutralisation of the suspended pellet, starch was determined and expressed in glucose equivalents per g DW (Hendriks et al., 2003). Absorbencies were read at 340 or 595 nm using an MP96 microplate reader (SAFAS, Monaco). For fluorescence, 405 nm excitation and 485 nm emission were used with a Xenius multifunction microplate reader (SAFAS, Monaco). All chemicals and substrates for targeted analyses were purchased from Sigma-Aldrich Ltd. (Gillingham, United Kingdom). All enzymes were purchased from Roche Applied Science (Meylan, France).

5.3 LC-MS based metabolomic profiling

LC-MS-based metabolomic profiling of extracts was performed using the same extracts as for targeted analyses. The sample injection order was randomized. The QC sample was injected every 12 samples to correct for mass spectrometer signal drift. The extracts were analysed using LC-MS (Ultimate 3000 − LTQ-Orbitrap Elite, ThermoScientific, Bremen, Germany), using a C18 chromatographic column (C18-Gemini 2.0 × 150 mm, 3 μm, 110 Å, Phenomenex, Torrance, CA, USA), a 18 min acetonitrile gradient in acidified water (solvent A: ultrapure water + 0.1% formic acid, solvent B: LC-MS grade acetonitrile) with a 300 μL.min⁻¹ flow rate and the following elution gradient: 0-0.5 min, 3% B; 0.5-1 min, 3-10% B; 1-9 min, 10-50% B; 9-13 min, 50-100% B; 13-14 min, 100% B; 14-14.5 min 100-3% B; 14.5-18 min, 3% B. The column temperature was 30 °C. The injection volume was 5 μL. The LC-MS instrument was equipped with an HESI source operated in the positive-ion mode. Source parameters were the following: source voltage, 3.2 kV; sheath gas, 45 arbitrary units (a.u.); auxiliary gas, 15 a.u.; sweep gas, 0 a.u.; capillary temperature, 350 °C; heater temperature, 350 °C. Full Scan MS spectra were acquired at 240k resolution power with a 50-1000 mass range. Data dependent MS/MS spectra were acquired at 60k resolution power. The selected ions were fragmented in CID mode at a 35% normalized collision energy. The MS data were processed using R (R Core Team, 2018) with XCMS (Smith et al., 2006) and MetNormalizer (Shen et al., 2016) packages. Briefly, the corresponding MS-based variables were named using their nominal masses in Da and retention time in s (MxxxTyyy). Variables detected in blank extracts were filtered out. Variables with m/z values varying by more than 0.005 Da or with retention time varying by more than 20 s between different samples were also filtered out. Variables with intensity coefficients of variation in QC greater than 20% were also removed. This resulted in a data matrix of 4843 variables. Intensity drift was corrected using support vector regression. Finally, intensities were normalized according to the sample powder mass used for extraction. Annotation of intense ions (Fernandez et al., 2019; Stelzner et al., 2019) was performed using RT, accurate m/z and fragment ions from an MS/MS acquisition of an aliquot of the QC sample. This resulted in the annotation of 18 compounds belonging to eight compound families (Tab. 1). All chemicals for LC-MS analyses were purchased from Sigma Aldrich (Saint-Quentin Fallavier, France) and Extrasynthèse (Genay, France).

Finally, due to plant death or the lack of leaf material for several plants, 121 and 125 samples out of the 144 initial ones were analysed by the targeted (6 variables) and LC-MS based metabolomic (4843 variables) approaches, respectively. To get an overview of each data set, a principal component analysis (PCA) was performed using BioStatFlow web tool (Jacob et al., 2020) on data mean-centred and scaled to unit variance. The two treatments tended to separate along PC2 explaining about 28% of total variability for the targeted analyses (Fig. 1A) and about 9% for the LC-MS based data (Fig. 1B). The lines and hybrids tended to separate along PC1 explaining about 10% of total variability for the LC-MS based data (Fig. 1B). These metabolome data can be combined with other omic and phenotypic data of the same samples (Blanchet et al., 2018; Gody et al., 2020; Balliau et al., 2021) to get deeper insights into drought effects and heterosis.

Table 1

Annotation of LC-MS signatures of sunflower leaf ethanolic extracts with LC-MS and LC-MS/MS data in positive ionization mode.

thumbnail
Fig. 1

Principal component analysis of sunflower leaf metabolomic data obtained using targeted measurements of major compounds and untargeted LC-MS-Orbitrap analyses of ethanolic extracts. Leaves were harvested on parental lines (closed symbols) and their hybrids (open symbols) cultivated in Heliaphen phenotyping facility in well-watered or water-deficit conditions. A. Scores plot on the PC1 × PC2 plan for targeted measurements (6 variables). B. Scores plot on the PC1 × PC2 plan for LC-MS profiles (4843 variables). Green, well-watered; Orange, water-deficit.

Conflicts of interest

The authors declare that they have no conflicts of interest in relation to this article.

Acknowledgments

We thank the Heliaphen team (especially Nicolas Blanchet) for plant culture. These data were produced with the funding of the French National Research Agency (SUNRISE ANR-11-BTBR-0005, MetaboHUB ANR-11-INBS-0010, PHENOME ANR-11-INBS-0012). This work was part of the “Laboratoire d’Excellence (LABEX)” TULIP (ANR-10-LABX-41).

References

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Cite this article as: Berton T, Bernillon S, Fernandez O, Duruflé H, Flandin A, Cassan C, Jacob D, Langlade NB, Gibon Y, Moing A. 2021. Leaf metabolomic data of eight sunflower lines and their sixteen hybrids under water deficit. OCL 28: 42.

Supplementary material

DATA-TargetedAnalyses-SunflowerLeaf.txt: This file contains targeted measurements of major compounds for each genotype and their three biological replicates (in columns) for WW and WD conditions.

DATA-LCMS-SunflowerLeaf.txt: This file contains the intensities of LC-MS-Orbitrap metabolite signatures for each genotype and their three biological replicates (in columns) for WW and WD conditions.

(Refer to website)

All Tables

Table 1

Annotation of LC-MS signatures of sunflower leaf ethanolic extracts with LC-MS and LC-MS/MS data in positive ionization mode.

In the text

All Figures

thumbnail
Fig. 1

Principal component analysis of sunflower leaf metabolomic data obtained using targeted measurements of major compounds and untargeted LC-MS-Orbitrap analyses of ethanolic extracts. Leaves were harvested on parental lines (closed symbols) and their hybrids (open symbols) cultivated in Heliaphen phenotyping facility in well-watered or water-deficit conditions. A. Scores plot on the PC1 × PC2 plan for targeted measurements (6 variables). B. Scores plot on the PC1 × PC2 plan for LC-MS profiles (4843 variables). Green, well-watered; Orange, water-deficit.

In the text

Table 1

Annotation of LC-MS signatures of sunflower leaf ethanolic extracts with LC-MS and LC-MS/MS data in positive ionization mode.

Variable
Metabolite Rt a (min)
Metabolite [M + H]⁺ a
m/z
LC-MS/MS fragments
m/z a,b
Putative name
Metabolite ID
Class
Neutral molecular formula
Calculated molecular formula [M + H]⁺
m/z
MSI levelc

M284T195
3.26
284.0997
152.0568
Guanosine
CHEBI:16750
Nucleosides
C₁₀H₁₃N₅O₅
284.09894
2

M166T209
3.30
166.0864
120.0806
Phenylalanine
CHEBI:28044
Amino acids
C₉H₁₁NO₂
166.08626
2

M382T210
3.50
382.1730
220.1195; 202.1091; 136,0618
Pantothenic acid-hexose
–
Amino compounds
C₁₅H₂₇NO₁₀
382.17077
3

M285T209
3.50
285.0837
ND
Xanthosine
CHEBI:18107
Nucleosides
C₁₀H₁₂N₄O₆
285.08296
3

M356T275 d
4.60
355.1024
ND
5-O-Caffeoylquinic acid
CHEBI:16384
Cinnamic acids
C₁₆H₁₈O₉
355.10236
2

M342T281 d
4.68
341.0869
179.0340
Esculin
CHEBI:4853
Coumarins
C₁₅H₁₆O₉
341.08671
2

M356T315_1 d
5.25
355.1017
163.0392; 145.0286
3-O-Caffeoylquinic acid
CHEBI:16112
Cinnamic acids
C₁₆H₁₈O₉
355.10236
2

M356T341 d
5.68
355.1023
ND
4-O-Caffeoylquinic acid
CHEBI:75491
Cinnamic acids
C₁₆H₁₈O₉
355.10236
2

M340T362 d
6.04
339.1080
321.1128; 147.0442
Coumaroylquinic acid
CHEBI:1945
Cinnamic acids
C₁₆H₁₈O₈
339.10744
2

M611T367
6.10
611.1630
465.1028; 303.0502
Rutin
CHEBI:28527
Flavonoids
C₂₇H₃₀O₁₆
611.16066
2

M465T374
6.21
465.1034
303.0503
Quercetin hexoside
–
Flavonoids
C₂₁H₂₀O₁₂
465.10275
3

M370T374 d
6.22
369.1179
207.0995; 177.0550; 145.0286
Feruloylquinic acid
CHEBI:86388
Cinnamic acid
C₁₇H₂₀O₉
369.11800
2

M642T389 d
6.47
641.1736
495.1133; 333.0603
Pentahydroxy, methoxyflavone hexoside-deoxyhexoside
–
Flavonoids
C₂₈H₃₂O₁₇
641.17122
3

M518T468 d
7.74
517.1334
ND
3,4-Dicaffeoylquinic acid
CID: 5281780
Cinnamic acids
C₂₅H₂₄O₁₂
517.13405
3

M549T502_2
8.36
549.1256
ND
Trihydroxy,methoxyflavone malonylhexoside
–
Flavonoids
C₂₅H₂₄O₁₄
549.12388
3

M379T524
8.71
379.1747
ND
Niveusin C or Hydroxyleptocarpin
–
Sesquiterpenoids
C₂₀H₂₆O₇
379.17513
3

M274T582
9.68
273.0759
255.0655; 163.0392; 137.0234
Butein
CHEBI:3237
Flavonoids
C₁₅H₁₂O₅
273.07574
2

M409T617
10.28
409.1862
ND
3-O-Methylniveusin A
CID: 131752540
Sesquiterpenoids
C₂₁H₂₈O₈
409.18569
3

a

Rt and m/z measured with the raw data files. Only the [M + H]⁺ ion was considered as it was the most abundant ion in each acquired MS spectrum.

b

MS/MS fragmentation data from data dependent scan in CID mode at a 35% normalized collision energy value; ND: spectrum was not acquired.

c

Level of metabolite identification (Sumner et al., 2007).

d

This variable corresponds to M1 of the [M + H]⁺ ion.

Fig. 1

Principal component analysis of sunflower leaf metabolomic data obtained using targeted measurements of major compounds and untargeted LC-MS-Orbitrap analyses of ethanolic extracts. Leaves were harvested on parental lines (closed symbols) and their hybrids (open symbols) cultivated in Heliaphen phenotyping facility in well-watered or water-deficit conditions. A. Scores plot on the PC1 × PC2 plan for targeted measurements (6 variables). B. Scores plot on the PC1 × PC2 plan for LC-MS profiles (4843 variables). Green, well-watered; Orange, water-deficit.