Functional Studies of Pineapple AcKNOX1 on Leaf Margin Development

doi:10.16420/j.issn.0513-353x.2024-0304

Acta Horticulturae Sinica ›› 2025, Vol. 52 ›› Issue (2): 292-308.doi: 10.16420/j.issn.0513-353x.2024-0304

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

Functional Studies of Pineapple AcKNOX1 on Leaf Margin Development

JING Yonglin¹^,², CHEN Langxin¹^,², LI Junguo¹^,², WANG Jiabin¹^,², WANG Xiaobing¹^,², YANG Qingquan¹^,², MENG Chunyang¹^,², XU Li¹^,²^,^*()

¹ Key Laboratory of Crop Gene Resources and Germplasm Enhancement in Southern，Ministry of Agriculture and Rual Affairs，Key Laboratory of Tropical Crops Germplasm Resources Genetic Improvement and Innovation of Hainan Province，Institute of Tropical Crop Genetic Resources，Chinese Academy of Tropical Agricultural Sciences，Haikou 571101，China
² National Gene Bank of Tropical Crops，Danzhou，Hainan 571700，China

Received:2024-08-21 Revised:2024-10-18 Online:2025-02-25 Published:2025-02-23
Contact: XU Li

Abstract

Abstract:

In this study，AcKNOX1 was cloned from pineapple‘Tainong 16’，which is a homeobox gene，belongs to the three amino acid loop extension superfamily. The gene’s open reading frame was 990 bp with 329 amino acids. The protein was localized in the nucleus. In addition to the core promoter elements TATA-box and CAAT-box，the AcKNOX1 promoter region contained many elements related to light，hormonal and abiotic stress responses. AcKNOX1 was expressed in different tissues with the highest expression in the stem，followed by leaves，which suggested a certain tissue specificity. AcKNOX1 showed higher expression in spiny leaf materials than in spineless leaf materials. The expression of AcKNOX1 was up-regulated by short-term GA₃，ABA，and 6-BA stress. Ectopic overexpression of AcKNOX1 in Arabidopsis thaliana suggested that transformants showed larger and deeper serrations. Analysis of interaction proteins obtained by Yeast one and two hybrid system showed that AcKNOX1 could interact and activate AcCUC1 and AcCUC2 promoter regions. And AcKNOX1 interacted with AcBLH1 and AcBLH4 to form a heterodimer. And the inteaction was verified by Luciferase complementation imaging assay（LCI）and Bimolecular fluorescence complementation（BIFC）. The results revealed that AcKNOX1 gene played a vital role in the regulation of pineapple leaf margin development.

Key words: Ananas comosus, leaf margin development, AcKNOX1, protein-interation

JING Yonglin, CHEN Langxin, LI Junguo, WANG Jiabin, WANG Xiaobing, YANG Qingquan, MENG Chunyang, XU Li. Functional Studies of Pineapple AcKNOX1 on Leaf Margin Development[J]. Acta Horticulturae Sinica, 2025, 52(2): 292-308.

Figures/Tables 15

Table 1 Primers for cloning and expression analysis of AcKNOX1

用途 Application	序列名称 Sequence name	上游引物序列（5′-3′） Forward primer	下游引物序列（5′-3′） Reverse primer
AcKNOX1克隆 Cloning of AcKNOX1	AcKNOX1	CCCTTTCCCAAAACCCTACC	CGCCTTCATCGATTGATCGT
AcKNOX1克隆 Cloning of AcKNOX1	AcKNOX1+	GGGGTACCCCCTTTCCCAAAACCCTACC	GCTCTAGACGCCTTCATCGATTGATCGT
亚细胞定位 Subcellular localization	pBI221-AcKNOX1	GACTCTAGAGGATCCATGGATGAGCAACTTTACCAAGTTA	CTCCGCGGCGGATCCTGCGCTTCTTGGTCACATCTATCG
启动子克隆 Cloning of promoter	AcKNOX1-promoter	CAAAATAATGAAATCTATGGCCAAA	AAAAGGAAACCACACCCGAAA
qRT-PCR	qPCR-AcKNOX1	TGCCGTTTTGATGTTCTCACTAAG	CGATTGATCGTATACCCAGTTTG

Fig. 1 Amplification of the AcKNOX1

Fig. 2 Multiple sequence alignment of AcKNOX1 and KNOX proteins from Arabidopsis thaliana（At）

Fig. 3 Analysis of phylogenetic of AcKNOX1 Ac：Ananas comosus；At：Arabidopsis thaliana；Os：Oryza sativa；Pt：Populus trichocarpa；Zm：Zea mays

Fig. 4 PCR amplification of AcKNOX1 promoter

Fig. 5 Expression analysis of AcKNOX1 in different tissues of pineapple‘Tainong 16’ Different lowercase indicate significant differences（P < 0.05）

Fig. 6 Expression level of AcKNOX1 in pineapple seedlings after exogenous GA3，ABA and 6-BA treatments * Indicates a significant difference between treatment and control at the 0.05 level at the same time

Fig. 7 Subcellular localization of AcKNOX1 protein in onion epidermal cells

Fig. 8 Molecular identification of transgenic Arabidopsis plants 1：Positive control；2：Wild type；3-5：Transgenic Arabidopsis

Fig. 9 The phenotype of wild-type（WT），trans-empty vector（Vector）and transgenic（AcKNOX1-OE）lines of Arabidopsis A：The leaf margin of cotyledon，the red triangle shows serration at the leaf margin；B：The hypocotyl of cotyledon，the white triangles indicate the junction of the hypocotyl and root；C：The hypocotyl length；D：The leaf phenotype during bolting；E：The phenotype of the sixth rosette leaf；F：Quantification of serration in the sixth rosette leaf；G：The leaf phenotype after podding. L1，L2，L3，L4 indicate four lines of transgenic plants

Fig. 10 Yeast one-hybrid assay of AcKNOX1 to the promoters of AcCUC Negative control：pAbAi-AcCUCpro + pGADT7，pAbAi + pGADT7

Fig. 11 AcKNOX activates the AcCUC promoters in dual-luciferase assays

Fig. 12 Interaction between AcKNOX1 and AcBLH1，AcBLH4 validated by yeast two-hybrid experiments

Fig. 13 BIFC assay between AcKNOX1 and AcBLH1

Fig. 14 BIFC assay between AcKNOX1 and AcBLH4

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Functional Studies of Pineapple AcKNOX1 on Leaf Margin Development

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