Lateral Root Induserbare System i<em> Arabidopsis</em> Og Maize
Summary
The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.
Abstract
Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.
Introduction
Rotsystemet er avgjørende for plantevekst, da det sikrer forankring og opptak av vann og næringsstoffer fra jorden. På grunn av at utvidelsen av et rotsystem hovedsakelig er avhengig av produksjonen av lateral røtter, har sin initiering og dannelse blitt mye studert. Lateral røtter er initiert i en bestemt undergruppe av pericycle celler, kalt grunnleggeren celler 1. I de fleste dicots, slik som Arabidopsis thaliana, er disse cellene plassert ved protoxylem polene 2, mens det i enfrøbladete planter, så som mais, er de funnet på barken polene 3. Grunnlegger-celler er merket med et øket auxin respons til 4, etterfulgt av ekspresjon av spesifikke cellesyklus-gener (for eksempel cyklin B1; 1 / CYCB1, 1), hvoretter de gjennomgår en første runde av asymmetriske anticlinal divisjoner 5. Etter en rekke koordinerte anticlinal og periclinal divisjoner, er en lateral root primordium dannet som til slutt vil fremstå som en enutonomous lateral root. Plasseringen og timing av lateral root initiering er imidlertid ikke forutsigbar, siden disse hendelsene er verken rik eller synkronisert. Dette vanskeliggjør bruk av molekylære metoder som transcriptomics å studere denne prosessen.
Å takle dette, har en Lateral Root induserbar System (LRIS) er utviklet 6, 7. I dette systemet, er frøplanter først behandlet med N -1-naphthylphthalamic syre (NPA), som hemmer auxin transport og oppbygging, dermed blokkerer lateral root initiering 8. Ved deretter å overføre frøplante til medium inneholdende det syntetiske auxin 1-naftalen-eddiksyre (NAA), svarer hele pericycle laget til de forhøyede nivåene auxin derved massivt induserende laterale rot initiere celledel 6. Som sådan, fører dette systemet til raske, synkrone og omfattende lateral røtter innvielser, noe som gir enkel samling av rot prøver beriket for et bestemt stadium av senral rot utvikling. Deretter kan disse prøvene brukes til å bestemme på genom uttrykk profil ved lateral rot formasjonen. Den LRIS har gitt allerede betydelig kunnskap om lateral root innvielse i Arabidopsis og mais 9-13, men behovet for å bruke dette systemet til andre plantearter blir mer tydelig etter hvert som flere genomer er sekvensert, og det er en økende interesse for å overføre kunnskap til økonomisk viktig arter.
Her er de detaljerte protokoller for Arabidopsis og mais LRISs gitt. Deretter blir et eksempel på bruk av systemet gitt, ved å illustrere hvordan transcriptomics data oppnådd fra mais LRIS kan brukes til å identifisere funksjonelle homologer som har en konservert funksjon under lateral rot initiering tvers av forskjellige plantearter. Endelig retningslinjer optimalisere LRIS for andre plantearter er foreslått.
Protocol
Representative Results
Discussion
I Arabidopsis LRIS protokollen, er det viktig å bare overføre spirene som har vokst helt i kontakt med NPA-inneholdende vekstmedium. Dette sikrer at lateral rot initiering blokkert over hele roten lengde. For å hindre at småplantene såret under overføringen, kan armene av de buede tang hektes under cotyledons av frøplante. Ved overføring, sørg for at de frøplante røtter er i tilstrekkelig kontakt med NAA holdige agar medium. Dette kan oppnås ved skumming roten over agaroverflaten over en liten dista…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank Davy Opdenacker for technical assistance and photography. We greatly thank Dr. Annick Bleys for helpful suggestions to improve the manuscript. This work was financed by the Interuniversity Attraction Poles Programme IUAP P7/29 ‘MARS’ from the Belgian Federal Science Policy Office, by the FWO grant G027313N and by the Agency for Innovation by Science and Technology, IWT (IR).
Materials
| ARABIDOPSIS LRIS | |||
| Seeds | |||
| Arabidopsis seeds | Col-0 ecotype | ||
| Gas sterilization of seeds | |||
| micro-centrifuge tubes 1.5 ml | SIGMA-ALDRICH | 0030 125.215 | Eppendorf microtubes 3810X, PCR clean |
| micro-centrifuge tubes 2 ml | SIGMA-ALDRICH | 0030 120.094 | Eppendorf Safe-Lock microcentrifuge tubes |
| hydrochloric acid | Merck KGaA | 1,003,171,000 | 37% (fuming) for analysis EMSURE ACS,ISO,Reag. Ph Eu |
| glass desiccator | SIGMA-ALDRICH | Pyrex | |
| glass beaker | |||
| plastic micro-centrifuge tubes box or holder | |||
| Bleach sterilization of seeds | |||
| ethanol | Chem-Lab nv | CL00.0505.1000 | Ethanol, abs. 100% a.r. dilute to 70% |
| sodium hypochlorite (NaOCl) | Carl Roth | 9062.3 | 12% |
| Tween 20 | SIGMA-ALDRICH | P1379 | |
| sterile water | |||
| Growth medium | |||
| Murashige and Skoog salt mixture | DUCHEFA Biochemie B.V. | M0221-0050 | |
| myo-inositol | SIGMA-ALDRICH | I5125-100G | |
| 2-(N-morpholino)ethanesulfonic acid (MES) | DUCHEFA Biochemie B.V. | M1503.0100 | |
| sucrose | VWR, Internation LLC | 27483.294 | D(+)-Sucrose Ph. Eur. |
| KOH | Merck KGaA | 1050211000 | pellets for analysis (max. 0.002% Na) EMSURE ACS,ISO,Reag. Ph Eur |
| Plant Tissue Culture Agar | LabM Limited | MC029 | |
| Lateral root induction chemicals | |||
| N-1-naphthylphthalamic acid (NPA) | DUCHEFA Biochemie B.V. | No. N0926.0250 | 10 µM (Arabidopsis) |
| 1-naphthalene acetic acid (NAA) | DUCHEFA Biochemie B.V. | No. N0903.0050 | 10 µM (Arabidopsis) |
| dimethylsulfoxide (DMSO) | SIGMA-ALDRICH | 494429-1L | |
| Making a mesh for transfer | |||
| nylon mesh | Prosep byba | Synthetic nylon mesh 20 µm | |
| Sowing and seedling handling | |||
| square petri dish plates | GOSSELIN | BP124-05 | 12 x 12 cm |
| 50 ml DURAN tubes | SIGMA-ALDRICH | CLS430304 | Corning 50 mL centrifuge tubes |
| drigalski | Carl Roth | K732.1 | |
| pipette | |||
| cut pipette tips | Daslab | 162001X | Universal 200, cut off 5 mm of tip before autoclaving |
| breathable tape | 3M Deutschland GmbH | cat. no. 1530-1 | |
| tweezers | Fiers nv/sa | K342.1; K344.1 | Dumont tweezers type a nr 5; Dumont tweezers type e nr 7 |
| Growth conditions | |||
| growth room | 21 °C, continuous light | ||
| Materials | Company | Catalog | Comments |
| MAIZE LRIS | |||
| Seeds | |||
| Maize kernels | B-73 | ||
| Bleach sterilization of kernels | |||
| glass beaker | |||
| magnetic stirrer | Fiers nv/sa | C267.1 | |
| sodium hypochlorite (NaOCl) | Carl Roth | 9062.3 | 12% |
| sterile water | |||
| Lateral root induction chemicals | |||
| N-1-naphthylphthalamic acid (NPA) | DUCHEFA Biochemie B.V. | No. N0926.0250 | 50 µM (maize primary root), 25 µM (maize adventitious root) |
| 1-naphthalene acetic acid (NAA) | DUCHEFA Biochemie B.V. | No. N0903.0050 | 50 µM (maize) |
| dimethylsulfoxide (DMSO) | SIGMA-ALDRICH | 494429-1L | |
| Sowing and seedling handling | |||
| paper hand towels | Kimberly-Clark Professional* | 6681 | SCOTT Hand Towels – Roll / White; sheet size (24 x 46 cm) |
| seed germination paper | Anchor Paper Company | 10 X 15 38# seed germination paper | |
| tweezers | Fiers nv/sa | K342.1; K344.1 | Dumont tweezers type a nr 5; Dumont tweezers type e nr 7 |
| 250 ml (centrifuge) tubes | SCHOTT DURAN | 2160136 | approx. 5.6 cm diameter and 14.7 cm height |
| 700 ml tubes | DURAN GROUP | 213994609 | cylinders, round foot tube, D 60 x 250 |
| rack | for maize tubes, home made | ||
| sterile water | |||
| Growth conditions | |||
| growth cabinet | 27 °C, continuous light, 70% relative humidity |
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