Comparative physiological, biochemical, metabolomic, and transcriptomic analyses reveal the formation mechanism of heartwood for Acacia melanoxylon

Abstract

Acacia melanoxylon is well known as a valuable commercial tree species owing to its high-quality heartwood (HW) products. However, the metabolism and regulatory mechanism of heartwood during wood development remain largely unclear. In this study, both microscopic observation and content determination proved that total amount of starches decreased and phenolics and flavonoids increased gradually from sapwood (SW) to HW. We also obtained the metabolite profiles of 10 metabolites related to phenolics and flavonoids during HW formation by metabolomics. Additionally, we collected a comprehensive overview of genes associated with the biosynthesis of sugars, terpenoids, phenolics, and flavonoids using RNA-seq. A total of ninety-one genes related to HW formation were identified. The transcripts related to plant hormones, programmed cell death (PCD), and dehydration were increased in transition zone (TZ) than in SW. The results of RT-PCR showed that the relative expression level of genes and transcription factors was also high in the TZ, regardless of the horizontal or vertical direction of the trunk. Therefore, the HW formation took place in the TZ for A. melanoxylon from molecular level, and potentially connected to plant hormones, PCD, and cell dehydration. Besides, the increased expression of sugar and terpenoid biosynthesis-related genes in TZ further confirmed the close connection between terpenoid biosynthesis and carbohydrate metabolites of A. melanoxylon. Furthermore, the integrated analysis of metabolism data and RNA-seq data showed the key transcription factors (TFs) regulating flavonoids and phenolics accumulation in HW, including negative correlation TFs (WRKY, MYB) and positive correlation TFs (AP2, bZIP, CBF, PB1, and TCP). And, the genes and metabolites from phenylpropanoid and flavonoid metabolism and biosynthesis were up-regulated and largely accumulated in TZ and HW, respectively. The findings of this research provide a basis for comprehending the buildup of metabolites and the molecular regulatory processes of HW formation in A. melanoxylon.

Keywords: Acacia melanoxylon; Flavonoids; Heartwood formation; Metabolomic; Phenolics; Terpenoids; Transcriptomic.

Fig. 1

Effect of HW formation on non-structural carbohydrates in A. melanoxylon. (a, b, c) As a control group without staining, (d, e, f) PAS staining showed phenols, (g, h, i) I₂-KI staining showed the remaining starches. Meanwhile, Figures a, d and e are SW. Figures b, e and h are transition areas. In addition, Figures c, f and i are HW. Note: Data are presented in the mean ± SE. Different capital letters indicate that the treatment effect is significantly different at the p < 0.05 level. Scale bars = 10 μm

Fig. 2

The content comparison of total flavonoids, phenolics, starches, sugars, and terpenoids in SW, TZ, and HW of A. melanoxylon. Note: Data are presented in the mean ± SE. Different capital letters indicate that the treatment effect is significantly different at the p < 0.05 level

Fig. 3

Enzymatic activity of SW, TZ and HW of A. melanoxylon. Note, POD: Peroxidase; PPO: Polyphenol Oxidase; ACO: 1-aminocyclopropane-1-carboxylate oxidase; HCT: hydroxycinnamoyl shikimate transferase; F3’H: flavanone 3’-hydroxylase; CAD: cinnamyl-alcohol dehydrogenase; PAL: Phe ammonia lyase; CCR: cinnamoyl-CoA reductase; AMY: alpha-amylase; SuSy: sucrose synthase

Fig. 4

Preliminary analysis of metabonomic data. (a–b) Principal component analysis of metabolites for SW, TZ, and HW (c) Correlation analysis for differential metabolites among SW, TZ, and HW. (d) Enrichment analysis of differential metabolites among SW, TZ, and HW (e) Z-score (standard score) analysis of 34 DEMs (f) Histogram analysis of KEGG metabolic pathway (g) Network diagram analysis of pathway correlation of differential metabolites

Fig. 5

Transcriptomic analysis of A. melanoxylon. (a) Volcano plots of DEGs of A. melanoxylon. (b) GO enrichment analysis of DEGs. (c-d) The KEGG classification and enrichment analysis of DEGs (e) Cluster thermogram of differentially expressed transcription factors (f) Correlation network diagram between differentially expressed transcription factors and differentially enriched phenolics and flavonoids

Fig. 6

Analysis of differentially enriched metabolites in four pathways related to flavonoids and phenylpropanoids Note, POD: Peroxidase; PPO: Polyphenol Oxidase; ACO: 1-aminocyclopropane-1-carboxylate oxidas; HCT: hydroxycinnamoyl shikimate transferase; F3’H: flavanone 3’-hydroxylase; CAD: cinnamyl-alcohol dehydrogenase; PAL: Phe ammonia lyase; CCR: cinnamoyl-CoA reductase; AMY: alpha-amylase; SuSy: sucrose synthase

Fig. 7

Schematic diagram of different pathways activated in A. melanoxylon. Note, SuSy: sucrose synthase; UGP: UTP–glucose-1-phosphate uridylyltransferase; APS: ADP-glucose; SS: starch synthase; SBE: starch branching enzyme/1,4-alpha-glucan branching enzyme; DBE: debranching enzyme 1/isoamylase; AMY: alpha-amylase; ADH: alcohol dehydrogenase; UGE: UDP-glucose 4-epimerase; UGD: UDP-glucose dehydrogenase; UXS: UDP-XYL synthase; CSLD: cellulose synthase-like D5; XYL: xylan 1,4-beta-xylosidase/Glycosyl hydrolase; XK: xylulose kinase-2/ xylulokinase; XI: xylose isomerase, RPE: ribulose-phosphate 3-epimerase; RSW: ribose 5-phosphate isomerase A; TSL: transketolase; PGM: Phosphoglucomutase/phosphomannomutase; PGI: phosphoglucose isomerase 1/ glucose-6-phosphate isomerase; AACT: acetyl-CoA C-acetyltransferase; HMGS: hydroxymethylglutaryl-CoA synthase, HMGR: hydroxymethylglutaryl-CoA reductase; MK: mevalonate kinase; PMK: phosphomevalonate kinase; MVD: diphosphomevalonate decarboxylase; GGDS: geranylgeranyl diphosphate synthase, type III; FPS: farnesyl-diphosphate; SME: squalene monooxygenase; GES: geranylgeranyl diphosphate synthase, type III; TTS: trimethyltridecatetraene/dimethylnonatriene synthase; ATS: (-)-alpha-terpineol synthase

Fig. 8

Expression profile of TFs and genes in different tree height/tree ages of A. melanoxylon. The expression level of other transcripts is based on the ribosomal protein L4E (RPL4) transcripts