Transcriptome Analysis of the Cytokinin Response in Medicago truncatula

Zhixiang Zhou,Haicong Liu,Cuina Ma,Yuehui Chao,Liebao Han 한국식물학회 2020 Journal of Plant Biology Vol.63 No.3

As an important legume plant, Medicago truncatula is a preeminent model for the study of the processes of nitrogen fxation, symbiosis, and legume genomics. The regulatory mechanism of the cytokinin response has been studied in many plants, such as rice, Arabidopsis, tomato, and barley, but information about regulatory pathways and genes involved in the cytokinin response in Medicago truncatula is notably limited. In this study, to better understand the cytokinin response in Medicago truncatula, transcriptome analysis of seedlings grown with 6-benzylaminopurine or lovastatin was performed using RNASeq. In this study, 3627 and 3093 transcripts were diferentially expressed in cytokinin-induced/control (Cyto/Ctrl) and cytokinin-inhibited/control (Inh/Ctrl) groups, respectively, and diferentially expressed genes were tested by quantitative real-time PCR (qRT-PCR). Analysis of the cytokinin response in Medicago truncatula revealed a large number of transcripts involved in signal transduction, metabolic process, secondary metabolite biosynthesis, transport and catabolism, growth and development, defense mechanisms, and transcription. There were 43 transcription factor families, including 1845 transcription factor (TF) genes with 2147 TF transcripts, as detected by RNA-Seq. Additionally, 216 TF genes with 220 transcripts were diferentially expressed in Cyto/Ctrl, and 185 TF genes with 189 transcripts were in Inh/Ctrl. A total of 289 and 260 DETs involved in biosynthesis, metabolism, and transduction of plant hormones were identifed in the Cyto/Ctrl and Inh/ Ctrl groups, respectively. Furthermore, 15 transcripts, including A-ARR, IPT, and CKX, were demonstrated to play roles in cytokinin regulation or signal transduction. These fndings were associated with the cytokinin response in other plants. The resulting data provide the frst cytokinin transcriptome analysis in Medicago truncatula. Further analysis and identifcation of cytokinin-regulated transcripts or signal transduction transcripts may help to elucidate the regulatory mechanisms governing the cytokinin response in Medicago truncatula.

Phosphorylation of A-Type ARR to function as negative regulator of cytokinin signal transduction.

Landes Bioscience 2008 Plant signaling & behavior Vol.3 No.5

<P>The plant hormone cytokinins regulate diverse aspects of plant growth and development. In Arabidopsis, a multi-step TCS system similar to bacterial and yeast TCS is used for cytokinin signaling. In a TCS system, a His sensor kinase perceives the signal by auto-phosphorylating on a His residue in response to an output signal, and the phosphate group is transferred to a conserved Asp residue in the receiver domain of the response regulator. The response regulator then modulates downstream signaling. Cytokinin multi-step TCS system utilizes an additional component, histidine-containing phosphotransfer domain protein (HPT) to transfer the phosphate group from a sensor kinase to a response regulator in the nucleus. The typical response regulators are classified into either type A or B. The type-B ARRs are transcription activators that act as positive regulators of cytokinin signaling, whereas most of the type-A ARRs are negative regulators of cytokinin signaling. Histidyl-aspartidyl phosphorelays are presumed to be essential for this cytokinin signal transduction in plants. Our studies have shown that ARR7, an A-type response regulator, negatively regulates cytokinin signaling in various aspects by acting as a transcriptional repressor and that the phosphorylation of ARR7 is required for these ARR7-regulated cytokinin-responses. Here I propose potential mechanisms by which the phosphorylation of ARRs is involved in regulating cytokinin- mediated gene expression, mainly based on biochemical and structural studies of bacterial response regulators. Protein-protein interaction and DNA-binding studies using the phosphorylated and the un-phosphorylated forms of the ARR proteins with their structural determination will provide molecular understanding of cytokinin-responsive gene regulation by ARRs.</P>

Cytokinins overproduction에 따른 담배형질전환체의 변화

정용윤,Chung, Yong-Yoon 배재대학교 자연과학연구소 1998 自然科學論文集 Vol.10 No.1

식물의 주요 phytohormone의 하나인 cytokinin은 식물체의 줄기와 뿌리성장 그 외에도 영양의 전달이나 노화방지, 열매숙성 등 식물의 성장과 발달에 미치는 영향은 크고 다양하다. Cytokinin 생합성에 관여하는 효소를 생산하는 것으로 알려져 있으며 토양박테리아 Agrobacterium tumefaciens에 존재하는 유전자인 isopentenyl transferase (jpt)를 이용한 많은 분자생물학적 연구가 진행되어 왔는데 그 중 하나로 이 연구에서 jpt 유전자에 의한 cytokinin의 overproduction이 식물체에 성장과 발달에 어떠한 영향을 주는지 관찰하고 그 결과가 제시할 수 있는 작물의 유전 공학적 이용가능성에 대하여 알아본다. Cytokinin is one of major growth regulators in plants. In this study, the gene isopentenyl transferase (jpt) which encodes a key enzyme involved in the biosynthesis of the growth regulator cytokinin isolated from Agrobacterium tumefaciens was introduced ito tobacco plant via Agrobacterium-mediated transformation. The jpt gene was modulated using the proteinase inhibitor II (PI-IIK) promotor. In general, this promoterlipt gene fusion resulted in overproduction of cytokinins throughout the transgenic plants. The overproduction of cytokinin caused dramatic changes in morphology of the plant, including stem thickness and reduced root development. The studies reported in this paper were initiated to examine the consequences of overproduction of cytokinin in plant.

<i>Arabidopsis</i> ABCG14 is essential for the root-to-shoot translocation of cytokinin

Ko, Donghwi,Kang, Joohyun,Kiba, Takatoshi,Park, Jiyoung,Kojima, Mikiko,Do, Jihye,Kim, Kyung Yoon,Kwon, Mi,Endler, Anne,Song, Won-Yong,Martinoia, Enrico,Sakakibara, Hitoshi,Lee, Youngsook National Academy of Sciences 2014 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.111 No.19

<P>Cytokinins are phytohormones that induce cytokinesis and are essential for diverse developmental and physiological processes in plants. Cytokinins of the <I>trans</I>-zeatin type are mainly synthesized in root vasculature and transported to the shoot, where they regulate shoot growth. However, the mechanism of long-distance transport of cytokinin was hitherto unknown. Here, we report that the <I>Arabidopsis</I> ATP-binding cassette (ABC) transporter subfamily G14 (<I>AtABCG14</I>) is mainly expressed in roots and plays a major role in delivering cytokinins to the shoot. Loss of <I>AtABCG14</I> expression resulted in severe shoot growth retardation, which was rescued by exogenous <I>trans</I>-zeatin application. Cytokinin content was decreased in the shoots of <I>atabcg14</I> plants and increased in the roots, with consistent changes in the expression of cytokinin-responsive genes. Grafting of <I>atabcg14</I> scions onto wild-type rootstocks restored shoot growth, whereas wild-type scions grafted onto <I>atabcg14</I> rootstocks exhibited shoot growth retardation similar to that of <I>atabcg14</I>. Cytokinin concentrations in the xylem are reduced by ∼90% in the <I>atabcg14</I> mutant. These results indicate that AtABCG14 is crucial for the translocation of cytokinin to the shoot. Our results provide molecular evidence for the long-distance transport of cytokinin and show that this transport is necessary for normal shoot development.</P>

Characterization of an Arabidopsis Gene that Mediates Cytokinin Signaling in Shoot Apical Meristem Development

Chung-Mo Park,정재훈,윤주,Yeon-Hee Seo 한국분자세포생물학회 2005 Molecules and cells Vol.19 No.3

Cytokinins are adenine derivatives that regulate numerous plant growth and developmental processes,including apical and floral meristem development,stem growth, leaf senescence, apical dominance, and stress tolerance. However, not much is known about how cytokinin biosynthesis and metabolism is regulated. We identified a novel Arabidopsis gene, ALL, encoding an aldolase-like enzyme that regulates cytokinin signaling. An Arabidopsis mutant, all-1D, in which ALL is activated by the nearby insertion of the 35S enhancer, exhibited extreme dwarfism with rolled, dark-green leaves and reduced apical dominance, symptomatic of cytokinin-overproducing mutants. Consistent with this, ARR4 and ARR5, two representative primary cytokinin-responsive genes, were significantly induced in all-1D. Whereas SHOOT MERISTEMLESS (STM) and KNAT1, which regulate meristem development, were also greatly induced, expression of REV and PHV that regulate lateral organ polarity was inhibited. ALL encodes an aldolase-like enzyme that belongs to the HpcH/HpaI aldolase family in prokaryotes and is downregulated by exogenous cytokinin, possibly through a negative feedback pathway. We propose that ALL is involved in cytokinin biosynthesis or metabolism and acts as a positive regulator of cytokinin signaling during shoot apical meristem development and determination of lateral organ polarity.

Cytokinin‐facilitated proteolysis of ARABIDOPSIS RESPONSE REGULATOR 2 attenuates signaling output in two‐component circuitry

Kim, Kangmin,Ryu, Hojin,Cho, Young‐,Hyun,Scacchi, Emanuele,Sabatini, Sabrina,Hwang, Ildoo Blackwell Publishing Ltd 2012 The Plant journal Vol.69 No.6

<P><B>Summary</B></P><P>Cytokinins propagate signals via multiple phosphorelays in a mechanism similar to bacterial two‐component systems. In Arabidopsis, signal outputs are determined by the activation state of transcription factors termed type‐B Arabidopsis response regulators (ARRs); however, their regulatory mechanisms are largely unknown. In this study, we demonstrate that the proteolysis of ARR2, a type‐B ARR, modulates cytokinin signaling outputs. ARR2‐hemagglutinin (HA) is rapidly degraded by cytokinin treatment, but other type‐B ARRs, such as ARR1‐HA, ARR10‐HA, ARR12‐HA and ARR18‐HA, are not. ARR2 degradation is mediated by the <I>26S</I> proteasome pathway, and requires cytokinin‐induced phosphorylation of Asp80 residue in the receiver domain. Through mutational analysis of amino acid residues in the receiver domain, we found that substitution of Lys90 with Gly inhibits ARR2 degradation. <I>ARR2</I><SUP><I>K90G</I></SUP><I>‐HA</I> in transgenic Arabidopsis conferred enhanced cytokinin sensitivity in various developmental processes, including primary root elongation, callus induction, leaf senescence and hypocotyl growth. ARR2<SUP>K90G</SUP>‐HA increased the expression of type‐A <I>ARR</I>s, primary cytokinin‐responsive genes and indicators of signaling output in two‐component circuits. Expression of <I>ARR2</I><SUP><I>K90G</I></SUP><I>‐HA</I> from the native <I>ARR2</I> promoter in the <I>arr2‐4</I> knock‐out mutant also increased cytokinin sensitivity. In conclusion, ARR2 proteolysis is involved in the maintenance of the primary signaling output for normal developmental processes mediated by cytokinin in Arabidopsis.</P>

Overexpression of Brassica rapa NGATHA1 Gene Confers De-Etiolation Phenotype and Cytokinin Resistance on Arabidopsis thaliana

So Hyun Kwon,장수철,고재흥,송종태,김정회 한국식물학회 2011 Journal of Plant Biology Vol.54 No.2

Brassica rapa NGATHA1 (BrNGA1) encodes a B3-type transcription factor. By analyzing Arabidopsis overexpressors of BrNGA1 (BrNGA1ox), we have previously demonstrated that BrNGA1 may be involved in negative regulation of cell proliferation during lateral organ and root growth. In the present study, we have found that BrNGA1ox seedlings grown in the dark display de-etiolation phenotypes,such as short hypocotyls, open and elongated cotyledons, and developing true leaves. BrNGA1ox seedlings as well as adult plants and calli are also resistant specifically to exogenous cytokinins. These data raise the possibility that the de-etiolation phenotypes of BrNGA1ox seedlings may result from an alteration in cytokinin response. We set out to test whether the de-etiolation phenotype is due to cytokinin overproduction or constitutively activated cytokinin response. First, BrNGA1ox was crossed to the CKX2ox plant, an overexpression line of CYTOKIN OXIDASE 2, which is responsible for degradation of active cytokinins. We found,however, no difference in the de-etiolation and shoot growth phenotypes between BrNGA1ox and BrNGA1ox CKX2ox plants. Next, we measured the transcripts level of ARR5 and ARR7, frequently employed as molecular markers for cytokinin signaling and yet found no difference in their transcripts levels of the wild-type and BrNGA1ox seedlings and shoots. These data indicate that biological role of BrNGA1 involved in de-etiolation seems to be associated with neither cytokinin overproduction nor its altered signaling. Possible molecular mechanisms by which BrNGA1 may interfere with cytokinin responses and etiolation are discussed.

사이토카이닌과 에틸렌, 프로피코나졸 복합처리에 따른 고년근 인삼 생육 및 진세노사이드 함량 변화

권나영,이정우,정성민,조익현,장인복,방경환,김영창,김동휘,황현아,임유경,조현우 한국약용작물학회 2023 한국약용작물학회지 Vol.31 No.1

Background: Korean ginseng (Panax ginseng C. A. Meyer) is recognized for its medicinal properties. Because ginseng is cultivated for 4 – 6 years in one place, the growing environment can affect ginseng growth. Specifically, root growth can be altered by plant hormones, which in turn, affects crop yields. Among plant hormones, cytokinin is known to increase secondary growth by promoting cambium cell division. In addition, ethylene and propiconazole can regulate root development. In this study, the effects and interactions of cytokinin, ethylene, and propiconazole on four-year-old ginseng plants were analyzed. Methods and Results: Ginseng plants were treated with each hormone every two weeks. Cytokinin (6-benzylaminopurine, 500 µM) was applied to the soil, and 100 nM of propiconazole or 100 µM of ethylene was applied in combination with cytokinin. Root width and weight, and ginsenoside content of each plant were the highest when cytokinin and propiconazole were applied together. The highest number of buds was observed under cytokinin treatment. However, no significant differences were observed in shoot growth, except for stem width. Conclusions: The greatest effect on root development was observed when cytokinin and propiconazole were applied together, and ginsenoside content increased following hormone treatment.

사이토카이닌과 에틸렌, 프로피코나졸 복합처리에 따른 고년근 인삼 생육 및 진세노사이드 함량 변화

권나영(Na Yeong Kwon),이정우(Jung Woo Lee),정성민(Sung Min Jung),조익현(Ick Hyun Jo),장인복(In Bok Jang),방경환(Kyong Hwan Bang),김영창(Young Chang Kim),김동휘(Dong Hwi Kim),황현아(Hyeon A Hwang),임유경(Yoo Kyung Lim),조현우(Hyun Woo 한국약용작물학회 2023 한국약용작물학회지 Vol.31 No.1

Background: Korean ginseng (Panax ginseng C. A. Meyer) is recognized for its medicinal properties. Because ginseng is cultivated for 4 – 6 years in one place, the growing environment can affect ginseng growth. Specifically, root growth can be altered by plant hormones, which in turn, affects crop yields. Among plant hormones, cytokinin is known to increase secondary growth by promoting cambium cell division. In addition, ethylene and propiconazole can regulate root development. In this study, the effects and interactions of cytokinin, ethylene, and propiconazole on four-year-old ginseng plants were analyzed. Methods and Results: Ginseng plants were treated with each hormone every two weeks. Cytokinin (6-benzylaminopurine, 500 μM) was applied to the soil, and 100 nM of propiconazole or 100 μM of ethylene was applied in combination with cytokinin. Root width and weight, and ginsenoside content of each plant were the highest when cytokinin and propiconazole were applied together. The highest number of buds was observed under cytokinin treatment. However, no significant differences were observed in shoot growth, except for stem width. Conclusions: The greatest effect on root development was observed when cytokinin and propiconazole were applied together, and ginsenoside content increased following hormone treatment.

내병성 목초 품종개량을 위한 PR4 유전자의 연구

차준영(Joon-Yung Cha),네티엘마와티(Netty Ermawati),정민희(Min Hee Jung),김기용(Ki-Yong Kim),손대영(Daeyoung Son) 한국초지조사료학회 2007 한국초지조사료학회지 Vol.27 No.4

Cytokinin은 식물의 성장과 발달에 중요한 역할을 하는 필수 호르몬이다. mRNA differential display 방법으로 애기장대 amp1 돌연변이체로부터 cytokinin에 의하여 발현이 유도되는 PR4 유전자를 분리하였다. AtPR4로 명명한 애기장대 PR4 유전자는 212개의 아미노산으로 구성되어 있었으며 분자량은 22,900이고 등전점은 7.89로 추정되었다. Genomic DNA 분석결과, AtPR4는 single copy 유전자인 것으로 나타났다. AtPR4의 mRNA는 cytokinin과 NaCl에 의해서는 발현이 유도되었지만 SA와 JA에 의해서는 발현이 억제되었다. PR 단백질은 내병성 등 생체방어기작에 관여하는 것으로 알려져 있다. 따라서 본 연구에서 분리한 애기장대 PR4 유전자는 내병성 목초 품종의 개발에 유용하게 사용될 것으로 사료된다. Cytokinins are essential plant hormones that play crucial roles in various aspects of plant growth and development. By using mRNA differential display, we isolated a cytokinine-inducible cDNA encoding pathogenesis-related (PR) 4 from Arabidopsis amp1 mutant. The full-length PR4 cDNA, designated AtPR4, contains an open reading frame of 212 amino acids with calculated molecular mass of 22,900 Da and isoelectric point (pI) of 7.89. Genomic DNA blotting showed that the Arabidopsis genome has one copy of AtPR4. AtPR4 mRNA was induced by cytokinin and NaCl, but decreased by SA or JA treatment. PR proteins are induced in response to pathogen attack. Thus the AtPR4 gene isolated in this study may be a useful candidate for genetic engineering of forage crops for increased tolerance against pathogen.