Decapitation Experiments Combined with the Transcriptome Analysis Reveal the Mechanism of High Temperature on Chrysanthemum Axillary Bud Formation

Temperature is an important factor that largely affects the patterns of shoot branching in plants. However, the effect and mechanism of temperature on axillary bud development in chrysanthemum remains poorly defined. The purpose of the present study is to investigate the effect of high temperature on the axillary bud growth and the mechanism of axillary bud formation in chrysanthemum. Decapitation experiments combined with the transcriptome analysis were designed. Results showed that the axillary bud length was significantly inhibited by high temperature. Decapitation of primary shoot (primary decapitation) resulted in slower growth of axillary buds (secondary buds) under 35 °C. However, secondary decapitation resulted in complete arrest of tertiary buds at high temperature. These results demonstrated that high temperature not only inhibited axillary bud formation but also retarded bud outgrowth in chrysanthemum. Comparative transcriptome suggested differentially expressed gene sets and identified important modules associated with bud formation. This research helped to elucidate the regulatory mechanism of high temperature on axillary bud growth, especially bud formation in chrysanthemum. Meanwhile, in-depth studies of this imperative temperature signaling can offer the likelihood of vital future applications in chrysanthemum breeding and branching control.

Control of axillary bud growth in tobacco through toxin gene expression system

The control of axillary bud development after removing the terminal buds (topping) of plants is a research hotspot, and the control of gene expression, like switching on and off, allows us to further study biological traits of interest, such as plant branching and fertility. In this study, a toxin gene control system for plants based on dexamethasone (DEX) induction was constructed, and the positive transgenic tobacco exhibited growth retardation in the application area (axillary bud). The expression level of the lethal Diphtheria toxin A (DTA) gene under different DEX concentrations at different application days was analyzed. The highest expression levels appeared at 5 days after the leaf injection of DEX. The DTA transcripts were induced by 5 µM DEX and peaked in response to 50 µM DEX at 5 days after leaf injection. Here, a chemical induction system, combined with a toxin gene, were used to successfully control the growth of tobacco axillary buds after topping. The DTA expression system under DEX induction was sensitive and efficient, therefore, can be used to control axillary bud growth and development in tobacco.

Carotenoid Cleavage Dioxygenase Genes of Chimonanthus praecox, CpCCD7 and CpCCD8, Regulate Shoot Branching in Arabidopsis

Strigolactones (SLs) regulate plant shoot development by inhibiting axillary bud growth and branching. However, the role of SLs in wintersweet (Chimonanthus praecox) shoot branching remains unknown. Here, we identified and isolated two wintersweet genes, CCD7 and CCD8, involved in the SL biosynthetic pathway. Quantitative real-time PCR revealed that CpCCD7 and CpCCD8 were down-regulated in wintersweet during branching. When new shoots were formed, expression levels of CpCCD7 and CpCCD8 were almost the same as the control (un-decapitation). CpCCD7 was expressed in all tissues, with the highest expression in shoot tips and roots, while CpCCD8 showed the highest expression in roots. Both CpCCD7 and CpCCD8 localized to chloroplasts in Arabidopsis. CpCCD7 and CpCCD8 overexpression restored the phenotypes of branching mutant max3-9 and max4-1, respectively. CpCCD7 overexpression reduced the rosette branch number, whereas CpCCD8 overexpression lines showed no phenotypic differences compared with wild-type plants. Additionally, the expression of AtBRC1 was significantly up-regulated in transgenic lines, indicating that two CpCCD genes functioned similarly to the homologous genes of the Arabidopsis. Overall, our study demonstrates that CpCCD7 and CpCCD8 exhibit conserved functions in the CCD pathway, which controls shoot development in wintersweet. This research provides a molecular and theoretical basis for further understanding branch development in wintersweet.

Carotenoid Cleavage Dioxygenase Genes of Chimonanthus praecox, CpCCD7 and CpCCD8, Regulate Shoot Branching in Arabidopsis

Strigolactones (SLs) regulate plant shoot development by inhibiting axillary bud growth and branching. However, the role of SLs in wintersweet (Chimonanthus praecox) shoot branching remains unknown. Here, we identified and isolated two wintersweet genes, CCD7 and CCD8, in-volved in the SL biosynthetic pathway. Quantitative real-time PCR revealed that CpCCD7 and CpCCD8 were down-regulated in wintersweet during branching. When new shoots were formed, expression levels of CpCCD7 and CpCCD8 were almost the same as the control (un-decapitation). CpCCD7 was expressed in all tissues, with the highest expression in shoot tips and roots, while CpCCD8 showed the highest expression in roots. Both CpCCD7 and CpCCD8 localized to chloroplasts in Arabidopsis. CpCCD7 and CpCCD8 overexpression restored the phenotypes of branching mutant max3-9 and max4-1, respectively. CpCCD7 overexpression reduced the rosette branch number, whereas CpCCD8 overexpression lines showed no phenotypic differences compared with wild-type plants. Additionally, the expression of AtBRC1 was significantly up-regulated in transgenic lines, indicating that two CpCCD genes functioned similarly to the homologous genes of the Arabidopsis. Overall, our study demonstrates that CpCCD7 and CpCCD8 exhibit conserved functions in the CCD pathway, which controls shoot development in wintersweet. This research provides a molecular and theoretical basis for further understanding branch development in wintersweet.

Nitrogen Nutrition Promotes Rhizome Bud Outgrowth via Regulation of Cytokinin Biosynthesis Genes and an Oryza longistaminata Ortholog of FINE CULM 1

Oryza longistaminata, a wild rice, can propagate vegetatively via rhizome formation and, thereby, expand its territory through horizontal growth of branched rhizomes. The structural features of rhizomes are similar to those of aerial stems; however, the physiological roles of the two organs are different. Nitrogen nutrition is presumed to be linked to the vegetative propagation activity of rhizomes, but the regulation of rhizome growth in response to nitrogen nutrition and the underlying biological processes have not been well characterized. In this study, we analyzed rhizome axillary bud growth in response to nitrogen nutrition and examined the involvement of cytokinin-mediated regulation in the promotion of bud outgrowth in O. longistaminata. Our results showed that nitrogen nutrition sufficiency promoted rhizome bud outgrowth to form secondary rhizomes. In early stages of the response to nitrogen application, glutamine accumulated rapidly, two cytokinin biosynthesis genes, isopentenyltransferase, and CYP735A, were up-regulated with accompanying cytokinin accumulation, and expression of an ortholog of FINE CULM1, a negative regulator of axillary bud outgrowth, was severely repressed in rhizomes. These results suggest that, despite differences in physiological roles of these organs, the nitrogen-dependent outgrowth of rhizome axillary buds in O. longistaminata is regulated by a mechanism similar to that of shoot axillary buds in O. sativa. Our findings provide a clue for understanding how branched rhizome growth is regulated to enhance nutrient acquisition strategies.

Foliar Thidiazuron Promotes the Growth of Axillary Buds in Strawberry

Strawberry (Fragaria × ananassa Duch.) can be easily propagated with daughter plants or through crown division, which are developed from the axillary bud at the axils of leaves. This study was conducted to investigate the effects of different cytokinins, auxins, and their combinations on the axillary bud growth in strawberry. Four cytokinins (6-benzyladenine, kinetin, zeatin, and thidiazuron (TDZ)) and three auxins (indole-3-acetic acid, indole-3-butyric acid, and naphthaleneacetic acid) at a concentration of 50 mg·L−1 were sprayed on the leaves three times in 10-day intervals. The expression levels of cytokinin, auxin, and meristem-related genes in the crowns were also investigated. The results showed that TDZ was the most effective hormone for the axillary bud growth, and also promoted plant growth. However, chlorophyll, soluble sugar, and starch contents in the leaves were lower after TDZ. TDZ activated the cytokinin signal transduction pathway, while repressing the auxin synthesis genes. Several meristem-related transcription factors were upregulated, which might be essential for the growth of the axillary buds. These results suggested that TDZ can improve the cultivation of strawberry, while further research is needed to explain the effect on phytochemistry.

BcHTT4 Inhibits Branching of Non-Heading Chinese Cabbage at the Vegetative Stage

Branching is speculated to contribute to the plant architecture and crop yield. As a quantitative trait, branching is regulated by multiple genes in non-heading Chinese cabbage (NHCC). Several related candidate genes have been discovered in previous studies on the branching of NHCC, but their specific functions and regulatory mechanisms still need to be verified and explored. In this study, we found that the expression of BcHTT4, the ortholog to HEAT-INDUCED TAS1 TARGET4 (HTT4) in Arabidopsis, was significantly different between ‘Suzhouqing’ (common type) and ‘Maertou’ (multiple shoot branching type) in NHCC, which was consistent with the previous transcriptome sequencing results. The silencing of BcHTT4 expression in non-heading Chinese cabbage promotes axillary bud growth at the vegetative stage. When BcHTT4 is overexpressed in Arabidopsis, branching will decrease. In further study, we found that BcHTT4 interacts with immunophilin BcFKBP13 in vivo and in vitro through yeast two-hybrid analysis and bimolecular fluorescence complementation (BiFC) assays. Moreover, quantitative real-time PCR analysis showed that when the expression of BcHTT4 was silenced in ‘Suzhouqing’, the expression of BcFKBP13 also decreased significantly. Our findings reveal that BcHTT4 is involved in the branching mechanism and interacts with immunophilin BcFKBP13 in NHCC.

Targeted Editing of SlMAPK6 Using CRISPR/Cas9 Technology to Promote the Development of Axillary Buds in Tomato Plants

The mitogen-activated protein kinase (MAPK) cascade signaling system has been relatively conserved throughout the evolution of eukaryotes and is involved in the regulation of growth and development and metabolism. In this study, dwarf tomato plants were used as the research material. First, the tissue-specific expression of SlMAPK6 was measured in wild-type plants by quantitative RT-PCR. The results showed that SlMAPK6 was highly expressed in the tissues of the stems, leaves and flowers but was expressed at low levels in the tissues of the roots, sepals and fruits. Second, SlMAPK6-knockout lines CRISPR-3 and CRISPR-7 were obtained by CRISPR-Cas9 technology and Agrobacterium-mediated transformation. Compared with wild-type, the mutant lines CRISPR-3 and CRISPR-7 showed significant phenotypic characteristics, such as increased numbers of axillary buds and true leaves, thickened stems, and longer leaflets. In addition, to explore the molecular mechanism by which MAPK regulates axillary bud growth, we also showed that SlMAPK6 positively regulates the strigolactone synthesis genes SlCCD7 and SlCCD8 and the gibberellin (GA) synthesis genes GA20ox3 and GA3ox1 and negatively regulates the axillary bud development-related genes Ls, BL and BRC1b/TCP8 and the GA synthesis inhibitory gene GAI. Therefore, SlMAPK6 appears to regulate the synthesis of strigolactone and GA to induce the growth and development of tomato axillary buds.

Blocking plasmodesmata in specific phloem cell types reduces axillary bud growth in Arabidopsis thaliana

The plasticity of above ground plant architecture depends on the regulated re-activation and growth of axillary meristems laid down in the axils of leaves along the stem, which often arrest as dormant buds. Plasmodesmata connecting plant cells might control the movement of regulators involved in this developmental switch. Constructs capable of occluding these structures were employed in phloem cell types, because of the importance of phloem in local and systemic trafficking. We show that over-accumulation of callose within companion cells of the Arabidopsis inflorescence reduces the growth rates of activated buds, but does not affect bud activation. Growth rate reductions were not dependent on the phloem-mobile strigolactone receptor, which regulates bud activation. Furthermore, there was no correlation with early bud sugar profiles, which can also affect bud activity and depend on phloem-mediated delivery. It is therefore possible that an as yet unknown mobile signal is involved in modulating branch growth rate.