Transcriptome profiling of Malus sieversii under freezing stress after being cold-acclimated

Abstract

Background: Freezing temperatures are an abiotic stress that has a serious impact on plant growth and development in temperate regions and even threatens plant survival. The wild apple tree (Malus sieversii) needs to undergo a cold acclimation process to enhance its freezing tolerance in winter. Changes that occur at the molecular level in response to low temperatures are poorly understood in wild apple trees.

Results: Phytohormone and physiology profiles and transcriptome analysis were used to elaborate on the dynamic response mechanism. We determined that JA, IAA, and ABA accumulated in the cold acclimation stage and decreased during freezing stress in response to freezing stress. To elucidate the molecular mechanisms of freezing stress after cold acclimation, we employed single molecular real-time (SMRT) and RNA-seq technologies to study genome-wide expression profiles in wild apple. Using the PacBio and Illumina platform, we obtained 20.79G subreads. These reads were assembled into 61,908 transcripts, and 24,716 differentially expressed transcripts were obtained. Among them, 4410 transcripts were differentially expressed during the whole process of freezing stress, and these were examined for enrichment via GO and KEGG analyses. Pathway analysis indicated that "plant hormone signal transduction", "starch and sucrose metabolism", "peroxisome" and "photosynthesis" might play a vital role in wild apple responses to freezing stress. Furthermore, the transcription factors DREB1/CBF, MYC2, WRKY70, WRKY71, MYB4 and MYB88 were strongly induced during the whole stress period.

Conclusions: Our study presents a global survey of the transcriptome profiles of wild apple trees in dynamic response to freezing stress after two days cold acclimation and provides insights into the molecular mechanisms of freezing adaptation of wild apple plants for the first time. The study also provides valuable information for further research on the antifreezing reaction mechanism and genetic improvement of M. sieversii after cold acclimation.

Keywords: Freezing stress; Malus sieversii; Photosynthesis; Plant hormone signal transduction; Sugar and starch metabolism; Transcription factor.

Fig. 1

Processing methods and physiological and biochemical indicators after cold treatments at various time points. (A) Two-month-old wild apple seedlings growing in tissue culture bottles (left); harvested leaf (right); (B) Treatment process and sampling time points; (C) sucrose content; (D) MDA content; (E) Fv/Fm values; (F) REC values; (G) endogenous ABA content; (H) endogenous JA content; and (I) endogenous IAA content. Bars represent the mean ± SD (n = 3). Accumulation levels were analyzed using a T-test. Within each figure, asterisks above the bars indicate statistical significance (*p < 0.05; **p < 0.01)

Fig. 2

Characterization of M. sieversii transcripts. (A) GMAP mapping statistics. (B) Classification of transcripts corrected based on the reference genome sequences. (C) The gene numbers involving AS events. TML represents mixed samples containing treated and control samples

Fig. 3

Analysis of differentially expressed transcripts (DETs) at different times (CK, 0, 1, 6, and 10 h) across the transcriptome. (A) Number of DETs in the pairwise analyses. Upregulated (orange) and downregulated (blue) transcripts were quantified. (B) Venn diagram of the DETs at the different time points compared with the CK; (C) KEGG pathway enrichment at 0 h vs CK; (D) KEGG pathway enrichment at 10 h vs CK; (E) KEGG pathway enrichment at 10 h vs 0 h. dpi. A rich factor was the ratio of an input number to the background number in a specific pathway. The size and color of the dots represent the transcript numbers and the q-values, respectively

Fig. 4

Heat map of the relative expression levels of DEGs involved in sugar metabolism and the antioxidant defense system; (A) Sugar and starch metabolism-related genes; (B) Antioxidant-related genes. The red and blue colors indicate high and low expression levels (log2 fragments per kilobase of transcript per million mapped reads, FPKM), respectively

Fig. 5

Plant signal transduction pathways and expression of related genes in M. sieversii. (A) Heatmap of the ABA signal transduction pathway. (B) Heatmap of the JA signal transduction pathway. (C) Heatmap of the BR signal transduction pathway. (D) Heatmap of the ET signal transduction pathway. (E) Heatmap of the auxin signal transduction pathway. The red and blue colors indicate high and low expression levels (log2 fragments per kilobase of transcript per million mapped reads, FPKM), respectively

Fig. 6

Identification and analysis of the transcript expression levels of the top 15 transcription factor families. (A) Statistics of the differentially expressed transcription factors. (B) Expression profiles of differentially expressed TFs among different samples. The heat map was generated from the FPKM values. The red and blue colors indicate high and low expression levels, respectively