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. 2025 May 29;15(1):18825.
doi: 10.1038/s41598-024-81004-x.

Genome-wide investigation of cytokinin oxidase/dehydrogenase (CKX) family genes in Brassica juncea with an emphasis on yield-influencing CKX

Balaji Balamurugan  1   2 , Mansi Singh  1 , Dwijesh C Mishra  3 , Sandhya Sharma  1 , Kishor Gaikwad  1 , Ganapati Mukri  2 , Mahesh Rao  1 , Navin C Gupta  4
Affiliations

Genome-wide investigation of cytokinin oxidase/dehydrogenase (CKX) family genes in Brassica juncea with an emphasis on yield-influencing CKX

Balaji Balamurugan et al. Sci Rep. .

Abstract

Rapeseed mustard (Brassica juncea) is an important edible oilseed crop whose yield is impacted not only by the biotic and abiotic stresses but the low productivity of the cultivars also has a great impact over it. In the past, cytokinin oxidase/dehydrogenase (CKX) family gene manipulation has increased yield and stress tolerance and has been cloned and characterized in different plant species. Cytokinin oxidase/dehydrogenase (CKX) is an important enzyme regulating cytokinin homeostasis and regulating the yield and tolerance traits in plants. This study demonstrated a comprehensive investigation of the CKX gene family in B. juncea cv. Varuna. As a result, a total of 24 CKX genes were identified across the 36 chromosomes (AABB) and classified into seven distinct subgroups. These seven subgroups were annotated based on their homology with their counterparts present in Arabidopsis thaliana. Expression pattern analysis by RT-qPCR in the high-and low-yielding B. juncea cultivars showed that all 24 CKX expressed differentially in various tissues; most were expressed in leaves, stems and developing siliques. The two functional domains, FAD-binding and Cytokinin-binding domains required for CKX activity, were conserved across the CKX family genes. Our findings systematically revealed the evolutionary dynamics of the BjCKX family genes. They led the foundation for subsequent validation of the CKX for their functional role in yield enhancement in B. juncea.

Keywords: Brassica juncea; BjCKX; CKX; Cytokinin; Cytokinin oxidase/dehydrogenase; Oilseed; Rapeseed-mustard.

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Conflict of interest statement

Declarations. Competing interests: The authors declare no competing interests. Guidelines statement: Experiments conducted in this study have been carried out following relevant institutional, national, and international guidelines and legislation. Permissions/licences for the use of plant materials: The authors declare that they have obtained due permission for the collection and use of the B. juncea cultivars (Pusa Jaikisan/PJK and RLM 198) for the current study at the ICAR-NIPB, New Delhi.

Figures

Fig. 1
Fig. 1
Schematic representation of the BjCKX gene family members identified in B. juncea cv. Varuna on the A and B sets of chromosomes.
Fig. 2
Fig. 2
The phylogenetic tree of B. juncea and A. thaliana CKX proteins. (a) The maximum likelihood phylogenetic tree was constructed using MEGA 11 with 1000 bootstrap replicates, (b) The phylogenetic tree was clustered into 7 subclades (I–VII). A distinct colour represents each subclade.
Fig. 3
Fig. 3
Gene structure of CKX genes in B. juncea. Schematic representation of the exon–intron distribution in the CKX genes of B. juncea. The blocks represent exons and the line joining the blocks represents introns. Numerical values above the bars and below the thin lines are the size of the exons, and introns, respectively in base pairs. The bar above the last exon represents the 100 bp scale.
Fig. 4
Fig. 4
Gene structure of CKX genes in B. juncea. (a) Conserved motif analysis was conducted using MEME Suite. A total of 20 motifs were predicted. (b) The domain organization of BjCKXs.
Fig. 5
Fig. 5
The CRE analysis in the BjCKX promoter sequences. The 2 kb upstream regulatory sequences of the BjCKX gene promoter regions were extracted from the whole genome sequence of the B. juncea cv. Varuna and analyzed using the PlantCARE database.
Fig. 6
Fig. 6
Circos plot of CKX gene duplication in B. juncea. The different colours represent the genes found in different (I-VII subclades) subgroups, and the lines in the middle show segmental and tandem duplications between different chromosomes.
Fig. 7
Fig. 7
Triple synteny plots between B. rapa, B. juncea, and B. nigra genomes, with orthologous CKX genes shown with connecting lines.
Fig. 8
Fig. 8
Morphological features of the various plant tissues of RLM 198 and PJK genotypes of the B. juncea. (a) Leaf, (b) Flower buds, (c) Open flower (d) Petal (e) Reproductive part (f) Androecium (g) Gynoecium (h) Inflorescence (i) Siliques in plants at maturity (j) Siliques in plants at the harvesting stage (k) Individual mature seed (l) Seeds harvested after maturity.
Fig. 9
Fig. 9
Heatmap showing the differentially expressed BjCKX gene in leaf, stem junction, flower buds and silique tissues of RLM 198 and PJK genotypes of B. juncea. The colour in the heatmap from green to red represents the expression values from low to high. The individual box shows the expression values of a particular BjCKX in the respective tissue of the RLM 198 and PJK genotypes.
Fig. 10
Fig. 10
The bar diagram shows the fold change in the expression values of the BjCKX genes in different tissues of the RLM 198 and PJK genotypes. (a) Leaf, (b) Stem junction, (c) Flower buds and (d) silique tissues.
Fig. 11
Fig. 11
The phylogenetic analysis of the BjCKX gene family with the functionally characterized yield influencing CKX of other plant species, (a) BjCKX association with known yield-responsive CKX genes (b) Subtree of BjCKX3.4 with other yield influencing CKX3 (c) Subtree of BjCKX5.2 with other yield influencing CKX5.
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