Transcriptome-Based Screening of Candidate Low-Temperature-Associated Genes and Analysis of the BocARR-B Transcription Factor Gene Family in Kohlrabi (Brassica oleracea L. var. caulorapa L.)

Abstract

Low temperature is a significant abiotic stress factor that not only impacts plant growth, development, yield, and quality but also constrains the geographical distribution of numerous wild plants. Kohlrabi (Brassica oleracea L. var. caulorapa L.) belongs to the Brassicaceae family and has a short growing period. In this study, a total of 196,642 unigenes were obtained from kohlrabi seedlings at low temperatures; of these, 52,836 unigenes were identified as differentially expressed genes. Transcription factor family members ARR-B, C3H, B3-ARF, etc. that had a high correlation with biochemical indicators related to low temperature were identified. A total of nineteen BocARR-B genes (named BocARR-B1-BocARR-B19) were obtained, and these genes were distributed unevenly across seven chromosomes. Nineteen BocARR-B genes searched four conserved motifs and were divided into three groups. The relative expression level analysis of 19 BocARR-B genes of kohlrabi showed obvious specificity in different tissues. This study lays a foundation and provides new insight to explain the low-temperature resistance mechanism and response pathways of kohlrabi. It also provides a theoretical basis for the functional analysis of 19 BocARR-B transcription factor gene family members.

Keywords: BocARR-B transcription factor; differentially expressed genes; gene relative expression level analysis; kohlrabi (Brassica oleracea L. var. caulorapa L.); low-temperature stress; metabolites networks.

Figure 1

Biochemical indicators of kohlrabi seedlings at low temperature (CK) and under low-temperature stress at 12 °C, 8 °C, and 4 °C. (A) Fresh and dry weight. (B) Water content. (C) Catalase (CAT) activity. (D) Superoxide dismutase (SOD) activity. (E) Malondialdehyde (MDA) content. (F) Proline (Pro) content. (G) Soluble protein content. (H) Soluble sugar content. ** Represents a significant difference when the p-value is 0.01.

Figure 2

Transcriptome data summary and analysis of differentially expressed unigenes in kohlrabi seedlings under low-temperature stress. (A) The transcript and unigene sequences’ length distribution. (B) Functional annotation of assembled unigenes. (C) Differentially expressed unigenes in different pairwise comparisons. (D) Venn diagram of differentially expressed unigene numbers in pairwise comparisons between control and treatment of 12 °C vs. CK, 8 °C vs. CK, and 4 °C vs. CK. White and red digits indicate the number of downregulated and upregulated genes, respectively. (E) Venn diagram of differentially expressed unigene numbers in pairwise comparisons of treatment of 12 °C vs. CK, 8 °C vs. 12 °C, and 4 °C vs. 8 °C. White and red digits indicate the number of downregulated and upregulated genes, respectively.

Figure 3

Availability analysis of transcriptome data using RNA-Seq and RT-qPCR was used to construct the expression-level bar graphs at 4 °C, 8 °C, 12 °C, and CK (22 °C). (A) BnaC05g00840D (Cluster-17807.81882); (B) SYD (Cluster-17807.82832); (C) Zinc finger protein CONSTANS-LIKE 4 (Cluster-17807.77797); (D) MYB1-R1 (Cluster-17807.81941); (E) C3H49 (Cluster-17807.85268); (F) C3H30 (Cluster-17807.81598); (G) Zinc finger protein CONSTANS-LIKE 4 (Cluster-17807.83850); (H) RGA2 (Cluster-17807.82153); (I) DOF3.3 (Cluster-17807.86516); (J) B-box zinc finger protein 25 (Cluster-17807.84461).

Figure 4

A putative interplay of kohlrabi seedlings under low-temperature stress. The DEG changes were represented by the log₂FPKM. Blue and red, respectively, represent a decrease and an increase in the expression level of genes.

Figure 5

Analysis of differentially expressed transcription factors and identification of low-temperature-related differentially expressed transcription factors. (A) Venn diagram of differentially expressed transcription factors in the comparison of 12 °C vs. CK, 8 °C vs. CK, and 4 °C vs. CK. White and red digits indicate the number of downregulated and upregulated genes, respectively. (B) Venn diagram of the differentially expressed transcription factor in the comparison of 12 vs. CK, 8 °C vs. 12 °C, and 4 °C vs. 8 °C. White and red digits indicate the number of downregulated and upregulated genes, respectively. (C) Co-expression network map between differentially expressed transcription factors and CAT activity, SOD activity, MDA content, Pro content, and soluble sugar content. Yellow squares indicate biochemical indicators related to the low temperature. Blue circles indicate differentially expressed transcription factors. (D) Expression profile of differentially expressed transcription factors’ related low-temperature stress in kohlrabi using RNA sequencing. The changes in the differentially expressed transcription factors are represented by the log₂FPKM.

Figure 6

Co-expression network map between differentially expressed transcription factors related to the low temperature and DEGs related to the metabolites pathway. Blue circles indicate DEGs related to the metabolites pathway; yellow squares indicate differentially expressed transcription factors related to the low temperature.

Figure 7

Chromosomal locations of BocARR-B genes in kohlrabi. Note: C1–C9 indicates nine chromosomes. The scale bar on the left shows the chromosome lengths (Mb). The blue and yellow regions of the chromosome represent a low and high gene density, respectively.

Figure 8

Evolutionary relationships, conserved protein motifs, and domains of the 19 BocARR-Bs. (A) Phylogenetic relationship analysis using amino acid sequences of BocARR-Bs by MEGA11. The size of the purple circle represents the bootstrap value. The display range indicates the branch metadata bootstrap. (B) Distribution of 4 motifs. (C) Distribution of REC_type_ARR-like domain, myb_SHAQKYF domain, PLN03162 superfamily domain, and HFD_SF superfamily domain. (D) Four different conserved motifs of BocARR-Bs.

Figure 9

Evolutionary tree of BocARR-B family genes in kohlrabi and Arabidopsis.

Figure 10

Expression analysis of BocARR-B family genes in kohlrabi. (A) Expression profile of BocARR-B genes in kohlrabi using RNA sequencing during low-temperature stress. Data in the heatmap box are normalized expression levels of three replicates. (B–L) RT-qPCR analysis of eleven BocARR-B transcription factor genes in different tissues of kohlrabi. * Represents a significant difference when the p-value is 0.05. ** Represents a significant difference when the p-value is 0.01.