Genome-wide identification and functional analysis of PEL gene family in Brassica napus L

Abstract

Background: The function of the PEL gene has recently been studied in rice and Arabidopsis. Overexpression of the PEL1 gene leads to the Pseudo-Etiolation in Light phenotype. PEL1 downregulates chlorophyll accumulation in Arabidopsis and rice, and knocking out genes that downregulate chlorophyll content may improve crop quality and yield. However, the role of the PEL gene family in Brassica crops has not yet been reported.

Results: This study identified 24 members of PEL gene family in Brassica napus (B. napus), Brassica rapa (B. rapa), Brassica oleracea (B. oleracea), and Arabidopsis thaliana. Among them, PEL1 and PEL3 encode acidic proteins, while PEL2 and PEL4 encode weakly basic proteins. Phylogenetic and collinearity analyses showed that PEL genes are highly conserved and share a common domain (A_thal_3526). We cloned the BnaPEL1 gene from B. napus, and overexpression resulted in yellowing leaves and reduced chlorophyll content. CRISPR/Cas9-mediated knockout of BnaPEL1 increased chlorophyll content, enhanced photosynthesis, and improved yield. Transcriptome analysis revealed that differential genes are involved in carbohydrate metabolism and translation, with key roles in nucleocytoplasmic transport and chlorophyll biosynthesis.

Conclusions: Our study concludes that BnaPEL1 negatively regulates chlorophyll content and its knockout enhances photosynthesis and yield in rapeseed. The present study lays the foundation for the functional research and application of the PEL gene in oilseed rape.

Keywords: Brassica napus L.; Pseudo-Etiolation in light gene; Chlorophyll; Photosynthesis; Transcriptome; Yield.

Fig. 1

The PEL gene family analysis of B. napus, B. rapa, B. oleracea, and Arabidopsis. A Phylogenetic tree analysis of PEL proteins; B Collinearity analysis, inner circle represents chromosomes, outer circle represents gene density;C Gene structures and MEME Motifs analysis.

Fig. 2

Cis-Regulatory Elements of the BnaPEL Gene Promoter and Heatmap of Its Expression in Different Tissues and Organs. A Cis-elements detected in the promoter of the BnaPEL genes. B Expression heatmap of BnaPEL genes in different tissues and organs. C Expression of BnaPEL1 at 14, 21, 28, 35, 42 and 49 days of silique walls

Fig. 3

Functional Validation of BnaPEL1 Gene in Arabidopsis. A and B Phenotypic observation of wild-type Arabidopsis, Atpel mutant, and BnaPEL1 overexpression lines. The Atpel mutant exhibited smaller leaves with a greener color, whereas the overexpression lines showed larger leaves with a yellowish color compared to the wild-type and mutant. Bar = 5 cm. C SPAD values of Arabidopsis leaves during the bolting stage. Higher SPAD values indicate higher chlorophyll content, which is an indicator of photosynthetic capacity. D Leaf area during the bolting stage of Arabidopsis, measured as length × width to reflect leaf size. E Thousand seed weight of Arabidopsis seeds

Fig. 4

Identification of Target Sites and Phenotypic Comparison of B. napus Knockout Lines. A Gene editing status of knockout target sites. B The leaves of BnaPEL1-A04 and BnaPEL1-C08 knockout lines, Bar = 10 cm. C Transmission electron microscopy observation of chloroplasts in the leaves of WT (710). D Transmission electron microscopy observation of chloroplasts in the leaves of the BnaPEL1-C08 knockout line. The bars in the images from left to right in panels C and D represent 10 μm and 1 μm, respectively

Fig. 5

Analysis of Chlorophyll Content and Photosynthetic Capacity. A-D Chlorophyll content in the leaves of WT and BnaPEL1 knockout lines at the seedling stage, five-leaf stage, budding and bolting stage, and flowering stage. E-H Chlorophyll content in the silique wall at 14, 21, 28, and 35 days after silique development

Fig. 6

Agronomic Traits of BnaPEL1 Gene Knockout Lines. A Plant height at the harvest stage in rapeseed. B Fresh weight of aboveground parts at the budding stage in rapeseed. C Dry weight of aboveground parts at the budding stage in rapeseed. D Number of effective branches in rapeseed. E Number of siliques per plant in rapeseed. F Thousand seed weight of rapeseed. G Seed oil content in rapeseed. H Seed weight per plant of rapeseed

Fig. 7

Statistical Analysis of Differentially Expressed Genes and GO and KEGG Analysis. A Number of Upregulated and Downregulated DEGs. B and C GO and KEGG Classification Statistics of Gene Sets. C Top 20 Enriched Pathways of KEGG Enrichment Analysis for DEGs

Fig. 8

Differential Gene Expression Analysis in the BnaPEL1 gene Knockout Strains for Enriched Pathways and Chlorophyll Biosynthesis Pathway. A, B and C Differential Gene Expression Analysis of Nucleocytoplasmic Transport, Galactose metabolism and Porphyrin and chlorophyll metabolism. D Enzymes Involved in the Chlorophyll Biosynthesis Pathway and DEGs