02741nas a2200145 4500008004100000245014300041210006900184260001200253490000600265520220300271100002402474700002202498700001802520856005702538 2020 eng d00aMetabolomics analysis reveals both plant variety and choice of hormone treatment modulate vinca alkaloid production in Catharanthus roseus0 aMetabolomics analysis reveals both plant variety and choice of h c09/20200 v43 a

The medicinal plant Catharanthus roseus produces numerous secondary metabolites of interest for the treatment of many diseases – most notably for the terpene indole alkaloid (TIA) vinblastine, which is used in the treatment of leukemia and Hodgkin's lymphoma. Historically, methyl jasmonate (MeJA) has been used to induce TIA production, but in the past, this has only been investigated in whole seedlings, cell culture, or hairy root culture. This study examines the effects of the phytohormones MeJA and ethylene on the induction of TIA biosynthesis and accumulation in the shoots and roots of 8‐day‐old seedlings of two varieties of C. roseus. Using LCMS and RT‐qPCR, we demonstrate the importance of variety selection, as we observe markedly different induction patterns of important TIA precursor compounds. Additionally, both phytohormone choice and concentration have significant effects on TIA biosynthesis. Finally, our study suggests that several early‐induction pathway steps as well as pathway‐specific genes are likely to be transcriptionally regulated. Our findings highlight the need for a complete set of'omics resources in commonly used C. roseus varieties and the need for caution when extrapolating results from one cultivar to another.

1 aFraser, Valerie, N.1 aPhilmus, Benjamin1 aMegraw, Molly uhttps://onlinelibrary.wiley.com/doi/10.1002/pld3.26701085nas a2200373 4500008004100000245010300041210006900144260001200213490000600225100002100231700002100252700001900273700001700292700001800309700002200327700001900349700001600368700002200384700002000406700002100426700001600447700001700463700001500480700002000495700002000515700001300535700001400548700001800562700001700580700001600597700001900613700001700632856006200649 2019 eng d00aArabidopsis bioinformatics resources: The current state, challenges, and priorities for the future0 aArabidopsis bioinformatics resources The current state challenge c01/20190 v31 aDoherty, Colleen1 aFriesner, Joanna1 aGregory, Brian1 aLoraine, Ann1 aMegraw, Molly1 aProvart, Nicholas1 aSlotkin, Keith1 aTown, Chris1 aAssmann, Sarah, M1 aAxtell, Michael1 aBerardini, Tanya1 aChen, Sixue1 aGehan, Malia1 aHuala, Eva1 aJaiswal, Pankaj1 aLarson, Stephen1 aLi, Song1 aMay, Sean1 aMichael, Todd1 aPires, Chris1 aTopp, Chris1 aWalley, Justin1 aWurtele, Eve uhttps://onlinelibrary.wiley.com/doi/full/10.1002/pld3.10902308nas a2200169 4500008004100000245013500041210006900176260001200245300001200257490000800269520169800277100002501975700002102000700002302021700001802044856007602062 2018 eng d00aIdentification of transcription factors from NF-Y, NAC, and SPL families responding to osmotic stress in multiple tomato varieties0 aIdentification of transcription factors from NFY NAC and SPL fam c09/2018 a441-4500 v2743 a

Identifying osmotic stress-responsive transcription factors (TFs) can facilitate discovery of master regulators mediating salt and/or drought tolerance. To date, few RNA-seq datasets for high resolution time course of salt or drought stress treatments are publicly available for certain crop species. However, such datasets may be available for other crops, and in combination with orthology analysis may be used to infer candidate osmotic stress regulators across distantly related species. Here, we demonstrate the utility of this approach for identification and validation of osmotic stress-responsive transcription factors in tomato. First, we developed physiologically calibrated salt and dehydration-responsive systems for tomato cultivars using real time measurements of transpiration rate and photosynthetic efficiency. Next, we identified differentially expressed TFs in rice using raw RNA-seq datasets for a publicly available salt stress time course. Putative salt stress-responsive TFs in tomato were then inferred based on their orthology with the transcription factors upregulated by salt in rice. Finally, using our osmotic stress system, we experimentally validated stress-responsive expression of predicted tomato candidates representing NUCLEAR FACTOR Y, SQUAMOSA PROMOTER BINDING, and NAC domain TF families. Quantification of transcript copy numbers confirmed that mRNAs encoding all three TFs were strongly upregulated not only by salt but also by drought stress. Induction by both salt and dehydration occurred in a temporal manner across diverse tomato cultivars, suggesting that the identified TFs may play important roles in regulating osmotic stress responses.

1 aFilichkin, Sergei, A1 aAnsariola, Mitra1 aFraser, Valerie, N1 aMegraw, Molly uhttps://www.sciencedirect.com/science/article/abs/pii/S016894521830353401445nas a2200145 4500008004100000020004600041245009100087210006900178300001200247490000900259520092600268100002201194700001501216856006801231 2017 eng d a1940-6029 (Electronic)1064-3745 (Linking)00aDNase I SIM: A Simplified In-Nucleus Method for DNase I Hypersensitive Site Sequencing0 aDNase I SIM A Simplified InNucleus Method for DNase I Hypersensi a141-1540 v16293 a

Identifying cis-regulatory elements is critical in understanding the direct and indirect interactions that occur within gene regulatory networks. Current approaches include DNase-seq, a technique that combines sensitivity to the nonspecific endonuclease DNase I with high-throughput sequencing to identify regions of regulatory DNA on a genome-wide scale. Yet, challenges still remain in processing recalcitrant tissues that have low DNA content. Here, we describe DNase I SIM (for Simplified In-nucleus Method), a protocol that simplifies and facilitates generation of DNase-seq libraries from plant tissues for high-resolution mapping of DNase I hypersensitive sites. By removing steps requiring the use of gel agarose plugs in DNase-seq, DNase I SIM reduces the time required to perform the protocol by at least 2 days, while also making possible the processing of difficult plant tissues including plant roots.

1 aFilichkin, S., A.1 aMegraw, M. uhttps://link.springer.com/protocol/10.1007/978-1-4939-7125-1_1002932nas a2200589 4500008004100000245010500041210006900146260001200215300001400227490000800241520124200249100002101491700002301512700001701535700002501552700001601577700002001593700001701613700002301630700002001653700001701673700002201690700002501712700002401737700002501761700002101786700002201807700002101829700002301850700001601873700001901889700001601908700002401924700001801948700002201966700002401988700002602012700001402038700001702052700002402069700002002093700002002113700001902133700001602152700002002168700002202188700001702210700002002227700002102247700002302268856005102291 2017 eng d00aThe Next Generation of Training for Arabidopsis Researchers: Bioinformatics and Quantitative Biology0 aNext Generation of Training for Arabidopsis Researchers Bioinfor c12/2017 a1499-15090 v1753 a
It has been more than 50 years since Arabidopsis (Arabidopsis thaliana) was first introduced as a model organism to understand basic processes in plant biology. A well-organized scientific community has used this small reference plant species to make numerous fundamental plant biology discoveries (Provart et al., 2016). Due to an extremely well-annotated genome and advances in high-throughput sequencing, our understanding of this organism and other plant species has become even more intricate and complex. Computational resources, including CyVerse,3 Araport,4 The Arabidopsis Information Resource (TAIR),5 and BAR,6 have further facilitated novel findings with just the click of a mouse. As we move toward understanding biological systems, Arabidopsis researchers will need to use more quantitative and computational approaches to extract novel biological findings from these data. Here, we discuss guidelines, skill sets, and core competencies that should be considered when developing curricula or training undergraduate or graduate students, postdoctoral researchers, and faculty. A selected case study provides more specificity as to the concrete issues plant biologists face and how best to address such challenges.
1 aFriesner, Joanna1 aAssmann, Sarah, M.1 aBastow, Ruth1 aBailey-Serres, Julia1 aBeynon, Jim1 aBrendel, Volker1 aBuell, Robin1 aBucksch, Alexander1 aBusch, Wolfgang1 aDemura, Taku1 aDinneny, Jose, R.1 aDoherty, Colleen, J.1 aEveland, Andrea, L.1 aFalter-Braun, Pascal1 aGehan, Malia, A.1 aGonzales, Michael1 aGrotewold, Erich1 aGutierrez, Rodrigo1 aKramer, Ute1 aKrouk, Gabriel1 aMa, Shisong1 aMarkelz, R.J., Cody1 aMegraw, Molly1 aMeyers, Blake, C.1 aMurray, James, A.H.1 aProvart, Nicholas, J.1 aRhee, Sue1 aSmith, Roger1 aSpalding, Edgar, P.1 aTaylor, Crispin1 aTeal, Tracy, K.1 aTor, Keiko, U.1 aTown, Chris1 aVaughn, Matthew1 aVierstra, Richard1 aWare, Doreen1 aWilkins, Olivia1 aWilliams, Cranos1 aBrady, Siobhan, M. uhttp://www.plantphysiol.org/content/175/4/149902614nas a2200517 4500008004100000020004600041245014500087210006900232260001200301300001200313490000700325520091000332653004701242653005901289653002401348653003801372653002901410653001701439653002001456653002101476653002001497653003601517653003301553653003001586653004301616653004101659653004801700653003301748100001901781700001501800700001801815700001101833700001801844700001301862700002001875700001401895700001401909700002101923700001401944700002201958700001501980700001501995700001802010700001902028856004902047 2016 eng d a1878-1551 (Electronic)1534-5807 (Linking)00aEstablishment of Expression in the SHORTROOT-SCARECROW Transcriptional Cascade through Opposing Activities of Both Activators and Repressors0 aEstablishment of Expression in the SHORTROOTSCARECROW Transcript c12/2016 a585-5960 v393 a

Tissue-specific gene expression is often thought to arise from spatially restricted transcriptional cascades. However, it is unclear how expression is established at the top of these cascades in the absence of pre-existing specificity. We generated a transcriptional network to explore how transcription factor expression is established in the Arabidopsis thaliana root ground tissue. Regulators of the SHORTROOT-SCARECROW transcriptional cascade were validated in planta. At the top of this cascade, we identified both activators and repressors of SHORTROOT. The aggregate spatial expression of these regulators is not sufficient to predict transcriptional specificity. Instead, modeling, transcriptional reporters, and synthetic promoters support a mechanism whereby expression at the top of the SHORTROOT-SCARECROW cascade is established through opposing activities of activators and repressors.

10aArabidopsis Proteins/ genetics/ metabolism10aArabidopsis/ genetics/growth & development/ metabolism10aComputer Simulation10aGene Expression Regulation, Plant10aGene Regulatory Networks10aGenes, Plant10aGenes, Reporter10aGenes, Synthetic10aModels, Genetic10aPlant Roots/cytology/metabolism10aPlants, Genetically Modified10aPromoter Regions, Genetic10aRepressor Proteins/genetics/metabolism10aTrans-Activators/genetics/metabolism10aTranscription Factors/ genetics/ metabolism10aTwo-Hybrid System Techniques1 aSparks, E., E.1 aDrapek, C.1 aGaudinier, A.1 aLi, S.1 aAnsariola, M.1 aShen, N.1 aHennacy, J., H.1 aZhang, J.1 aTurco, G.1 aPetricka, J., J.1 aForet, J.1 aHartemink, A., J.1 aGordan, R.1 aMegraw, M.1 aBrady, S., M.1 aBenfey, P., N. uhttps://doi.org/10.1016/j.devcel.2016.09.03102264nas a2200181 4500008004100000022001400041245010600055210006900161260001300230300001200243490000700255520168200262100001801944700002101962700002501983700002502008856004902033 2016 eng d a1532-298X00aSmall Genetic Circuits and MicroRNAs: Big Players in Polymerase II Transcriptional Control in Plants.0 aSmall Genetic Circuits and MicroRNAs Big Players in Polymerase I c2016 Feb a286-3030 v283 a

RNA Polymerase II (Pol II) regulatory cascades involving transcription factors (TFs) and their targets orchestrate the genetic circuitry of every eukaryotic organism. In order to understand how these cascades function, they can be dissected into small genetic networks, each containing just a few Pol II transcribed genes, that generate specific signal-processing outcomes. Small RNA regulatory circuits involve direct regulation of a small RNA by a TF and/or direct regulation of a TF by a small RNA and have been shown to play unique roles in many organisms. Here, we will focus on small RNA regulatory circuits containing Pol II transcribed microRNAs (miRNAs). While the role of miRNA-containing regulatory circuits as modular building blocks for the function of complex networks has long been on the forefront of studies in the animal kingdom, plant studies are poised to take a lead role in this area because of their advantages in probing transcriptional and posttranscriptional control of Pol II genes. The relative simplicity of tissue- and cell-type organization, miRNA targeting, and genomic structure make the Arabidopsis thaliana plant model uniquely amenable for small RNA regulatory circuit studies in a multicellular organism. In this Review, we cover analysis, tools, and validation methods for probing the component interactions in miRNA-containing regulatory circuits. We then review the important roles that plant miRNAs are playing in these circuits and summarize methods for the identification of small genetic circuits that strongly influence plant function. We conclude by noting areas of opportunity where new plant studies are imminently needed.

1 aMegraw, Molly1 aCumbie, Jason, S1 aIvanchenko, Maria, G1 aFilichkin, Sergei, A uhttps://megraw.cgrb.oregonstate.edu/node/31201715nas a2200253 4500008004100000022001400041245011200055210006900167260001300236300001100249490000700260520091500267653002701182653002501209653002101234653003401255653001501289653002601304100002201330700002101352700001801373700002101391856004901412 2015 eng d a1879-035600aAlternative splicing in plants: directing traffic at the crossroads of adaptation and environmental stress.0 aAlternative splicing in plants directing traffic at the crossroa c2015 Apr a125-350 v243 a

In recent years, high-throughput sequencing-based analysis of plant transcriptomes has suggested that up to ∼60% of plant gene loci encode alternatively spliced mature transcripts. These studies have also revealed that alternative splicing in plants can be regulated by cell type, developmental stage, the environment, and the circadian clock. Alternative splicing is coupled to RNA surveillance and processing mechanisms, including nonsense mediated decay. Recently, non-protein-coding transcripts have also been shown to undergo alternative splicing. These discoveries collectively describe a robust system of post-transcriptional regulatory feedback loops which influence RNA abundance. In this review, we summarize recent studies describing the specific roles alternative splicing and RNA surveillance play in plant adaptation to environmental stresses and the regulation of the circadian clock.

10aAdaptation, Biological10aAlternative Splicing10aCircadian Clocks10aPlant Physiological Phenomena10aRNA, Plant10aStress, Physiological1 aFilichkin, Sergei1 aPriest, Henry, D1 aMegraw, Molly1 aMockler, Todd, C uhttps://megraw.cgrb.oregonstate.edu/node/31402854nas a2200373 4500008004100000022001400041245012300055210006900178260001600247300001100263490000800274520174600282653001602028653002502044653001802069653002302087653002802110653001902138653001602157653001702173100002502190700001802215700001802233700001902251700001602270700002102286700002102307700001802328700002202346700002502368700001802393700002002411856004902431 2015 eng d a1477-912900aThe cyclophilin A DIAGEOTROPICA gene affects auxin transport in both root and shoot to control lateral root formation.0 acyclophilin A DIAGEOTROPICA gene affects auxin transport in both c2015 Feb 15 a712-210 v1423 a

Cyclophilin A is a conserved peptidyl-prolyl cis-trans isomerase (PPIase) best known as the cellular receptor of the immunosuppressant cyclosporine A. Despite significant effort, evidence of developmental functions of cyclophilin A in non-plant systems has remained obscure. Mutations in a tomato (Solanum lycopersicum) cyclophilin A ortholog, DIAGEOTROPICA (DGT), have been shown to abolish the organogenesis of lateral roots; however, a mechanistic explanation of the phenotype is lacking. Here, we show that the dgt mutant lacks auxin maxima relevant to priming and specification of lateral root founder cells. DGT is expressed in shoot and root, and localizes to both the nucleus and cytoplasm during lateral root organogenesis. Mutation of ENTIRE/IAA9, a member of the auxin-responsive Aux/IAA protein family of transcriptional repressors, partially restores the inability of dgt to initiate lateral root primordia but not the primordia outgrowth. By comparison, grafting of a wild-type scion restores the process of lateral root formation, consistent with participation of a mobile signal. Antibodies do not detect movement of the DGT protein into the dgt rootstock; however, experiments with radiolabeled auxin and an auxin-specific microelectrode demonstrate abnormal auxin fluxes. Functional studies of DGT in heterologous yeast and tobacco-leaf auxin-transport systems demonstrate that DGT negatively regulates PIN-FORMED (PIN) auxin efflux transporters by affecting their plasma membrane localization. Studies in tomato support complex effects of the dgt mutation on PIN expression level, expression domain and plasma membrane localization. Our data demonstrate that DGT regulates auxin transport in lateral root formation.

10aArabidopsis10aBiological Transport10aCyclophilin A10aIndoleacetic Acids10aLycopersicon esculentum10aPlant Proteins10aPlant Roots10aPlant Shoots1 aIvanchenko, Maria, G1 aZhu, Jinsheng1 aWang, Bangjun1 aMedvecká, Eva1 aDu, Yunlong1 aAzzarello, Elisa1 aMancuso, Stefano1 aMegraw, Molly1 aFilichkin, Sergei1 aDubrovsky, Joseph, G1 aFriml, Jiří1 aGeisler, Markus uhttps://megraw.cgrb.oregonstate.edu/node/31602503nas a2200325 4500008004100000022001400041245012000055210006900175260001300244300001100257490000600268520148900274653002501763653001601788653002501804653002101829653003801850653001201888653003301900100002501933700002101958700002801979700002002007700001902027700002502046700001802071700001802089700002102107856004902128 2015 eng d a1752-986700aEnvironmental stresses modulate abundance and timing of alternatively spliced circadian transcripts in Arabidopsis.0 aEnvironmental stresses modulate abundance and timing of alternat c2015 Feb a207-270 v83 a

Environmental stresses profoundly altered accumulation of nonsense mRNAs including intron-retaining (IR) transcripts in Arabidopsis. Temporal patterns of stress-induced IR mRNAs were dissected using both oscillating and non-oscillating transcripts. Broad-range thermal cycles triggered a sharp increase in the long IR CCA1 isoforms and altered their phasing to different times of day. Both abiotic and biotic stresses such as drought or Pseudomonas syringae infection induced a similar increase. Thermal stress induced a time delay in accumulation of CCA1 I4Rb transcripts, whereas functional mRNA showed steady oscillations. Our data favor a hypothesis that stress-induced instabilities of the central oscillator can be in part compensated through fluctuations in abundance and out-of-phase oscillations of CCA1 IR transcripts. Taken together, our results support a concept that mRNA abundance can be modulated through altering ratios between functional and nonsense/IR transcripts. SR45 protein specifically bound to the retained CCA1 intron in vitro, suggesting that this splicing factor could be involved in regulation of intron retention. Transcriptomes of nonsense-mediated mRNA decay (NMD)-impaired and heat-stressed plants shared a set of retained introns associated with stress- and defense-inducible transcripts. Constitutive activation of certain stress response networks in an NMD mutant could be linked to disequilibrium between functional and nonsense mRNAs.

10aAlternative Splicing10aArabidopsis10aArabidopsis Proteins10aCircadian Clocks10aGene Expression Regulation, Plant10aIntrons10aNonsense Mediated mRNA Decay1 aFilichkin, Sergei, A1 aCumbie, Jason, S1 aDharmawardhana, Palitha1 aJaiswal, Pankaj1 aChang, Jeff, H1 aPalusa, Saiprasad, G1 aReddy, A, S N1 aMegraw, Molly1 aMockler, Todd, C uhttps://megraw.cgrb.oregonstate.edu/node/31501941nas a2200169 4500008004100000022001400041245013400055210006900189260000900258300000700267490000700274520137700281100002101658700002501679700001801704856004901722 2015 eng d a1746-481100aImproved DNase-seq protocol facilitates high resolution mapping of DNase I hypersensitive sites in roots in Arabidopsis thaliana.0 aImproved DNaseseq protocol facilitates high resolution mapping o c2015 a420 v113 a

BACKGROUND: Identifying cis-regulatory elements is critical in understanding the direct and indirect regulatory mechanisms of gene expression. Current approaches include DNase-seq, a technique that combines sensitivity to the nonspecific endonuclease DNase I with high throughput sequencing to identify regions of regulatory DNA on a genome-wide scale. While this method was originally developed for human cell lines, later adaptations made the processing of plant tissues possible. Challenges still remain in processing recalcitrant tissues that have low DNA content.

RESULTS: By removing steps requiring the use of gel agarose plugs in DNase-seq, we were able to significantly reduce the time required to perform the protocol by at least 2 days, while also making possible the processing of difficult plant tissues. We refer to this simplified protocol as DNase I SIM (for simplified in-nucleus method). We were able to successfully create DNase-seq libraries for both leaf and root tissues in Arabidopsis using DNase I SIM.

CONCLUSION: This protocol simplifies and facilitates generation of DNase-seq libraries from plant tissues for high resolution mapping of DNase I hypersensitive sites.

[Link to Protocol, Additional Data, and Supplementary Materials]

1 aCumbie, Jason, S1 aFilichkin, Sergei, A1 aMegraw, Molly uhttps://megraw.cgrb.oregonstate.edu/node/31002171nas a2200217 4500008004100000022001400041245012000055210006900175260001500244520145100259100002501710700002101735700002701756700002001783700001901803700002501822700001801847700001801865700002101883856004901904 2014 eng d a1752-986700aEnvironmental Stresses Modulate Abundance and Timing of Alternatively Spliced Circadian Transcripts in Arabidopsis.0 aEnvironmental Stresses Modulate Abundance and Timing of Alternat c2014 Nov 33 a

Environmental stresses profoundly altered accumulation of nonsense mRNAs including intron retaining (IR) transcripts in Arabidopsis. Temporal patterns of stress-induced IR mRNAs were dissected using both oscillating and non-oscillating transcripts. Broad range thermal cycles triggered a sharp increase in the long intron retaining CCA1 isoforms and altered their phasing to different times of day. Both abiotic and biotic stresses such as drought or P. syringae infection induced similar increase. Thermal stress induced a time delay in accumulation of CCA1 I4Rb transcripts whereas functional mRNA showed steady oscillations. Our data favor a hypothesis that stress-induced instabilities of the central oscillator can be in part compensated through fluctuations in abundance and out of phase oscillations of CCA1 IR transcripts. Altogether, our results support a concept that mRNA abundance can be modulated through altering ratios between functional and nonsense/IR transcripts. SR45 protein specifically bound to the retained CCA1 intron in vitro, suggesting that this splicing factor could be involved in regulation of intron retention. Transcriptomes of NMD-impaired and heat-stressed plants shared a set of retained introns associated with stress- and defense-inducible transcripts. Constitutive activation of certain stress response networks in an NMD mutant could be linked to disequilibrium between functional and nonsense mRNAs.

1 aFilichkin, Sergei, A1 aCumbie, Jason, S1 aDharmawadhana, Palitha1 aJaiswal, Pankaj1 aChang, Jeff, H1 aPalusa, Saiprasad, G1 aReddy, A, S N1 aMegraw, Molly1 aMockler, Todd, C uhttps://megraw.cgrb.oregonstate.edu/node/317