Three Protein Kinases from the Etiolated Oat Seedlings Phosphorylate Oat Phytochrome A In Vitro

Park, Young-Il,Kim, Jae-Hun,Lee, Jae-Deok,Kim, Yong-Woo,Kim, In-Soo Korean Society for Biochemistry and Molecular Biol 1998 Journal of biochemistry and molecular biology Vol.31 No.3

Phosphorylation of phytochrome may play important functional roles to control plant photomorphogenesis. Many attempts have failed to identify the protein kinase that phosphorylates phytochrome in vivo. It has been reported that a polycation-stimulated protein kinase activity was associated with the purified phytochrome. However, it is not known if the kinase activity is an intrinsic property of phytochrome or whether it comes from a contaminant of the purified phytochrome. In the present study, three protein kinases that phosphorylate phytochrome have been identified from etiolated oat seedlings. A polycationstimulated protein kinase that had very similar enzymatic properties with that associated with the purified phytochrome was identified in the cytosolic extract. It phosphorylated several contaminant proteins in the kinase preparation as well as phytochrome and had a broad substrate specificity. A CK II-type protein kinase phosphorylated phytochrome and the exogenously added casein. It is likely that this kinase may not be a feasible candidate for the kinase phosphorylating phytochrome in vivo since the content of the kinase seemed to well exceed the content of phytochrome in the etiolated oat seedlings. Another protein kinase that had unique enzymatic properties phosphorylated phytochrome very specifically and seemed to be present in a small quantity in the etiohlted seedlings. It is expected that one of three kinases may be responsible for the phytochrome phosphorylation in vivo.

Three Protein Kinases from the Etiolated Oat Seedlings Phosphorylate Oat Phytochrome A in Vitro

Kim, In-Soo,Park, Young-Il,Kim, Jae-Hun,Lee, Jae-Deok,Kim, Yong-Woo The Korea Science and Technology Center 1998 BMB Reports Vol.31 No.3

Phosphorylation of phytochrome may play important functional roles to control plant photomorphogenesis. Many attempts have failed to identify the protein kinase that phosphorylates phytochrome in vivo. It has been reported that a polycation-stimulated protein kinase activity was associated with the purified phytochrome. However, it is not known if the kinase activity is an intrinsic property of phytochrome or whether it comes from a contaminant of the purified phytochrome. In the present study, three protein kinases that phosphorylate phytochrome have been identified from etiolated oat seedlings. A polycation-stimulated protein kinase that had very similar enzymatic properties with that associated with the purified phytochrome was identified in the cytosolic extract. It phosphorylated several contaminant proteins in the kinase preparation as well as phytochrome and had a broad substrate specificity. A CK II-type protein kinase phosphorylated phytochrome and the exogenously added casein. It is likely that this kinase may not be a feasible candidate for the kinase phosphorylating phytochrome in vivo since the content of the kinase seemed to well exceed the content of phytochrome in the etiolated oat seedlings. Another protein kinase that had unique enzymatic properties phosphorylated phytochrome very specifically and seemed to be present in a small quantity in the etiolated seedlings. It is expected that one of three kinases may be reasonable for the phytochrome phosphorylation in vivo.

Decoding of Light Signals by Plant Phytochromes and Their Interacting Proteins

Bae, Gabyong,Choi, Giltsu Annual Reviews 2008 Annual review of plant biology Vol.59 No.-

<P>Phytochromes are red/far-red light photoreceptors that convert the information contained in external light into biological signals. The decoding process starts with the perception of red light, which occurs through photoisomerization of a chromophore located within the phytochrome, leading to structural changes that include the disruption of intramolecular interactions between the N- and C-terminal domains of the phytochrome. This disruption exposes surfaces required for interactions with other proteins. In contrast, the perception of far-red light reverses the photoisomerization, restores the intramolecular interaction, and closes the interacting surfaces. Light information represented by the concentration of opened interacting surfaces is converted into biological signals through the modulating activity of interacting proteins. This review summarizes plant phytochromes, phytochrome-interacting proteins, and signal transmission from phytochromes to their interacting proteins.</P>

Phytochrome B inhibits binding of phytochrome‐interacting factors to their target promoters

Park, Eunae,Park, Jeongmoo,Kim, Junghyun,Nagatani, Akira,Lagarias, J. Clark,Choi, Giltsu Blackwell Publishing Ltd 2012 The Plant journal Vol.72 No.4

<P><B>Summary</B></P><P>Phytochromes are red and far‐red light receptors in plants that mediate critical responses to light throughout the lifecycle. They achieve this in part by targeting negatively acting bHLH transcription factors called phytochrome‐interacting factors (PIFs) for degradation within the nucleus. However, it is not known whether protein degradation is the primary mechanism by which phytochromes inhibit these repressors of photomorphogenesis. Here, we use chromatin immunoprecipitation to show that phyB inhibits the regulatory activity of PIF1 and PIF3 by releasing them from their DNA targets. The N‐terminal fragment of phyB (NG‐GUS‐NLS; NGB) also inhibits binding of PIF3 to its target promoters. However, unlike full‐length phyB, NGB does not promote PIF3 degradation, establishing the activity of NGB reflects its ability to inhibit PIF binding to DNA. We further show that Pfr forms of both full‐length phyB and NGB inhibit DNA binding of PIF1 and PIF3 <I>in vitro</I>. Taken together, our results indicate that phyB inhibition of PIF function involves two separate processes: sequestration and protein degradation.</P>

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

최다민,조재용,김우연,한윤정,김정일 한국식물학회 2021 Journal of Plant Biology Vol.64 No.2

Phytochrome-interacting factors (PIFs) are a basic helix-loop-helix family of transcriptional regulators that maintain skotomorphogenesis and suppress photomorphogenesis. PIFs are regulated by plant photoreceptors, especially phytochromes. In general, PIFs physically interact with phytochromes, and this interaction induces PIF’s phosphorylation and subsequent degradation, contributing to the initiation of photomorphogenic development. Among the eight members of PIF (PIF1 to PIF8) reported in Arabidopsis thaliana, PIF3 is the first discovered member and plays central roles in de-etiolation and chlorophyll biosynthesis. More recently, PIF3 has been also reported to regulate hormone signaling and cold tolerance in plants. Although PIF3 protein shows dynamic behaviors in plants, its study has been limited due to the lack of an authentic PIF3 antibody. In this study, we produced polyclonal antibodies using inclusion bodies and characterized the PIF3 antibody based on specificity and sensitivity. In addition, we investigated PIF3 phosphorylation and degradation during phytochrome-mediated light signaling in plants. Furthermore, we successfully performed in vitro protein–protein interaction and co-immunoprecipitation assays between phytochrome B (phyB) and PIF3 using the antibody. Therefore, we obtained an authentic PIF3 antibody that could be used as a valuable tool to study the multi-faceted functions of PIF3.

Identification of phytochrome-interacting protein candidates in Arabidopsis thaliana by co-immunoprecipitation coupled with MALDI-TOF MS

Phee, Bong-Kwan,Shin, Dong Ho,Cho, Jin-Hwan,Kim, Seong-Hee,Kim, Jeong-Il,Lee, Youn-Hyung,Jeon, Jong-Seong,Bhoo, Seong Hee,Hahn, Tae-Ryong WILEY-VCH 2006 Proteomics Vol. No.

<P>Phytochrome-interacting proteins have been extensively studied to elucidate light-signaling pathway in plants. However, most of these proteins have been identified by yeast two-hybrid screening using the C-terminal domain of phytochromes. We used co-immunoprecipitation followed by proteomic analysis in plant cell extracts in an attempt to screen for proteins interacting either directly or indirectly with native holophytochromes including the N-terminal domain as well as C-terminal domain. A total of 16 protein candidates were identified, and were selected from 2-DE experiments. Using MALDI-TOF MS analysis, 7 of these candidates were predicted to be putative phytochrome A-interacting proteins and the remaining ones to be phytochrome B-interacting proteins. Among these putative interacting proteins, protein phosphatase type 2C (PP2C) and a 66-kDa protein were strong candidates as novel phytochrome-interacting proteins, as knockout mutants for the genes encoding these two proteins had impaired light-signaling functions. A transgenic knockout Arabidopsis study showed that a 66-kDa protein candidate regulates hypocotyl elongation in a light-specific manner, and altered cotyledon development under white light during early developmental stages. The PP2C knockout plants also displayed light-specific changes in hypocotyl elongation. These results suggest that co-immunoprecipitation, followed by proteomic analysis, is a useful method for identifying novel interacting proteins and determining real protein-protein interactions in the cell.</P>

Regulation of Ethylene Biosynthesis in Phytochrome Mutants of the Arabidopsis Root

Ji Hye Park(박지혜),Soon Young Kim(김순영) 한국생명과학회 2012 생명과학회지 Vol.22 No.4

식물생장과 발달에 중요한 역할을 하는 phytochrome이 ethylene 생합성에 미치는 영향을 조사하기 위하여 여러 빛 조건에서 키운 phyA, phyB, phyAB에서 ethylene 생합성과 생합성에 관여하는 enzyme activity를 측정하였다. White light에서 키웠을 때 모든 mutant에서 ethylene 생합성이 감소되었다. 특히 double mutant에서는 wild type과 비교하여 37%가 감소하였다. Dark에서 키웠을 때에는 wild type만 감소하였고, mutant에서는 감소효과가 나타나지 않았다. Red light에서 키웠을 때 double mutant에서 급격한 감소가 일어났다. Far-red light 에서 키웠을 때는 phyB만 감소가 일어나지 않았다. Ethylene 생합성에 관여하는 enzyme인 ACO 활성 패턴과는 달리ACS 활성 패턴은 ethylene 생성 패턴과 유사하게 나타났다. 이 결과를 바탕으로 ethylene 생합성에는 phytochromeA와 B 모두 중요한 작용을 하며 특히 Pr 형태의 phytochrome이 ethylene 생성량을 조절한다는 것을 제시한다. 또한 phytochrome은 ethylene 생합성 단계에서 AdoMet가 ACC로 전환되는 단계에서 조절하는 것을 제시한다. In order to investigate the effect of phytochromes on the regulation of ethylene biosynthesis, we measured the ethylene production and the activities of enzymes involved in ethylene biosynthesis using phytochrome mutants such as phyA, phyB, and phyAB of Arabidopsis. The ethylene production was decreased in mutants grown in white light. In particular, double mutants showed a 37% decrease compared to the wild type in ethylene production. When Arabidopsis roots were grown in the dark, mutants did not show a decrease in ethylene production; however, production was significantly decreased in the double mutant grown in red light. Only phyB did not show the decrease in the ethylene production in far-red light. Unlike the ACO activities, the ACS activities of mutants showed the same pattern as the ethylene production under several light conditions. The results of ACS activities confirmed the expression of the ACS gene by RT-PCR analysis. The decrease of ethylene production in mutants was due to the lower activity of ACC synthase, which converts the S-adenosyl-L-methionine (AdoMet) to 1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene. These results suggested that both phytochrome A and B play an important role in the regulation of ethylene biosynthesis in Arabidopsis roots in the conversion step of AdoMet to ACC, which is regulated by ACS.

Phytochrome-Interacting Factors Have Both Shared and Distinct Biological Roles

정진길,최길주 한국분자세포생물학회 2013 Molecules and cells Vol.35 No.5

Phytochromes are plant photoreceptors that perceive red and far-red light. Upon the perception of light in Arabidopsis, light-activated phytochromes enter the nucleus and act on a set of interacting proteins, modulating their activities and thereby altering the expression levels of ~10% of the organism’s entire gene complement. Phytochrome-interacting factors (PIFs) belonging to Arabidopsis basic helix-loop-helix (bHLH) subgroup 15 are key interacting proteins that play negative roles in light responses. Their activities are post-translationally countered by light-acti-vated phytochromes, which promote the degradation of PIFs and directly or indirectly inhibit their binding to DNA. The PIFs share a high degree of similarity, but examinations of pif single and multiple mutants have indicated that they have shared and distinct functions in various devel-opmental and physiological processes. These are believed to stem from differences in both intrinsic protein properties and their gene expression patterns. In an effort to clarify the basis of these shared and distinct functions, we compared recently published genome-wide ChIP data, developmental gene expression maps, and responses to various stimuli for the various PIFs. Based on our observations, we propose that the biological roles of PIFs stem from their shared and distinct DNA binding targets and specific gene expression patterns.

Light activates the degradation of PIL5 protein to promote seed germination through gibberellin in Arabidopsis

Oh, Eunkyoo,Yamaguchi, Shinjiro,Kamiya, Yuji,Bae, Gabyong,Chung, Won-Il,Choi, Giltsu Blackwell Publishing Ltd 2006 The Plant journal Vol.47 No.1

<P>Summary</P><P>Angiosperm seeds integrate various environmental signals, such as water availability and light conditions, to make a proper decision to germinate. Once the optimal conditions are sensed, gibberellin (GA) is synthesized, triggering germination. Among environmental signals, light conditions are perceived by phytochromes. However, it is not well understood how phytochromes regulate GA biosynthesis. Here we investigated whether phytochromes regulate GA biosynthesis through PIL5, a phytochrome-interacting bHLH protein, in Arabidopsis. We found that <I>pil5</I> seed germination was inhibited by paclobutrazol, the <I>ga1</I> mutation was epistatic to the <I>pil5</I> mutation, and the inhibitory effect of <I>PIL5</I> overexpression on seed germination could be rescued by exogenous GA, collectively indicating that <I>PIL5</I> regulates seed germination negatively through GA. Expression analysis revealed that PIL5 repressed the expression of GA biosynthetic genes (<I>GA3ox1</I> and <I>GA3ox2</I>), and activated the expression of a GA catabolic gene (<I>GA2ox</I>) in both PHYA- and PHYB-dependent germination assays. Consistent with these gene-expression patterns, the amount of bioactive GA was higher in the <I>pil5</I> mutant and lower in the <I>PIL5</I> overexpression line. Lastly, we showed that red and far-red light signals trigger PIL5 protein degradation through the 26S proteasome, thus releasing the inhibition of bioactive GA biosynthesis by PIL5. Taken together, our data indicate that phytochromes promote seed germination by degrading PIL5, which leads to increased GA biosynthesis and decreased GA degradation.</P>

High Ambient Temperature Accelerates Leaf Senescence via PHYTOCHROME-INTERACTING FACTOR 4 and 5 in Arabidopsis

Kim, Chanhee,Kim, Sun Ji,Jeong, Jinkil,Park, Eunae,Oh, Eunkyoo,Park, Youn-Il,Lim, Pyung Ok,Choi, Giltsu Korean Society for Molecular and Cellular Biology 2020 Molecules and cells Vol.43 No.7

Leaf senescence is a developmental process by which a plant actively remobilizes nutrients from aged and photosynthetically inefficient leaves to young growing ones by disassembling organelles and degrading macromolecules. Senescence is accelerated by age and environmental stresses such as prolonged darkness. Phytochrome B (phyB) inhibits leaf senescence by inhibiting phytochrome-interacting factor 4 (PIF4) and PIF5 in prolonged darkness. However, it remains unknown whether phyB mediates the temperature signal that regulates leaf senescence. We found the light-activated form of phyB (Pfr) remains active at least four days after a transfer to darkness at 20℃ but is inactivated more rapidly at 28℃. This faster inactivation of Pfr further increases PIF4 protein levels at the higher ambient temperature. In addition, PIF4 mRNA levels rise faster after the transfer to darkness at high ambient temperature via a mechanism that depends on ELF3 but not phyB. Increased PIF4 protein then binds to the ORE1 promoter and activates its expression together with ABA and ethylene signaling, accelerating leaf senescence at high ambient temperature. Our results support a role for the phy-PIF signaling module in integrating not only light signaling but also temperature signaling in the regulation of leaf senescence.