The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.
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.
Roden er afgørende for planternes vækst, da det sikrer forankring og optagelse af vand og næringsstoffer fra jorden. Fordi udvidelse af et rodsystem hovedsageligt er afhængig af produktionen af laterale rødder, har deres indvielse og dannelse blevet bredt undersøgt. Lateral rødder indledt i en specifik undergruppe af pericycle celler, kaldet grundlægger celler 1. I de fleste tokimbladede, såsom Arabidopsis thaliana, er disse celler placeret på protoxylem poler 2, mens der i enkimbladede planter, såsom majs, findes de ved Phloem poler 3. Stiftende celler er markeret med en øget auxin respons 4, efterfulgt af ekspression af specifikke gener cellecyklus (f.eks cyclin B1, 1 / CYCB1; 1), hvorefter de underkastes en første runde af asymmetriske anticlinal divisioner 5. Efter en række koordinerede anticlinal og periclinal divisioner, er en lateral rod primordium dannet, der vil til sidst fremstå som en etutonomous lateral rod. Placeringen og timingen af lateral rod indvielse er dog ikke forudsigelig, da disse begivenheder er hverken rigelige eller synkroniseret. Dette vanskeliggør brugen af molekylære metoder såsom transcriptomics at studere denne proces.
For at løse dette, har en lateral Root Inducerbar System (lris) er udviklet 6, 7. I dette system er frøplanter først behandles med N -1-naphthylphthalamic syre (NPA), som hæmmer auxin transport og akkumulering dermed blokere lateral rodinitiering 8. Ved efterfølgende at overføre sætteplante til medium, der indeholder det syntetiske auxin 1-naphthalen eddikesyre (NAA), hele pericycle laget reagerer på de forhøjede auxin niveauer derved massivt inducerende laterale root indlede celledelinger 6. Som sådan, dette system fører til hurtige, synkrone og omfattende laterale rødder indvielser, giver nem samling af rod prøver beriget til et bestemt tidspunkt i slutningen afral rodudvikling. Efterfølgende kan disse prøver anvendes til at bestemme genom-dækkende udtryk profiler under lateral roddannelse. Den lris har givet allerede betydelige viden om lateral rod initiering i Arabidopsis og majs 9-13, men det er nødvendigt at anvende dette system til andre plantearter bliver tydeligere, efterhånden som flere genomer er sekventeret og der er en stigende interesse for at overføre viden til økonomisk vigtige arter.
Her er de detaljerede protokoller fra Arabidopsis og majs LRISs givet. Dernæst et eksempel på brugen af ordningen, ved at illustrere, hvordan transcriptomics data opnået fra majs lris kan bruges til at identificere funktionelle homologer, der har en bevaret funktion under lateral rodinitiering tværs af forskellige plantearter. Endelig retningslinjer optimere lris for foreslås andre plantearter.
I Arabidopsis lris protokollen, er det vigtigt kun at overføre de planter, der er vokset helt i kontakt med NPA-holdige vækstmedium. Dette sikrer, at lateral rodinitiering blokeres over hele root længde. For at undgå sårdannelse småplanterne under overførslen, kan armene på de buede pincet være tilsluttet under kimbladene af kimplanter. Ved overførsel, sørg for, at sætteplante rødder er i tilstrækkelig kontakt med NAA-holdige agar medium. Dette kan opnås ved at skumme roden over agar overflade ov…
The authors have nothing to disclose.
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).
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 |