The cold responsive mechanism of the paper mulberry: decreased photosynthesis capacity and increased starch accumulation

Abstract

Background: Most studies on the paper mulberry are mainly focused on the medicated and pharmacology, fiber quality, leaves feed development, little is known about its mechanism of adaptability to abiotic stress. Physiological measurement, transcriptomics and proteomic analysis were employed to understand its response to cold stress in this study.

Methods: The second to fourth fully expanded leaves from up to down were harvested at different stress time points forthe transmission electron microscope (TEM) observation. Physiological characteristics measurement included the relative electrolyte leakage (REL), SOD activity assay, soluble sugar content, and Chlorophyll fluorescence parameter measurement. For screening of differentially expressed genes, the expression level of every transcript in each sample was calculated by quantifying the number of Illumina reads. To identify the differentially expressed protein, leaves of plants under 0, 6, 12, 24, 48 and 72 h cold stress wereharvested for proteomic analysis. Finally, real time PCR was used to verify the DEG results of the RNA-seq and the proteomics data.

Results: Results showed that at the beginning of cold stress, respiratory metabolism was decreased and the transportation and hydrolysis of photosynthetic products was inhibited, leading to an accumulation of starch in the chloroplasts. Total of 5800 unigenes and 38 proteins were affected, including the repressed expression of photosynthesis and the enhanced expression in signal transduction, stress defense pathway as well as secondary metabolism. Although the transcriptional level of a large number of genes has been restored after 12 h, sustained cold stress brought more serious injury to the leaf cells, including the sharp rise of the relative electrolyte leakage, the declined Fv/Fm value, swelled chloroplast and the disintegrated membrane system.

Conclusion: The starch accumulation and the photoinhibition might be the main adaptive mechanism of the paper mulberry responded to cold stress. Most of important, enhancing the transport and hydrolysis of photosynthetic products could be the potential targets for improving the cold tolerance of the paper mulberry.

Fig. 1

Electron micrographs of paper mulberry leaf cells from control and treated plants. a Cells from control plant (0 h); b 3 h at 4 °C, showing early stage, chloroplasts with starch grains were noted; c 6 h at 4 °C, starch grains were present in many; d 12 h at 4 °C, showing invagination of the plasma membrane (arrow) accompanied with amounts of membrane vesicles (arrowhead), note the deformation of chloroplast; e 24 h at 4 °C, chloroplast envelope membrane swell (arrow); f 48 h at 4 °C, invagination of the plasma membrane and protrusion of vesicles into the vacuoles were noted (arrowhead), swelling continued until plastids were round (arrow). Abbreviations in the figures: V: vacuole, Chl: chloroplast, S: starch grain. Bar = 2 μm

Fig. 2

Changes of four physiological traits in leaves of paper mulberry under cold stress. a REL. b Maximum photochemical efficiency Fv/Fm. c Soluble sugar content. d SOD activity. Values are presented as mean ± SD of three biological replicates. Duncan’s test (p < 0.05) was used for data statistics at different time points and letters a, b, c, d, and e indicated statistical significance

Fig. 3

Differentially expressed unigenes in each sample. a Venn diagram of differentially expressed unigenes distributed in every sample. The time was represented the sample that were treated under cold stress. The expression pattern and the statistics of the differentially expressed genes in every pairwise comparison. b The up-regulated and down-regulated DEGs in every pairwise comparison. Taken pairwise comparison of 2 h vs 0 h as an example, up-regulated was referred to the expression was higher than that after 2 h of cold stress treatment while down-regulated referred to the expression was lower than that after 2 h of cold stress treatment. c The expression profile of all the 5800 DEGs during the cold stress. According to their expression pattern, all of them could be divided into 8 groups by using the Mev 4.0 software

Fig. 4

Annotation and functional classification of the DEGs. a Histogram of Gene Ontology (GO) classifications of the DEGs in paper mulberry under cold stress. Total 5043 DEGs could be annotated by GO and they mainly distributed in metabolic process, response to stimulus, catalytic, binding and organelle belonged to Biological process, Cellular components and Molecular function, respectively. b The number of DEGs in each COG functional classification group. A total of 1,851 DEGs had been annotated by COG. Except the function prediction only and function unkown, group O (Posttranslational modification, protein turnover, and chaperones), J (Translation, ribosomal structure and biogenesis), G (carbohydrate metabolism), C (energy production), E (amino metabolism), P (inorganic ion transport and metabolism), I (lipid metabolism) and Q (secondary metabolism) took up more than half of the total DEGs annotated in COG. c The statistics analysis of DEGs mapped to KEGG pathways

Fig. 5

The number of DEGs related to phytohormones signaling pathway and transcriptional factor. a Statistics of DEGs involved in the phytohormones signaling pathway. b No. of differentially expressed TFs in each family

Fig. 6

The supposed responsive mechanism of the paper mulberry exposed to cold stress. After signal reception, stress-activated Ca²⁺ signaling, ROS and hormone signaling modulate the expression of stress-responsive transcription factors, kinases, GST and so on. And then activated the expression of genes related to primary and secondary metabolism. The physiological changes mainly included chloroplast malfunction, starch accumulation, cell wall and membrane reconfiguration. Taken together, they endowed the paper mulberry with the new growth homeostasis under cold stress

Fig. 7

The exhibition of the Physiological process under normal condition and cold stress. Under normal condition, there was a dynamic equilibrium between starch syntheses and transformation into triose phosphate from the photosynthesis product. The sustained cold stress led to the photoinhibition, which affected the synthesis of the sucrose and these conversely led to the accumulation of the starch in the chloroplast. The dashed was used to illustrate the slower physical process and the inverted T-shaped line showed the repressed physical process. Double dotted line showed that the dynamic equilibrium of the triose phosphate transportation and the sucrose synthesis had been broken