A Simple Auxin Transcriptional Response System Regulates Multiple Morphogenetic Processes in the Liverwort Marchantia polymorpha methods

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    "name": "A Simple Auxin Transcriptional Response System Regulates Multiple Morphogenetic Processes in the Liverwort Marchantia polymorpha methods",
    "description": "Protocol for A Simple Auxin Transcriptional Response System Regulates Multiple Morphogenetic Processes in the Liverwort Marchantia polymorpha methods",
    "step": [
      {
        "@type": "HowToStep",
        "position": 1,
        "name": "MpARF1 is a modulator of the auxin signaling pathway",
        "text": "(A) Each panel show six independent lines, transformed with constructs as indicated above the panels. Plants are treated as indicated to the left. Arrows indicate apical notches. (B) Left panel: Rhizoids from control Hyg R lines. Right panel: pro EF1: MpARF1 ΔD34 lines develop elongated and profuse rhizoids. (C) Apical notch and gemmae cup measurements for control, pro EF1: MpARF1 and pro EF1: MpARF1 ΔD34 primary transformants after 50 days of growth (30 independent lines were scored per genotype). MpARF1 overexpression results in increased branching rates. Deletion of D34 MpARF1 recovers the gemmae cup production lost by over-expressing full length MpARF1 transcripts. (D) Semi-quantitative RT-PCR showing six independent pro EF1: MpARF1 lines exhibiting an inverse correlation between MpARF1 and MpIAA transcript levels. Three of the six lines are shown in the panel, with..."
      },
      {
        "@type": "HowToStep",
        "position": 2,
        "name": "MpTPL is involved in patterning multiple tissues in M. polymorpha development",
        "text": "(A) Mature dormant gemma of pro MpTPL: 3xVENUSN7 with expression in two apical notches (asterisks). (B) 5-day-old gemmae of pro MpTPL: 3xVENUSN7. Expression has expanded from apical notches (asterisk) to zones of newly formed air chambers (arrowheads). Inset illustrates background fluorescence in the wild type with identical settings. (C) Mature 25-day-old pro MpTPL: 3xVENUSN7 line showing expression in meristematic regions (asterisks). (D) Young gemmae cup (gc) primordia in mature pro MpTPL: 3xVENUSN7 line. (E) Expression pattern of mature pro MpTPL: 3xVENUSN7 thallus on mock and 10µM 2,4-D media. Plants were transferred to exogenous auxin after 16 days on B5 media. (F) pro EF1: VENUS indicating expression in apical notches (asterisk) and gemmae cups (gc). (G) Dorsal side of wild-type thallus covered with photosynthetic air chambers and two apical notches from which gemma..."
      },
      {
        "@type": "HowToStep",
        "position": 3,
        "name": "Auxin insensitive M. polymorpha plants fail to establish a dorsiventral thallus",
        "text": "(A) pro EF1: MpTPL-D34 IAA lines suffer from severe dwarfism compared to pro EF1: MpTPL plants. (B) Strong pro EF1: MpTPL-D34 IAA line showing a lack of organized patterning, dorsiventral polarity and a limited degree of differentiation. Scales (arrowhead), air pores (asterisk) and rhizoids (r) are visible in random places and growth is severely affected. (C) pro EF1: MpTPL-D34 MpARF1 representative line showing a degree of differentiation similar to the prothallus stage of wild-type development. (D) pro EF1: MpTPL-D34 MpARF1 showing single cell layer prothalli-like tisssue. (E) pro EF1: MpTPL-D34 MpARF2 representative line. (F) Wild-type sporeling (10 days) transitioning into a single cell layered prothallus (14 days) and fully established multiple prothalli 18 days after plating. (G) Strong pro EF1: MpTPL-D34 BDL line lacking growth, differentiation and dorsiventrality, but s..."
      },
      {
        "@type": "HowToStep",
        "position": 4,
        "name": "Auxin patterns the development of many tissues in M. polymorpha",
        "text": "Auxin distribution influences establishment of the dorsiventral body plan. Consistent with earlier reports, growth of gemmae from germination on auxin containing media resulted in rhizoid formation from both dorsal and ventral surfaces. In addition, transfer of gemmae with established dorsiventrality to auxin plates caused several other developmental abnormalities, such as distorted growth of air chambers, air pores, and gemmae cups, but not changes in dorsiventrality (Fig - ). This is consistent with the observation that dorsiventrality is established within the first 24 hours of gemmaling growth and is irreversible after this time [,,,,,, ]."
      },
      {
        "@type": "HowToStep",
        "position": 5,
        "name": "Materials and Methods",
        "text": "Sporangia from an Australian population of Marchantia polymorpha (from now on referred to as wild type) were collected from a southeastern Melbourne field location (37°57'48.36\"S, 145° 6'20.41\"E). Sporangia were sterilized in 1x sterilization solution (2% sodium hypochlorite, 0.1% TritonX-100) for 1 minute and subsequently washed with water. Sporangia were then dried and stored at 4°C."
      },
      {
        "@type": "HowToStep",
        "position": 6,
        "name": "Pharmacological assays",
        "text": "Dormant gemmae, or gemmalings grown on B5 media for 2, 4, 6, 8, 10, 12, 14 and 16 days, were plated on B5 media with 10 µM 2,4-D or NAA (Sigma-Aldrich). L-Kyneurenine (Sigma-Aldrich) was used in a similar fashion as exogenous auxin."
      },
      {
        "@type": "HowToStep",
        "position": 7,
        "name": "Supporting Information",
        "text": "(A) Wild-type gemmaling grown for 18 days on mock (DMSO). (B) 18-day-old wild-type gemmaling grown on 10 µM 2,4-D from day six of development. (C) 18-day-old wild-type gemmaling grown on auxin from day eight of development. (D) 18-day-old wild-type gemmaling grown on auxin from day 12 of development. Arrowheads indicate elongated gemma cups. (E) 18-day-old thallus grown on mock. (F) 18-day-old thallus grown on 250 µM L-Kyn; fused gemmae cups are observed. (G) Wild-type thalli after a month of growth. (H) pro MpSHI: iaaL plants fail to elongate laterally, forming narrow thalli after a month of growth. All scale bars are 1 mm, except for A to D, 1cm."
      },
      {
        "@type": "HowToStep",
        "position": 8,
        "name": "Branching rates measurements",
        "text": "Primary sporeling-transformants expressing either pro EF1: MpTPL N176H or a Hyg Res control T-DNA were simultaneously plated and notches were counted by eye after 25 days of growth in B5 media (N = 17)."
      }
    ],
    "tool": [],
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    "isBasedOn": {
      "@type": "ScholarlyArticle",
      "headline": "A Simple Auxin Transcriptional Response System Regulates Multiple Morphogenetic Processes in the Liverwort Marchantia polymorpha",
      "datePublished": "2015",
      "author": [
        {
          "@type": "Person",
          "name": "Eduardo Flores-Sandoval"
        },
        {
          "@type": "Person",
          "name": "D. Magnus Eklund"
        },
        {
          "@type": "Person",
          "name": "John L. Bowman"
        }
      ],
      "identifier": "10.1371/journal.pgen.1005207"
    }
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