Transcriptome profiling of low temperature-treated cassava apical shoots showed dynamic responses of tropical plant to cold stress

Abstract

Background: Cassava is an important tropical root crop adapted to a wide range of environmental stimuli such as drought and acid soils. Nevertheless, it is an extremely cold-sensitive tropical species. Thus far, there is limited information about gene regulation and signalling pathways related to the cold stress response in cassava. The development of microarray technology has accelerated the study of global transcription profiling under certain conditions.

Results: A 60-mer oligonucleotide microarray representing 20,840 genes was used to perform transcriptome profiling in apical shoots of cassava subjected to cold at 7°C for 0, 4 and 9 h. A total of 508 transcripts were identified as early cold-responsive genes in which 319 sequences had functional descriptions when aligned with Arabidopsis proteins. Gene ontology annotation analysis identified many cold-relevant categories, including 'Response to abiotic and biotic stimulus', 'Response to stress', 'Transcription factor activity', and 'Chloroplast'. Various stress-associated genes with a wide range of biological functions were found, such as signal transduction components (e.g., MAP kinase 4), transcription factors (TFs, e.g., RAP2.11), and reactive oxygen species (ROS) scavenging enzymes (e.g., catalase 2), as well as photosynthesis-related genes (e.g., PsaL). Seventeen major TF families including many well-studied members (e.g., AP2-EREBP) were also involved in the early response to cold stress. Meanwhile, KEGG pathway analysis uncovered many important pathways, such as 'Plant hormone signal transduction' and 'Starch and sucrose metabolism'. Furthermore, the expression changes of 32 genes under cold and other abiotic stress conditions were validated by real-time RT-PCR. Importantly, most of the tested stress-responsive genes were primarily expressed in mature leaves, stem cambia, and fibrous roots rather than apical buds and young leaves. As a response to cold stress in cassava, an increase in transcripts and enzyme activities of ROS scavenging genes and the accumulation of total soluble sugars (including sucrose and glucose) were also detected.

Conclusions: The dynamic expression changes reflect the integrative controlling and transcriptome regulation of the networks in the cold stress response of cassava. The biological processes involved in the signal perception and physiological response might shed light on the molecular mechanisms related to cold tolerance in tropical plants and provide useful candidate genes for genetic improvement.

Figure 1

Phenotypic and physiological changes in cold-stressed cassava. (A) 3-month-old cassava subjected to low temperature stress (7°C) for 0, 4, and 9 h in a chamber under weak light showing phenotypic changes. (B) Fully expanded cassava leaf (Left panel) and TEM analysis of a chloroplast (CP1, Right panel). (C) Low-temperature treated cassava leaf showing dehydration and necrosis (Left panel) and TEM analysis showing chloroplast abnormalities (CP2, Right panel). (D) MDA contents in cassava apical leaves exposed to 7°C for 0, 4, 9, and 24 h. (E) Proline accumulation in cassava apical leaves exposed to 7°C for 0, 4, 9, and 24 h. The double asterisks indicate a statistically significant difference (p < 0.01) for the data of the stress-treated samples compared to those of the unstressed samples. The mean values are calculated from three biological replicates; the error bars represent the standard error of the mean (SEM). Bar = 500 nm.

Figure 2

Expression profiling of cold-regulated genes in cassava apical shoots. (A) Hierarchical cluster analysis. (B) Venn diagrams showing cold-regulated genes across three comparisons (4 h/0 h, 9 h/0 h, and 9 h/4 h). The red numbers are the total numbers of differentially expressed genes (DEGs); the percentages in parentheses were calculated as the ratio of regulated genes to the total number of cold-regulated genes (508). It should be noted that one gene was up-regulated at 4 h/0 h but down-regulated at 9 h/4 h on our array.

Figure 3

TAIR percent of gene ontology (GO) terms for (A) 'Biological Process', (B) 'Molecular Function', and (C) 'Cellular Component' of cold up-regulated and down-regulated genes.

Figure 4

Expression patterns of important transcripts in response to cold stress at 0, 4, and 9 h by real-time RT-PCR analysis. (A) Members of AP2-EREBP transcription factor family. (B) CBF-like gene and its upstream regulons. (C) ProDH, P5CS, and ASP3 engaged in 'Proline metabolism'. (D) AMY1 and TPS participated in 'Starch and sucrose metabolism'. The double asterisks indicate a statistically significant difference (p < 0.01) for the data of the stress-treated samples compared to those of the unstressed samples.

Figure 5

KEGG pathway maps of cold-responsive genes. A total of 44 pathways were identified through KEGG mapping. Different colors represent the pathway entries and pathway names. The number of genes represented by AGI loci involved in each pathway is labeled in the parentheses.

Figure 6

Contents of total soluble sugars (A) and sucrose and glucose (B) in cassava leaves subjected to cold (7°C) for 0, 4, 9, and 24 h. The single and double asterisks indicate a statistically significant difference at p < 0.05 and p < 0.01, respectively, for the data of the stress-treated samples compared to those of the unstressed samples. The mean values are calculated from three biological replicates; the error bars represent the standard error of the mean (SEM).

Figure 7

Detection of H₂O₂, transcript level and enzymatic activities of ROS scavenging genes in cassava subjected to cold (7°C) for 0, 4, 9, and 24 h. (A) DAB polymerization of in vitro and greenhouse-grown leaves subjected to cold. (B) Quantification of H₂O₂ content in the leaves from the greenhouse-grown plants. (C) The transcript levels of genes encoding for ROS scavenging enzymes in response to cold stress. (D, E) Enzyme activities of catalase and superoxide dismutase (SOD) in the leaves of greenhouse-grown plants. The single and double asterisks indicate a statistically significant difference at p < 0.05 and p < 0.01, respectively, for the data of the stress-treated samples compared to those of the unstressed samples. The mean values are calculated from three biological replicates; the error bars represent the standard error of the mean (SEM). Bar = 1 cm.

Figure 8

Molecular model of the early cold response in cassava. Two biological processes, namely stress perception and the physiological response, are illustrated. After signal reception, stress-activated Ca²⁺ signaling, ROS signaling, and hormone signaling modulate the expression of stress-responsive genes, which include metabolic enzymes, transcription factors, kinases, and ion transporters. The physiological changes that manifested as membrane modification, chloroplast malfunction, and starch and sucrose metabolism, as well as amino acid metabolism, cause cassava to either have increased cold tolerance or to enter into accelerated programmed cell death. Their balance determines the outcome of the stressed cassava plant. Selected up- and down-regulated genes are in red or blue, respectively. The black arrows indicate a positive effect, and the black dashed arrows indicate a negative effect. The thick gray arrows show different biological processes in the stress response. AMY1: alpha-amylase like 1, AP2-EREBP: APETALA2-Ethylene Responsive Element Binding Protein, AREB3: ABA-responsive element binding protein 3, ASP3: Aspartate aminotransferase 3, AUX: auxin, CaM: calmodulin, EBF1: EIN3-binding F box protein 1, EIN4: ethylene insensitive 4, ERF2: ethylene response transcription activator, ETH: ethylene, FAB1: fatty acid biosynthesis 1, GA: gibberellins, GAI: gibberellic acid insensitive, GRAS: GAI, RGA, and SCR, GSTs: glutathione transferases, HSF: heat shock factor, JA: jasmonate, JAZ7: jasmonate-ZIM-domain protein 7, MAPK: Mitogen-activated protein kinase, PDS1: Phytoene desaturation 1, SAUR: auxin-responsive protein, SOD: superoxide dismutase, SSI2: fatty acid biosynthesis 2, TPS7: trehalose-phosphatase/synthase 7.