Generation and Characterization of a Specific Polyclonal Antibody against Arabidopsis thaliana Phytochrome-Interacting Factor 3

Choi, Da-Min; Cho, Jae-Yong; Kim, Woe‐Yeon; Han, Yun‐Jeong; Kim, Jeong-Il

doi:10.1007/s12374-021-09302-9

Cited by 3 publications

(6 citation statements)

References 43 publications

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“…Considering that the K411R and T418V mutants exhibit a higher kinase activity on PIF3 (Figure 1A), we hypothesized that these mutants phosphorylate PIF3 more efficiently, thus inducing a subsequent rapid degradation. To test this hypothesis, we first investigated PIF3 degradation under R and FR light conditions by western blotting with a recently developed PIF3specific antibody (Choi et al, 2021). As observed in AsA-OX, PIF3 was degraded rapidly in the K411R and T418V plants under R light (Figure 4A).…”

Section: Fr Light-induced Degradation and Phosphorylation Of Pif3 In Transgenic Plantsmentioning

confidence: 98%

“…After collecting the seedling samples, proteins were extracted immediately using a buffer (100 mM Tris-HCl, pH 7.8, 4 M urea, and protease inhibitors) and each extracted protein (60 µg) was separated on 6% SDS-polyacrylamide gel. The PIF3 protein levels in plants were then determined by western blotting using 1:3,000 PIF3-specific polyclonal antibody (Choi et al, 2021) or 1:500 GFP-specific monoclonal antibody (sc-9996; Santa Cruz Biotechnology).…”

Section: In Vivo Phosphorylation and Degradation Of Pif3 And Pif1mentioning

confidence: 99%

“…•s −1 ) for the time indicated (5, 10, and 30 min). The PIF1 levels were analyzed by western blotting using PIF1-specific polyclonal antibody (1:1,000) produced by the same method used for the PIF3-specific antibody (Choi et al, 2021).…”

Section: In Vivo Phosphorylation and Degradation Of Pif3 And Pif1mentioning

confidence: 99%

“…To analyze phosphorylation and degradation of PIF1 in vivo, we produced a PIF1-specific antibody using the method used for the PIF3-specific antibody (Choi et al, 2021). The purified α-PIF1 antibody reacted specifically with only full-length and 71 aa-deleted PIF1 proteins among seven PIFs in Arabidopsis (Supplementary Figure 9A).…”

Section: Regulation Of Pif1 By the Asphya Mutants With Increased Kinase Activitymentioning

confidence: 99%

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Protein Kinase Activity of Phytochrome A Positively Correlates With Photoresponses in Arabidopsis

et al. 2021

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Plant phytochromes are known as autophosphorylating serine/threonine protein kinases. However, the functional importance of their kinase activity is not fully elucidated. Previously, the kinase activity is shown to be necessary for the function of Avena sativa phytochrome A (AsphyA) using transgenic plants with mutants displaying reduced kinase activity, such as K411L and T418D. In this study, we isolated and analyzed two AsphyA mutants, K411R and T418V, that showed increased kinase activity. Transgenic phyA-201 plants with these mutants showed hypersensitive responses to far-red (FR) light, such as shorter hypocotyls and more expanded cotyledons than those of control plant (i.e., transgenic phyA-201 plant with wild-type AsphyA). Contrary to the mutants with reduced kinase activity, these mutants accelerated FR-induced phosphorylation and subsequent degradation of phytochrome-interacting factor 3 (PIF3) in Arabidopsis. Moreover, elongated hypocotyl 5 (HY5), a critical positive regulator of photoresponses in plants, accumulated in higher amounts in the transgenic plants under FR light than in the control plant. In addition, PIF1 degradation was accelerated in the transgenic plants. Consequently, the transgenic plants exhibit higher germination frequencies than the control plant. Collectively, our results demonstrate that the AsphyA mutants with increased kinase activity are hyperactive in plants, supporting a positive relationship between the kinase activity of phytochromes and photoresponses in plants.

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Section: Fr Light-induced Degradation and Phosphorylation Of Pif3 In Transgenic Plantsmentioning

confidence: 98%

Section: In Vivo Phosphorylation and Degradation Of Pif3 And Pif1mentioning

confidence: 99%

Section: In Vivo Phosphorylation and Degradation Of Pif3 And Pif1mentioning

confidence: 99%

Section: Regulation Of Pif1 By the Asphya Mutants With Increased Kinase Activitymentioning

confidence: 99%

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Protein Kinase Activity of Phytochrome A Positively Correlates With Photoresponses in Arabidopsis

et al. 2021

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“…In down-regulated genes present in two cultivars, only PIF3 was involved in both plant circadian subtitles and plant hormone signal transduction. Recent studies have shown that PIF3 is involved in regulating plant hormone synthesis and response to low temperature stress [46,47]. It is necessary to further study the role of this gene in the cold tolerance mechanism of Brassica oleracea L..…”

Section: Circadian Rhythm-plantmentioning

confidence: 99%

Comparative Transcriptome Analysis Between Cold-Tolerant and Sensitive Brassica Oleracea L. in Response to Low Temperature Stress

Jiang

Xia

Dai

et al. 2021

Preprint

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Background: Brassica oleracea L. occupies an important position in the annual production of vegetables. But during winter Brassica oleracea L. often suffers from low temperatures and even sub-zero temperatures. Through transcriptome analysis and identification, the pathways involved in cold tolerance of Brassica oleracea L. were analyzed and candidate genes related to cold tolerance of Brassica oleracea L. were identified.Results: Under low temperature stress, a large number of significantly different genes were found in Zhonggan1229 (ZG, low temperature tolerance) and Yingchun (YN, low temperature sensitive). There were 3902 significantly up-regulated genes and 5309 significantly down-regulated genes in ZG, and 4253 significantly up-regulated genes and 5938 significantly down-regulated genes in YN. Among them, 1844 different genes are the specific different genes in ZG and 6089 genes are the common different genes to response the low temperature stress. By annotating the specific different genes in ZG, 26 of the top 30 enriched GO terms belonged to biological processes, 4 terms belonged to molecular functions. By annotating the common different genes, 23 GO terms belonged to biological processes, 1 GO term belonged to molecular functions, and 6 GO terms belonged to cellular components. Circadian rhythms of plants and Plant hormone signal transduction were not only significantly enriched in the two analyzed genes, but also the effects of low temperature stress were most significant. Among the unique different genes in ZG, 154 genes were annotated into transcription factor families, and 79 genes were up-regulated and 75 genes were down-regulated, the encoding of MYB-related proteins was the largest group. Among the different genes shared by the two varieties, 516 genes were annotated into corresponding transcription factor families, 211 genes were up-regulated and 296 genes were down-regulated, however, there were 4 genes that were up-regulated in ZG but down-regulated in YN, and 5 genes that were down-regulated in ZG but up-regulated in YN, the largest group was the protein encoding ERF.Conclusions: The results identified important genes, pathways, and transcription factors that respond to low temperature stress, provided cold tolerance gene resources for the subsequent cold tolerance breeding research of Brassica oleracea L..

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Suppression of Phytochrome-Interacting Factors Enhances Photoresponses of Seedlings and Delays Flowering With Increased Plant Height in Brachypodium distachyon

et al. 2021

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Phytochromes are red and far-red photoreceptors that regulate plant growth and development under ambient light conditions. During phytochrome-mediated photomorphogenesis, phytochrome-interacting factors (PIFs) are the most important signaling partners that regulate the expression of light-responsive genes. However, the function of PIFs in monocots has not been studied well. In this study, using RNA interference (RNAi), we investigated the functions of BdPIL1 and BdPIL3, two PIF-like genes identified in Brachypodium distachyon, which are closely related to Arabidopsis PIF1 and PIF3. The expression of their genes is light-inducible, and both BdPIL1 and BdPIL3 proteins interact with phytochromes in an active form-specific manner. Transgenic Brachypodium seedlings with the RNAi constructs of BdPIL1 and BdPIL3 showed decreased coleoptile lengths and increased leaf growth when exposed to both red and far-red light. In addition, the transgenic plants were taller with elongated internodes than wild-type Bd21-3 plant, exhibiting late flowering. Moreover, RNA-seq analysis revealed downregulation of many genes in the transgenic plants, especially those related to the regulation of cell number, floral induction, and chlorophyll biosynthesis, which were consistent with the phenotypes of increased plant height, delayed flowering, and pale green leaves. Furthermore, we demonstrated the DNA-binding ability of BdPIL1 and BdPIL3 to the putative target promoters and that the DNA-binding was inhibited in the presence of phytochromes. Therefore, this study determines a molecular mechanism underlying phytochrome-mediated PIF regulation in Brachypodium, i.e., sequestration, and also elucidates the functions of BdPIL1 and BdPIL3 in the growth and development of the monocot plant.

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Generation and Characterization of a Specific Polyclonal Antibody against Arabidopsis thaliana Phytochrome-Interacting Factor 3

Cited by 3 publications

References 43 publications

Protein Kinase Activity of Phytochrome A Positively Correlates With Photoresponses in Arabidopsis

Protein Kinase Activity of Phytochrome A Positively Correlates With Photoresponses in Arabidopsis

Comparative Transcriptome Analysis Between Cold-Tolerant and Sensitive Brassica Oleracea L. in Response to Low Temperature Stress

Suppression of Phytochrome-Interacting Factors Enhances Photoresponses of Seedlings and Delays Flowering With Increased Plant Height in Brachypodium distachyon

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