Functional Analysis of LaPLT5 in Bulblet Enlargement of Lilium

doi:10.7668/hbnxb.20196709

Abstract

Abstract:

Bulblet enlargement is of significant importance for lily cultivation and breeding.The PLETHORA(PLT)transcription factors play a key regulatory role in plant organ expansion.This study used Lilium Tresor as experimental material to investigate the mechanism of the LaPLT5 gene during its bulblet enlargement process.By analyzing transcriptome data from small bulblets at stages S1 to S5 of Lilium Tresor,the LaPLT5 gene,which is potentially involved in bulblet enlargement,was identified.The gene was cloned from bulblet cDNA using molecular cloning techniques,and the structural characteristics of its encoded protein were predicted through bioinformatics analysis.The expression pattern of LaPLT5 during bulblet enlargement was examined using qRT-PCR,and its function was validated via virus-induced gene silencing(VIGS)and transient overexpression assays.The results showed that the LaPLT5 gene had a full length of 1 500 bp,encoding 499 amino acids,including 39 positively charged residues and 38 negatively charged residues.The protein had a relative molecular weight of 53 754.25,an instability index of 40.77,and an average hydrophilicity value of 0.56,indicating that it is an unstable hydrophobic protein.qRT-PCR analysis revealed that LaPLT5 expression was highest in the middle scale layers of Lilium Tresor bulblets,followed by that in small bulblets,and its expression level continuously increased from stages S2 to S4 during the enlargement of small bulblets.After VIGS-mediated silencing of LaPLT5,the diameter,circumference,and weight of small bulblets were highly significantly greater than those in the control group,whereas transient overexpression of LaPLT5 yielded opposite results.This indicated that LaPLT5 negatively regulated the enlargement of small bulblets in Lilium Tresor.Based on the phenotype of increased cell number but reduced cell area in small bulblets after LaPLT5 silencing,qRT-PCR analysis showed that the expression of cell wall relaxation-related genes,LaPME34,LaXTH1 and LaXTH6,was highly significantly downregulated.Meanwhile,the positive cell wall regulatory genes LaEBP1 and LaANT1 were highly significantly upregulated, whereas the negative regulatory gene LaCKX4 was highly significantly downregulated.These findings suggested that LaPLT5 likely regulated the expansion and division of small bulblet cells by coordinating the expression of genes involved in cell wall remodeling and cell division.In summary,LaPLT5 participates in the enlargement of small bulblets in Lilium Tresor and functions as a negative regulator of this process.

Key words: Lily, LaPLT5, Function analysis, Bulblet, Dilated

CLC Number:

S682.2

LIU Zhongxu, AN Rui, CHU Hongtao, WANG Mengdi, CHEN Yanzhu, LI Xiaorui, PAN Wenqiang, CAO Li, DU Yunpeng. Functional Analysis of LaPLT5 in Bulblet Enlargement of Lilium[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(4): 87-98. doi: 10.7668/hbnxb.20196709.

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http://www.hbnxb.net/EN/Y2026/V41/I4/87

Figures/Tables 11

Tab.1 The primers used in the qRT-PCR

引物名称 Primer name	序列(5'—3') Sequence (5'—3')
LaPME34-qPCR-F LaPME34-qPCR-R LaXTH1-qPCR-F LaXTH1-qPCR-R LaXTH6-qPCR-F LaXTH6-qPCR-R LaCKX4-qPCR-F LaCKX4-qPCR-R LaEBP1-qPCR-F LaEBP1-qPCR-R LaANT1-qPCR-F LaANT1-qPCR-R LaCYCD3-2-qPCR-F LaCYCD3-2-qPCR-R LaARR1-qPCR-F LaARR1-qPCR-R LaCYCB1-qPCR-F LaCYCB1-qPCR-R	CAACCGAGTCCATTTCAACC TGGTGTTGAGGGAGATGTG TAATTCCTCTGCACCCATCC GCATCTTTCCGGAACTTCAA TGGATCACATGAGCTTCGAC CGGCTACCAGCTTAATCTCT GTTGACCATGCCACCACCCA TGAGAGGGAGAAACTCAAACAGGGT AGTTGCAAAGCCACACTCTC TCACCTTCTTTCCCGAAGCA TAGATTACGCCCGAACTCGT GTAGAGACCGCGGTAGACAA ACTGGAAATAAGCGGAAGCA CGCAGTTGAACCGTTTGAAAG ACACGACTTGGACAAGGGAT TCTGTCTGAGTTGCCGGAAT TCTTCACCAGCAGAGAGATG GTGAACGAACACATTTCCGA

Fig.1 Divisions of the process of bulblets enlargement in Lilium Tresor

Fig.2 Screening of LaPLT5 and phylogenetic tree analysis A.Heatmap of PLT family gene expression;B.Phylogenetic tree and conserved motif analysis of LaPLT5 and its homologous genes.S3-1,2,3 and S5-1,2,3 represent biological replicates of different lines at the initial swelling stage and mature stage of bulblets in Lilium Tresor,respectively.

Fig.3 Physicochemical property analysis of LaPLT5 protein A.Amino acid composition of the LaPLT5 protein;B.Hydropathy plot analysis of the LaPLT5 protein;C.Transmembrane domain prediction of the LaPLT5 protein;D.Phosphorylation site prediction of the LaPLT5 protein.

Fig.4 Prediction of the secondary and tertiary structures of LaPLT5 protein A.Secondary structure prediction of LaPLT5;B.Tertiary structure prediction of LaPLT5.

Fig.5 Analysis of LaPLT5 expression in different parts of the Lilium Tresor and at different stages A.Expression levels of LaPLT5 in different tissues of lily;B.Expression levels of LaPLT5 in bulblets at stages S1 to S5.The data are presented as mean±SEM,n=3;the data are analyzed using Student's t-test.Different lowercase letters indicate statistically significant differences at P<0.05.

Fig.6 Phenotypic statistics and gene quantitative analysis of LaPLT5 silencing lines of small bulblets of Lilium Tresor A.Phenotypes of small bulb scales in LaPLT5-silenced lines(LaPLT5-TRV2)and the control(TRV2);B.Relative expression levels of LaPLT5 in three independent LaPLT5-silenced lines(LaPLT5-TRV2-1,2,3)and the control(TRV2);C.Circumference of small bulblets in LaPLT5-silenced lines(LaPLT5-TRV2)and the control(TRV2);D.Weight of small bulblets in LaPLT5-silenced lines(LaPLT5-TRV2)and the control(TRV2);E.Diameter of small bulblets in LaPLT5-silenced lines(LaPLT5-TRV2)and the control(TRV2).All data are expressed as mean±standard deviation,with n=3 in panel B and n=25 in panels C—E.Different uppercase letters indicate statistically significant differences at P<0.01, the same as Fig.7—10.

Fig.7 Tissue sections and data analysis of the LaPLT5-silenced line in small bulblets of Lilium Tresor A.Longitudinal sections of bulblets from LaPLT5-silenced lines(LaPLT5-TRV2)and control lines(TRV2);B.Cell number in bulblets of LaPLT5-silenced lines(LaPLT5-TRV2)and control lines(TRV2);C.Cell area in bulblets of LaPLT5-silenced lines(LaPLT5-TRV2)and control lines(TRV2).Violin plots were used to display the cell area of bulblets under the two treatments.All data are presented as mean standard deviation,for panels B and C,n=25.

Fig.8 Phenotypic statistics and gene quantitative analysis of LaPLT5 overexpressed lines in small bulblets of Lilium Tresor A.Phenotypes of small bulb scales in LaPLT5 overexpression lines(LaPLT5-OE)and the empty vector control(EV);B.Relative expression levels of LaPLT5 in three independent LaPLT5 overexpression lines(LaPLT5-OE-1,2,3)and the empty vector control(EV);C.Perimeter of bulblets in LaPLT5 overexpression lines(LaPLT5-OE)and the empty vector control(EV);D.Weight of bulblets in LaPLT5 overexpression lines(LaPLT5-OE)and the empty vector control(EV);E.Diameter of bulblets in LaPLT5 overexpression lines(LaPLT5-OE)and the empty vector control(EV).All data are presented as mean±standard deviation,with n=3 in panel B and n=25 in panels C-E.

Fig.9 Tissue sections and data analysis of the LaPLT5-overexpressing line in small bulblets of Lilium Tresor A.Sections of bulblets from LaPLT5 overexpression lines(LaPLT5-OE)and empty vector control lines(EV);B.Cell number in bulblets of LaPLT5 overexpression lines(LaPLT5-OE)and empty vector control lines(EV);C.Cell area in bulblets of LaPLT5 overexpression lines(LaPLT5-OE)and empty vector control lines(EV).Violin plots are used to show the cell area of bulblets under the two treatments.All data are presented as mean standard deviation.For panels B and C,n=25.

Fig.10 Relative expression levels of bulb enlargement-related downstream genes in LaPLT5-silenced lines Relative expression levels of various genes in three independent LaPLT5-silenced lines(LaPLT5-TRV2-1,2,3)and the control line(TRV2).

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