A sunflower WRKY transcription factor stimulates the mobilization of seed-stored reserves during germination and post-germination growth

Abstract

Key message

The sunflower transcription factor HaWRKY10 stimulates reserves mobilization in Arabidopsis. Gene expression and enzymes activity assays indicated that lipolysis and gluconeogenesis were increased. Microarray results suggested a parallelism in sunflower.

Abstract

Germinating oilseeds converts stored lipids into sugars, and thereafter in metabolic energy that is used in seedling growth and establishment. During germination, the induced lipolysis linked to the glyoxylate pathway and gluconeogenesis produces sucrose, which is then transported to the embryo and driven through catabolic routes. Herein, we report that the sunflower transcription factor HaWRKY10 regulates carbon partitioning by reducing carbohydrate catabolism and increasing lipolysis and gluconeogenesis. HaWRKY10 was regulated by abscisic acid and gibberellins in the embryo leaves 48 h after seed imbibition and highly expressed during sunflower seed germination and seedling growth, concomitantly with lipid mobilization. Sunflower leaf disks overexpressing HaWRKY10 showed repressed expression of genes related to sucrose cleavage and glycolysis compared with controls. Moreover, HaWRKY10 constitutive expression in Arabidopsis seeds produced higher decrease in lipid reserves, whereas starch and sucrose were more preserved compared with wild type. Gene transcripts abundance and enzyme activities involved in stored lipid mobilization and gluconeogenesis increased more in transgenic than in wild type seeds 36 h after imbibition, whereas the negative regulator of lipid mobilization, ABI4, was repressed. Altogether, the results point out a functional parallelism between tissues and plant species, and reveal HaWRKY10 as a positive regulator of storage reserve mobilization in sunflower.

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Acknowledgments

This work was supported by Agencia Nacional de Promoción Científica y Tecnológica [ANPCyT, PICT-PAE-37100, PICT-2011-850, PICT-2012-2439], Consejo Nacional de Investigaciones Científicas y Técnicas [CONICET, PIP 2011 11420100100278] and Universidad Nacional del Litoral [UNL, CAID 2011 50120110100349 and 50120110100399]. JR was a Fellow and MDH is a Fellow of CONICET and members of UNL; RLC, AAI and KFR are career members of CONICET and UNL.

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Correspondence to Karina F. Ribichich.

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Communicated by R. J. Rose.

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Supplementary material 1 (DOCX 13 kb)

Supplementary material 2 (XLSX 2796 kb)

299_2016_2002_MOESM3_ESM.eps

Supplementary Figure S1. Combined GO annotation graph. The sequences differentially regulated in HaWRKY10 OX leaf disks were functional annotated by GO terms and the complete annotation represented at the same time. Left column and transverse bars indicate GO level. GO term orange coloring intensity is proportional to the node score and, then, to the annotation density (EPS 3465 kb)

299_2016_2002_MOESM4_ESM.eps

Supplementary Figure S2. Mapman overview of selected differential expressed genes in HaWRKY10 OX. Data were extracted from the microarray experiment and validated by RT-qPCR. Red and green boxes represent upregulated and downregulated genes, respectively. Plots represent genes validated by RT-qPCR. The expression differences were confirmed in seven out of nine selected genes. Different letters mean significant differences with P < 0.05. Normalization was made with endogenous ACTIN (ACTIN2 and ACTIN8) and, thereafter, with the control sample (without HaWRKY10), arbitrarily assigned a value of one. W10 and 121 indicate sunflower tissue samples with or without overexpressed HaWRKY10, respectively (EPS 290 kb)

299_2016_2002_MOESM5_ESM.eps

Supplementary Figure S3. HaWRKY10 expression in sunflower plants. HaWRKY10 expression was measured in (a) organs of 7- and 14-day-old plants and (b) embryo leaves or cotyledons of 7 day-old plants under hormonal treatments. Normalization was made with endogenous ACTIN (ACTIN2 and ACTIN8) and, thereafter, with the R or the A samples (a, 7- and 14-day-old-plants, respectively) or with the control sample (b), arbitrarily assigned a value of one. R = root, H = hypocotyl, C = cotyledons, A = shoot apex, L = leaves; CTL = control, ABA = abscisic acid, ACC = 1-aminocyclopropane-1-carboxylic acid, GA3: gibberellic acid 3, IAA = indole-3-acetic acid, JA = methyl-jasmonic acid, SA = salicylic acid (EPS 1035 kb)

299_2016_2002_MOESM6_ESM.eps

Supplementary Figure S4. Expression levels of HaWRKY10 in several independent homozygous transgenic lines of Arabidopsis. Transcript levels of HaWRKY10 were quantified by RT-qPCR, normalized with endogenous ACTIN (ACTIN2 and ACTIN8) and, thereafter, with respect to the lower expression line arbitrarily assigned a value of one. Asterisks indicate the three lines chosen to continue the study, named W10-A, W10-B, and W10-C, respectively. Four biological replicates were done for each experiment of a total of two (EPS 686 kb)

299_2016_2002_MOESM7_ESM.eps

Supplementary Figure S5. Fatty acids profile (a) and oleosins (b) in HaWRKY10 Arabidopsis transgenic seeds versus WT. SAT: saturated, UNSAT: unsaturated with 1, 2, or 3 double bonds between the carbon atoms as indicated. OLEO: oleosin 1, 2, or 4 as indicated. Lane named MM corresponds to molecular mass markers, with sizes specified in kDa. W10-A, W10-B, and W10-C: three independent transgenic 35S:HaWRKY10 lines (EPS 289 kb)

299_2016_2002_MOESM8_ESM.eps

Supplementary Figure S6. HaWRKY10 induces the expression of a reporter gene through the binding to a promoter with cis-acting W box elements. Nicotiana benthamiana leaf disks were infiltrated with a A. tumefaciens expressing:GUS (negative control), pOs:GUS, 35S:GUS (positive control), pOs:GUS plus 35S:TF1, pOs:GUS plus 35S:TF2, pOs:GUS plus 35S:HaWRKY10. GUS: betaglucuronidase cDNA, pOs: promoter of the Os03g16950 rice gene with five W box elements, 35S: constitutive CaMV promoter, TF1: a non-W box recognizing TF cDNA, TF2: a W box recognizing TF cDNA (EPS 496 kb)

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Raineri, J., Hartman, M.D., Chan, R.L. et al. A sunflower WRKY transcription factor stimulates the mobilization of seed-stored reserves during germination and post-germination growth. Plant Cell Rep 35, 1875–1890 (2016). https://doi.org/10.1007/s00299-016-2002-2

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Keywords

  • Sunflower
  • Reserves mobilization
  • Germination
  • Arabidopsis
  • Transcription factor
  • WRKY