Phytosulfokine contributes to suspension culture of Cunninghamia lanceolata through its impact on redox homeostasis

Abstract

Background: Suspension culture is widely used in the establishment of efficient plant regeneration systems, as well as in the mass production of plant secondary metabolites. However, the establishment of a suspension culture system of Cunninghamia lanceolata is genotype-dependent given that proembryogenic masses (PEMs) are prone to browning during this process in recalcitrant genotypes. Previously, we reported that the plant peptide hormone phytosulfokine (PSK) can tremendously decrease the hydrogen peroxide (H₂O₂) level and help to initiate somatic embryogenesis (SE) in recalcitrant C. lanceolata genotypes. However, to date, no studies have revealed whether or how PSK may contribute to the establishment of a suspension culture system in these recalcitrant genotypes.

Results: Here, we demonstrated that exogenous application of PSK effectively inhibited PEM browning during suspension culture in a recalcitrant genotype of C. lanceolata. Comparative time-series transcriptome profiling showed that redox homeostasis underwent drastic fluctuations when PEMs were cultured in liquid medium, while additional PSK treatment helped to maintain a relatively stable redox homeostasis. Interestingly, PSK seemed to have a dual effect on peroxidases (PRXs), with PSK simultaneously transcriptionally repressing ROS-producing PRXs and activating ROS-scavenging PRXs. Furthermore, determination of H₂O₂ and MDA content, as well as cell viability, showed that exogenous PSK treatment inhibited PEM browning and safeguarded PEM suspension culture by decreasing the H₂O₂ level and increasing PEM activity.

Conclusions: Collectively, these findings provide a valuable tool for the future establishment of large-scale C. lanceolata PEM suspension culture without genotype limitations.

Keywords: Bowning; C. lanceolate; PSK; ROS; Suspension culture.

Fig. 1

PSK inhibits PEM browning during suspension culture in the recalcitrant genotype of C. lanceolata. C. lanceolata PEMs of the recalcitrant genotype 4098–5 were cultured in liquid medium with or without exogenous PSK a The H₂O₂ accumulation and cell viability of PEM cultures were examined via DAB staining b and Evans blue staining c

Fig. 2

Transcriptional comparison of C. lanceolata PEM cultures between the PSK treatment and nontreatment control. Differentially expressed unigenes were hierarchically clustered using the expression values a K-means clustering analysis classified these differentially expressed unigenes into four clusters according to their expression profiles under control conditions b A paired t test was performed at each time point between the PSK treatment and nontreatment control

Fig. 3

Comparison of the functional enrichment of four clusters with distinct expression patterns. GO a and KEGG b functional enrichment analyses were performed and compared across four unigene clusters with distinct expression patterns

Fig. 4

Transcriptional comparisons of redox homeostasis-related enzymes. The expression heatmap for the main enzymes that participate in enzymatic browning a ROS production b and ROS scavenging c PPO, polyphenol oxidase; PLD, phospholipase D; LOX, lipoxygenase; SOD, superoxide dismutase; PAO, polyamine oxidase; RBOH, respiratory burst oxidase homologue; CAT, catalase; GPX, glutathione peroxidase; APX, ascorbate peroxidase; GR, glutathione reductase; MDAR, monodehydroascorbate reductase

Fig. 5

A potential dual effect of PSK on the regulation of ClPRX transcription. Heatmaps of absolute a and relative b gene expression of ClPRXs in C. lanceolata PEM cultures with or without PSK treatment. The phylogenetic tree was reconstructed based on the multialignment of ClPRX and AtPRX proteins using the ML method with a bootstrap value of 1,000 c Comparisons of gene expression across suspension cultures are shown for four selected unigenes

Fig. 6

PSK contributes to the establishment of a PEM suspension culture system in C. lanceolata. The H₂O₂ a and MDA b contents of PEMs were quantified during suspension culture under both conditions. A t test was performed to examine the difference between the PSK treatment and the nontreatment control at each time point. PEMs were subcultured on solid medium after suspension culture c PEMs on solid medium were stained using Evans blue stain and Trypan blue stain d