PcMYBs responded to 6-BA to regulate PcCKXs to promote germination of primary rhizome buds of Polygonatum cyrtonema Hua

Abstract

Polygonatum cyrtonema Hua is valued both as a precious traditional Chinese medicinal herb and as a prime example of a plant that bridges medicinal and culinary applications. Renowned for its significant medicinal and edible qualities, this botanical exemplifies a unique convergence of therapeutic and nutritional benefits. However, the primary rhizome of Polygonatum cyrtonema Hua development is difficult to germinate into seedlings in the same year. The germination of primary rhizome buds of P. cyrtonema can be promoted by treatment with exogenous hormone 6-BA, but the related regulatory mechanism is not clear. In this study, we found that the cytokinin oxidase (CKX) plays a key role in the germination of primary rhizome buds of P. cyrtonema. PcCKX1,2,3 promoted the expression of dormancy positively regulated genes, and repressed the expression of dormancy negatively regulated genes, which in turn inhibited Arabidopsis seed germination, and PcCKX2 was the major gene. PcCKX1,2,3 promoted the expression of dormant positively regulated genes such as sweet potato IbZEP, IbNCED3, IbDOG1, IbABI5, IbCKX3, and IbCKX7, which in turn delayed the sprouting of sweet potato rhizomes, and that PcCKX2 played a major role. We further screened three MYB transcription factors significantly associated with PcCKX1,2,3. Yeast one-hybrid, Dual-LUC, and EMSA experiments showed that PcMYB4, PcKUA1, and PcCSA all bind to and repress the expression of elements of the PcCKX1,2,3 promoter. Heterologous transformation of Arabidopsis experiments showed that PcMYB4, PcKUA1, and PcCSA repressed the expression of dormancy-associated genes such as DOG1, NCED3, ABI5, CKX3, and CKX7, which, in turn, facilitated Arabidopsis seed germination. Taken together, we found that PcMYBs are involved in the transcriptional regulation of PcCKXs to promote the germination of primary rhizome buds of P. cyrtonema. The results of this study lay the foundation for analyzing the molecular mechanism of primary rhizome bud germination in P. cyrtonema.

Keywords: Polygonatum cyrtonema Hua; 6-benzylaminopurine; Bud germination; Cytokinin oxidase; Primary rhizome; Transcriptional regulation.

Fig. 1

Determination of buds emergence from primary rhizomes of P. cyrtonema. A Effect of different exogenous hormones on the emergence rate of primary rhizomes of P. cyrtonema. ** indicates highly significant difference compared to control (0 mg/L), P < 0.01, three biological replicates per treatment. B Effect of 6-BA treatment on the growth of seedlings of P. cyrtonema. The lower row shows the developmental status of primary rhizomes of P. cyrtonema at different times after 6-BA treatment, and the upper row shows the control at the same time, in which the primary rhizomes at 0 d were the same in the treatment and control groups. C Longitudinal sections of dormant buds from 0 d primary rhizomes. D The primary rhizome buds were cut longitudinally at 7 d and 21 d. E Longitudinal section of primary rhizome buds at 7 d and 21 d after 6-BA treatment. F-J are the ratios of DZ/IAA, tZ/IAA, tZR/IAA, cZ/IAA and iPA/IAA in the primary rhizome of P. cyrtonema. DZ is dihydrozeatin, cZ is cis-zeatin, tZ is trans-zeatin, tZR is trans-zeatin nucleoside and iPA is prenylated adenosine. C was the control group and T was the 6-BA treatment group among F-J

Fig. 2

Identification of PcCKX1,2.3. A Phylogenetic analysis of PcCKX1,2.3. B Expression levels of PcCKX1,2,3 in different tissues of P. cyrtonema. C-E Expression levels of PcCKX1,2,3 at different times after 6-BA treatment. F Enzymatic activity assay for the recombinant PcCKX2 protein. G Enzyme kinetic analysis for the recombinant PcCKX2 protein. H Subcellular localization of PcCKX1,2,3

Fig. 3

Transient overexpression of PcCKX1,2,3 in primary rhizomes of P. cyrtonema.A Fluorescence observation of transient overexpression of P. cyrtonema strains.B Gene expression of PcCKX1,2,3 after transient overexpression.C-E are the analysis of gene expression levels of overexpression and silencing of PcCKX1, PcCKX2, and PcCKX3, respectively, on the primary rhizomes of P. cyrtonema.EV indicates null loaded, VIGS indicates RNAi interference silencing, and OE indicates overexpressed gene. (*P < 0.05, **P < 0.01, ns differences are not significant; Different lowercase letters indicate significant differences, while the same letters denote no significant difference)

Fig. 4

Effects of PcCKX1,2,3 in overexpressed and backfilled mutant in Arabidopsis. A-B Analysis of seed germination rates of heterologously expressed PcCKX1,2,3 in Arabidopsis. The four regions"A-D"indicate wild-type, overexpression, mutant, and backfill Arabidopsis mutant seeds, respectively. C-E represent the expression of genes related to the heterologous expression of PcCKX1,2,3 and mutants in Arabidopsis, respectively. (*P < 0.05, **P < 0.01, ns differences are not significant; Different lowercase letters indicate significant differences, while the same letters denote no significant difference)

Fig. 5

Effect of PcCKX1,2,3 overexpression in transgenic sweet potato plants. A Germination rate analysis of heterologous expression of PcCKX1,2,3 in sweet potato. B qRT-PCR analysis of heterologous expression of PcCKX1,2,3 in sweet potato. (*P < 0.05, **P < 0.01, ns differences are not significant; Different lowercase letters indicate significant differences, while the same letters denote no significant difference)

Fig. 6

Screening and expression analysis of PcMYBs. A Promoter element analysis of PcCKX1,2,3. B Correlation analysis of PcCKX1,2,3 and PcMYBs transcription factors in P. cyrtonema. C Phylogenetic analysis of the PcMYBs family of P. cyrtonema. D Expression analysis of PcMYB4, PcKUA1, and PcCSA at different times, where C denotes the control group and T denotes the treatment group. (*P < 0.05, **P < 0.01)

Fig. 7

Subcellular localization of PcMYB4, PcKUA1 and PcCSA and analysis of their interaction with the PcCKX1,2,3 gene promoters.A Subcellular localization of PcMYBs. B Yeast one-hybrid validation of the specific elements of PcMYB4, PcKUA and PcCSA that bind to the PcCKX1, PcCKX2 and PcCKX3 promoters. C-D Dual-LUC chemiluminescence colorimetric analysis and enzyme activity analysis of PcMYB4, PcKUA1 and PcCSA with PcCKX1,2,3 gene promoter fragments. ** represents highly significant difference P < 0.01. E EMSA analysis of PcMYB4, PcKUA1 and PcCSA with PcCKX1,2,3 gene promoter fragments, respectively

Fig. 8

Effects of PcCKX1,2,3 overexpressing and backfilling their mutants in Arabidopsis. A-B Seed germination rate analysis of heterologously expressed PcMYBs in Arabidopsis. The four regions in the figure, including"A-D", represent wild-type (WT), overexpressed (OE), mutant (atmyb7, atkua1, and atcsa1), and backfilled Arabidopsis (RE) seeds, respectively. C-E Expression of genes associated with heterologous expression of PcMYB4, PcKUA1 and PcCSA and mutants in Arabidopsis, respectively. (*P < 0.05, **P < 0.01, ns not significantly different; Different lowercase letters indicate significant differences, while the same letters denote no significant difference)

Fig. 9

Pattern of molecular mechanism of 6-BA to promote the germination of primary rhizome buds of P. cyrtonema