Single-Molecule Fluorescence Methods to Study Plant Hormone Signal Transduction Pathways

Song, Song; Chang, Jian; Ma, Chongjun; Tan, Yan‐Wen

doi:10.3389/fpls.2017.01888

Cited by 7 publications

(9 citation statements)

References 72 publications

(81 reference statements)

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“…This result supports the synthesis of secondary metabolites such as flavonoids and alkaloids during the reproduction process and is consistent with the higher piperine content reported for KC compared to SA and S1 [9]. Interestingly, as plant hormones have been previously shown to be necessary to signal the flower formation, fruit set, and fruit maturation in black pepper [24,35], the high numbers of genes in the category of signal transduction and cell-cell signalling in S1 are consistent with the importance of expression of small molecule like plant hormones during fruit development [36]. Therefore, it would be useful to further study these groups of genes, related to growth and to secondary metabolism for future crop improvement.…”

supporting

confidence: 85%

Transcriptional Sequencing and Gene Expression Analysis of Various Genes in Fruit Development of Three Different Black Pepper (Piper nigrumL.) Varieties

Khew

Harikrishna

Wee

et al. 2020

International Journal of Genomics

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Black pepper (Piper nigrum) is a vital spice crop with uses ranging from culinary to pharmacological applications. However, limited genetic information has constrained the understanding of the molecular regulation of flower and fruit development in black pepper. In this study, a comparison among three different black pepper varieties, Semengok Aman (SA), Kuching (KC), and Semengok 1 (S1), with varying fruit characteristics was used to provide insight on the genetic regulation of flower and fruit development. Next-generation sequencing (NGS) technology was used to determine the flower and fruit transcriptomes by sequencing on an Illumina HiSeq 2500 platform followed by de novo assembly using SOAPdenovo-Trans. The high-quality assembly of 66,906 of unigenes included 64.4% of gene sequences (43,115) with similarity to one or more protein sequences from the GenBank database. Annotation with Blast2Go assigned 37,377 genes to one or more Gene Ontology terms. Of these genes, 5,874 genes were further associated with the biological pathways recorded in the KEGG database. Comparison of flower and fruit transcriptome data from the three different black pepper varieties revealed a large number of DEGs between flower and fruit of the SA variety. Gene Ontology (GO) enrichment analysis further supports functions of DEGs between flower and fruit in the categories of carbohydrate metabolic processes, embryo development, and DNA metabolic processes while the DEGs in fruit relate to biosynthetic process, secondary metabolic process, and catabolic process. The enrichment of DEGs in KEGG pathways was also investigated, and a large number of genes were found to belong to the nucleotide metabolism and carbohydrate metabolism categories. Gene expression profiling of flower formation-related genes reveals that other than regulating the flowering in black pepper, the flowering genes might also be implicated in the fruit development process. Transcriptional analysis of sugar transporter and carbohydrate metabolism genes in different fruit varieties suggested that the carbohydrate metabolism in black pepper fruit is developmentally regulated, and some genes might serve as potential genes for future crop quality improvement. Study on the piperine-related gene expression analysis suggested that lysine-derived products might present in all stages of fruit development, but the transportation was only active at the early stage of fruit development. These results indicate several candidate genes related to the development of flower and fruit in black pepper and provide a resource for future functional analysis and potentially for future crop improvement.

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supporting

confidence: 85%

Transcriptional Sequencing and Gene Expression Analysis of Various Genes in Fruit Development of Three Different Black Pepper (Piper nigrumL.) Varieties

Khew

Harikrishna

Wee

et al. 2020

International Journal of Genomics

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“…These results imply that oxygen maybe required for BIN2 activation. We tested the binding ratios of BES1–BIN2 in a buffer with a constant BES1 concentration but different concentrations of O 2 ([O 2 ]) using an air‐tight flow cell system (Song et al ., ). The spots in each view were counted and displayed in a box chart (Fig.…”

Section: Resultsmentioning

confidence: 73%

“…In a supersaturating [O 2 ] (9.85 ppm) condition, O 2 is released from the buffer and forms bubbles in the flow cell chamber during observation. The fluorescence of eGFP/A206K was not affected by the [O 2 ] in the buffer (Song et al ., ); therefore, we concluded that the binding ratio of BIN2 with BES1 increased as [O 2 ] increased until oxygen saturation ([O 2 ] = 7.88 ppm) at room temperature (22°C) and 101 kPa (Fig. c,d).…”

Section: Resultsmentioning

confidence: 97%

“…This association is stable enough for the two proteins to remain tightly bound until BIN2 phosphorylates BES1 at the rate constant 2.3 ± 1.4 s −1 . After phosphorylation, pBES1 dissociates with BIN2 and is captured by dimeric 14‐3‐3κ (Song et al ., ). pBES1 either cannot bind with the DNA or is transferred out of the nucleus; thus, BR signal transduction in the nucleus is halted.…”

Section: Discussionmentioning

confidence: 99%

“…In this study, we first used a single‐molecule assay (Song et al ., ; Fig. ) to study the interaction of BES1 with BIN2 of Arabidopsis thaliana .…”

Section: Introductionmentioning

confidence: 97%

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Reactive oxygen species‐mediated BIN2 activity revealed by single‐molecule analysis

et al. 2019

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Summary Much evidence has shown that reactive oxygen species (ROS) regulate several plant hormone signaling cascades, but little is known about the real‐time kinetics and the underlying molecular mechanisms of the target proteins in the brassinosteroid (BR) signaling pathway. In this study, we used single‐molecule techniques to investigate the true signaling timescales of the major BR signaling components BRI1‐EMS‐SUPPRESSOR 1 (BES1) and BRASSINOSTEROID INSENSITIVE 2 (BIN2) of Arabidopsis thaliana. The rate constants of BIN2 associating with ATP and phosphorylating BES1 were determined to be 0.7 ± 0.4 mM−1 s−1 and 2.3 ± 1.4 s−1, respectively. Interestingly, we found that the interaction of BIN2 and BES1 was oxygen‐dependent, and oxygen can directly modify BIN2. The activity of BIN2 was switched on via modification of specific cysteine (Cys) residues, including C59, C95, C99 and C162. The mutation of these Cys residues inhibited the BR signaling outputs. These findings demonstrate the power of using single‐molecule techniques to study the dynamic interactions of signaling components, which is difficult to be discovered by conventional physiological and biochemical methods.

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Single-Molecule Fluorescence Imaging Reveals Coassembly of CTPS and P5CS

Chang,

Yuan,

Gao

et al. 2024

J. Phys. Chem. B

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The cellular compartmentation induced by self-assembly of natural proteins has recently attracted widespread attention due to its structural−functional significance. Among them, as a highly conserved metabolic enzyme and one of the potential targets for cancers and parasitic diseases in drug development, CTP synthase (CTPS) has also been reported to self-assemble into filamentous structures termed cytoophidia. To elucidate the dynamical mechanism of cytoophidium filamentation, we utilize singlemolecule fluorescence imaging to observe the real-time self-assembly dynamics of CTPS and the coordinated assembly between CTPS and its interaction partner, Δ 1 -pyrroline-5-carboxylate synthase (P5CS). Significant differences exist in the direction of growth and extension when the two proteins selfassemble. The oligomer state distribution analysis of the CTPS minimum structural subunit under different conditions and the stoichiometry statistics of binding CTPS and P5CS by single-molecule fluorescence photobleach counting further confirm that the CTPS cytoophidia are mainly stacked with tetramers. CTPS can act as the nucleation core to induce the subsequent growth of the P5CS filaments. Our work not only provide evidence from the molecular level for the self-assembly and coordinated assembly (coassembly) of CTPS with its interaction partner P5CS in vitro but also offer new experimental perspectives for the dynamics research of coordinated regulation between other protein polymers.

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Single-Molecule Fluorescence Methods to Study Plant Hormone Signal Transduction Pathways

Cited by 7 publications

References 72 publications

Transcriptional Sequencing and Gene Expression Analysis of Various Genes in Fruit Development of Three Different Black Pepper (Piper nigrumL.) Varieties

Transcriptional Sequencing and Gene Expression Analysis of Various Genes in Fruit Development of Three Different Black Pepper (Piper nigrumL.) Varieties

Reactive oxygen species‐mediated BIN2 activity revealed by single‐molecule analysis

Single-Molecule Fluorescence Imaging Reveals Coassembly of CTPS and P5CS

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