Physio-Biochemical Integrators and Transcriptome Analysis Reveal Nano-Elicitation Associated Response during Dendrocalamus asper (Schult. and Schult. F.) Backer ex K. Heyne Micropropagation

Abstract

Bamboos are perennial, arborescent, monocarpic and industrially important non-timber plants. They are important for various purposes, such as carbon sequestration, biodiversity support, construction, and food and fiber production. However, traditional vegetative propagation is insufficient for bamboo multiplication. Moreover, little is known about the mechanism of gold nanoparticles (AuNPs) in vitro proliferation and regulation of physiological and biochemical properties. In this study, we investigated the impacts of citrate and cetyltrimethylammonium bromide (CTAB) coated AuNPs on in vitro proliferation, photosynthetic pigment content and antioxidant potential of Dendrocalamus asper (Schult. and Schult. F.) Backer ex K. Heyne. Various morpho-physiological and biochemical parameters were differentially affected along the citrate- and CTAB-coated AuNPs concentration gradients (200-600 µM). In vitro shoot proliferation, photosynthetic pigment content and antioxidant activities were higher in D. asper grown on Murashige and Skoog medium supplemented with 2 mg·L^-1 benzyladenine and 400 µM citrate-coated AuNPs than in those grown on Murashige and Skoog medium supplemented with 600 µM CTAB- coated AuNPs. Identification of genes regulating in vitro D. asper proliferation will help understand the molecular regulation of AuNPs-mediated elicitation for modulating various physiological and biochemical activities during micropropagation. Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway analyses identified differentially expressed genes associated with in vitro modulation of AuNPs-regulated biological processes and molecular functions. The findings of this study provide new insight into AuNPs-mediated elicitation of in vitro mass scale bamboo propagation.

Keywords: RNA sequencing; bamboo; gold nanoparticles; tissue culture; transcription factors; transcriptome.

Figure 1

In vitro shoot proliferation and total biomass production in proliferated D. asper under control (MS + 2 mg·L⁻¹ BAP) and AuNP treated plants after 30 days. Biomass proliferation in (A) control; (B) 400 µM citrate-AuNP treated; and (C) 600 µM CTAB-AuNP treated. Scale bar = 1 cm.

Figure 2

Effect of citrate and CTAB-AuNPs on growth and biomass enhancement of D. asper. (A) effect on number of stems; (B) effect on number of leaves; (C) effect on leaf length; (D) effect on plant height; (E) effect on fresh weight; and (F) effect on dry weight. The error bar represents standard error of three biological replicates. * Indicates significantly reduced and enhanced parameters in comparison to the control.

Figure 3

Effect of citrate and CTAB-AuNPs on photosynthetic pigments, sugar and starch content of D. asper. Effect on chlorophyll a (A), chlorophyll b (B) and total chlorophyll (C) content, respectively; (D) effect on total starch content; (E) effect on total soluble sugar content. The error bar represents the standard error of three biological replicates. * Indicates significantly reduced and enhanced concentrations in comparison to the control.

Figure 4

Effect of citrate and CTAB-AuNPs on antioxidant activity and lipid peroxidation of D. asper. (A) effect of total phenolic content; (B) effect of malondialdehyde content; and (C) effect on superoxide dismutase enzyme activity. The error bar represents standard error of three biological replicates. * Indicates significantly reduced and enhanced concentrations in comparison to the control.

Figure 5

Differentially expressed genes in different treatment conditions in D. asper. * q-value is a corrected p-value # Upregulated Genes-↑, Downregulated Genes-↓. BAC represents control, BAT1 represents 400 µM citrate AuNPs and BAT2 represents 600 µM CTAB AuNPs.

Figure 6

Venn diagram showing number of differentially expressed genes in different treatment conditions (400 µM citrate vs. Control, 600 µM CTAB vs. Control, 400 µM citrate vs. 600 µM CTAB) (A) Upregulated, (B) Downregulated genes. BAC represents control, BAT1 represents 400 µM citrate AuNPs and BAT2 represents 600 µM CTAB AuNPs. (C) Volcano plot showing the significant differentially expressed genes.

Figure 7

Heat maps showing differentially upregulated genes between control and AuNPs treated plants of D. asper. Heat map prepared at a log2fold scale of −4 to +4. BAC represents control; BAT1 represents 400 µM citrate AuNPs; and BAT2 represents 600 µM CTAB AuNPs.

Figure 8

Differentially downregulated genes between control and AuNPs treated plants in D. asper. Heat map prepared at a log2fold scale of −4 to +4. BAC represents control; BAT1 represents 400 µM citrate AuNPs; and BAT2 represents 600 µM CTAB AuNPs.

Figure 9

Differentially expressed transcription factors among control, citrate and CTAB coated gold nanoparticles.

Figure 10

GO enrichment analysis for upregulated (A) and downregulated (B) genes. Biological processes, cellular components, molecular functions and pathway enrichment analysis in BAT1-BAC (i), BAT2-BAC (ii) and BAT1-BAT2 (iii).

Figure 11

KEGG pathway analysis. KEGG enriched plant hormone signal transduction, transport, plant pathogen interaction and photosynthesis in BAC vs. BAT1, BAC vs. BAT2 and BAT2 vs. BAT1.