Transcriptional landscape of soybean (Glycine max) embryonic axes during germination in the presence of paclobutrazol, a gibberellin biosynthesis inhibitor

Abstract

Gibberellins (GA) are key positive regulators of seed germination. Although the GA effects on seed germination have been studied in a number of species, little is known about the transcriptional reprogramming modulated by GA during this phase in species other than Arabidopsis thaliana. Here we report the transcriptome analysis of soybean embryonic axes during germination in the presence of paclobutrazol (PBZ), a GA biosynthesis inhibitor. We found a number of differentially expressed cell wall metabolism genes, supporting their roles in cell expansion during germination. Several genes involved in the biosynthesis and signaling of other phytohormones were also modulated, indicating an intensive hormonal crosstalk at the embryonic axis. We have also found 26 photosynthesis genes that are up-regulated by PBZ at 24 hours after imbibition (HAI) and down-regulated at 36 HAI, which led us to suggest that this is part of a strategy to implement an autotrophic growth program in the absence of GA-driven mobilization of reserves. Finally, 30 transcription factors (mostly from the MYB, bHLH, and bZIP families) were down-regulated by PBZ and are likely downstream GA targets that will drive transcriptional changes during germination.

Figure 1

Gene expression profiling during seed germination. (A) Number of expressed genes (RPKM ≥1) and their estimated expression levels in each sample. (B) Number of DEGs at 12, 24 and 36 HAI. Numbers above the vertical bars stand for the ratio between down- and up-regulated genes. In the x-axes labels, C and P stand for control and PBZ, respectively.

Figure 2

Schematic model of hormonal crosstalk with gibberellin during G. max seed germination. The model was derived from a careful literature curation based on differentially expressed genes discussed along the manuscript. Positive interactions are indicated by arrows and T bars indicate repression. Abbreviations: Pyrabactin Resistance (PYR); PYR-like (PYL); Protein Phosphatase 2 C (PP2C); Nine-cis-epoxycarotenoid dioxygenase 3 (NCED3); Abscisic acid (ABA); Aminocyclopropane-1-carboxylic acid synthase (ACS); Paclobutrazol (PBZ); Gibberellin (GA); GIBBERELLIN INSENSITIVE DWARF 1 (GID1); GA 20-oxidase (GA20ox); GA 3-oxidase (GA3ox); Tryptophan aminotransferases 2 (TAR2); Auxin/Indole-3-Acetic Acid(AUX/IAA); Brassinosteroid (BR); BR 6-oxidase (BR6ox); BRI1 kinase inhibitor (BKI1); Type-A response regulator (A-RR); JASMONATE ZIM DOMAIN (JAZ); Jasmonic acid (JA).

Figure 3

Hormone biosynthesis pathways. Some GA deactivation and signaling genes discussed are also included. Up- and down-regulated genes are shown with up and down arrows. Black, red and blue arrows represent differential expression at 12, 24, and 36 HAI, respectively. Genes without arrows are expressed in at least one condition, although not classified as differentially expressed by our statistical thresholds. Genes are numbered as follows: 1) nine-cis-epoxycarotenoid dioxygenase 3 (NCED3); 2) SUR2; 3) tryptophan aminotransferase related 2 (TAR2); 4) DWARF4 (DWF4); 5) DWARF3 (DWF3); 6) brassinosteroid-6-oxidase 2 (BR6ox2); 7) 1-amino-cyclopropane-1-carboxylate synthase (ACS); 8) ent-copalyl diphosphate synthase (CPS); 9) ent-kaurene synthase (KS); 10) ent-kaurene oxidase (KO); 11) ent-kaurenoic acid oxidase (KAO); 12) GA 20-oxidase (GA20ox); 13) GA 2-oxidase (GA2ox); 14) GA 3-oxidase (GA3ox); 15) GIBBERELLIN INSENSITIVE DWARF1 (GID1) [Glyma.02G151100 (GID1b1), Glyma.10G022900 (GID1b2), Glyma.03G148300 (GID1b3), Glyma.10G158000 (GID1c1), and Glyma.20G230600 (GID1c2); 16) DELLA. Abbreviations: Abscisic Acid (ABA); indole-3-pyruvic acid (IPyA); Indol-3-acetaldoxime (IAOx); Indol-3-acetaldoxime N-oxide (IA Ox N-oxide); indole glucosinolates (IG); Indole-3-acetic acid (IAA); Brassinosteroid (BR); 1-aminocyclopropane-1-carboxylic acid (ACC); geranyl geranyl diphosphate (GGDP); ent-copalyl diphosphate (ent-CDP).

Figure 4

Hormone signal transduction. Rectangles with red lines represent gene families with at least one DEG. Up and down arrows represent PBZ up- and down-regulated genes. Numbers of DEGs are shown in circles adjacent to the red rectangles. Grey, light orange, and light blue arrows represent DEGs at 12, 24, and 36 HAI, respectively. Abbreviations: transport inhibitor response 1 (TIR1); Auxin/Indole-3-Acetic Acid (Aux/IAA); auxin-responsive Gretchen Hagen3 (GH3); small auxin upregulated RNA (SAUR); CYTOKININ RESPONSE 1 (CRE 1); Cytokinin (CK); His-containing phosphotransfer protein (AHP); Type-B response regulator (B-RR); Type-A response regulator (A-RR); Pyrabactin Resistance (PYR); PYR-like (PYL); Abscisic acid (ABA); Protein Phosphatase 2C (PP2C); Sucrose non-fermenting 1-related protein kinases subfamily 2 (SnRK2s); Abscisic acid responsive element-binding factor (ABF); Brassinosteroid-insensitive 1 (BRI1); BRI1-associated receptor kinase 1 (BAK1); Brassinosteroid (BR); BRI1 kinase inhibitor (BKI1); Brassinosteroid signaling kinases (BSK); BRI1-suppressor (BSU); brassinosteroid-insensitive 2 (BIN2); Brassinazole-resistant 1 (BZR1); BRI1-ethyl methanesulfonate-suppressor 1 (BES1); JASMONATE RESISTANT 1 (JAR1); Jasmonic acid (JA); Coronatine Insensitive 1 (COI1); JASMONATE ZIM DOMAIN (JAZ); JASMONATE INSENSITIVE 1 (JIN1).

Figure 5

Genes encoding differentially expressed cell-wall remodeling enzymes (A) and transcription factors (B). Up- and down-regulated genes are shown with up and down arrows. Black, red, and blue arrows represent DEGs at 12, 24, and 36 HAI, respectively. Abbreviations: alpha amylase-like (AMY); alpha-L-arabinofuranosidase (ARAF); arabinogalactan protein (AGP); beta glucosidase (BGLU); beta-xylosidase (BXL); expansin (EXP); FASCICLIN-like arabinogalactan-protein (FLA); glycine-rich protein (GRP); Glycosyl hydrolase family protein (GH); hydroxyproline-rich glycoprotein family protein (HRGP); laccase (LAC); Pectin lyase-like superfamily protein (PL); Peroxidase superfamily protein (PER); pectin methylesterase inhibitor superfamily protein (PMEI); proline-rich protein (PRP); wall associated kinase (WAK); xyloglucan endotransglucosylase/hydrolase (XTH); basic helix-loop-helix (bHLH); Basic Leucine Zipper (bZIP); C2H2 zinc finger (C2H2); Ethylene response factor (ERF); GRAS (gibberellin insensitive (GAI), Repressor of ga1-3 (RGA), SCARECROW-LIKE 3 (SCR) gene family; Homeodomain-leucine zipper (HD-ZIP); LESION SIMULATING DISEASE (LSD); Myelobastosis (MYB); Zinc finger Homeodomain (ZF-HD); No apical meristem (NAM), ATAF, and CUC (cup-shaped cotyledon) (NAC) family.