Outline of Final Project

 

INTRO:

 

Regeneration is regrowth of structures from a collection of stem cells

• Regeneration occurs in both animals and plants
  • Limbs of geckos (Alibardi 2009)
  • Crassulaceae and Cactaceae families can regrow roots from detached leaves on soil (Xu & Huang 2014)
• Dedifferentiation is rare—and much more common in plants
  • Can cut off apical tips that harbour pluripotent cells and the plant will de-differentiate ends to make new pluripotent cells (Xu et al. 2006)
  • But interconversions generally happen between roots-roots or shoots-shoots. There are some limitaitons

 

Most organisms do not have de novo dedifferentiation though

• plants can be induced to dedifferentiate, but it requires the intervention of plants hormones (Xu & Huang 2014)
  • high auxin, low cytokininà forms callous from which new shoots can grow
• dedifferentiation is not complete—need certain ‘collection’ of regenerative cells
  • procambium and cambium can give rise to new roots and shoots, but other cells can’t
• Bryophytes, on the other hand, CAN dedifferentiate.

 

Quick recap of bryophyte lifecycle:

• Begins as chloronema. Uniseriate, photosynthetic, round chloroplasts (Vanderpooten and Goffinet 2010, Kofuji 2014)
• Then, will grow into fast-spreading caulonema (Kofuji 2014)
  • Caulonema and chloronema and collectively known as protonema
• Form buds, which form gametophyte (1N) stage
• Gametophyte is main stage of life—it produces gametes (sperm and egg) and forms a sporophyte (2N)
• Short sporophyte phase will produce spores, which are spread and germinate into new protonema

 

Bryophytes can dedifferentiate from all states—a trait observed a long time ago.

• leafy gametophyte, rhizoids, and sporophyte all do this (Giles 1971, Westerdijk 1907, Von Wettstein 1924, von Maltzahn 1959)
• if they stay attached, they will not dedifferentiate (Mnium affine) (Giles 1971).
• Once cut, they will show changes in certain cells
  • Chloroplasts grow and divide (Giles 1971, Ishikawa et al 2011, Sakakibara et al 2014)
  • Divide asymmetrically; top cell becomes new protonema (Ishikawa et al 2001,
• Grows a whole new plant

 

BUT it gets complicated.

• some species will grow tonnes of protonema (Funaria); some will grow protonema JUST from detached boundary (Physcomitrella patens); and others won’t dedifferentiated at all (Dawsonia) (Giles 1971, Westerdijk 107; Von Wettstein 1924)
• not all cells are equal either; there is often polarity associated with dedifferentiatbility
  • sporophytes dedifferentiate easier near the apex (Von Wettstein 1924)
  • protonema will dedifferentiate better at basal area than tip
• Cells are arrested in G2—which mean they have a duplicated haploid genome! (Ishikawa 2011)

 

Possible mechanisms for triggering dedifferentiation:

• there is overall instability in system—so when you tear leaf off, you throw off the polarity of cells
  • von Maltzahn (1959) suggested leaves get signals from apex to tell them not to dedifferentiate
  • plants have similar signal for wounding: they have a constant auxin flow and when there is a wound, auxin builds up on the basal side and lacks on the apical side, so it changes transcription to initiate healing (asahina et al 2011, read and ross 2011)
  • thus, likely due to expression of thousands of different genes (Xiao et al 2012)

 

Other possible triggers:

• external cues and hormones
  • regular protonmea communicate to eachother via Factor H and Factor F— it would not be impossible for similar things to happen in gametophyte
  • light and dark appears to sometimes affect which way the plant will dedifferentiate, so perhaps you need external cues too?

 

Finally, it’s possible that the trigger is due to the direct production of wounding-proteins

• this is in contrast to the ‘lack’ of polarity theory, which involves the ABSENCE of certain factors
• this might make sense because there have also been reports of mosses that redifferentiate while still attached to main plantà perhaps the leaves got damaged
  • or, the species that show lots of dedifferentiation maybe got ‘damaged’ more and thus shows more protonemal growth

Goal is to determine the TRIGGER for dedifferentiation in physcomitrella patens

• two main papers are about the pathways causing cell dedifferentiation
  • Ishikawa et al (2011) discovers that CDKA is always present in cells (polar argument evidence?) but something triggers it to activate CDKD when leaf cells are cutà results in dedifferentiation
  • Sakakibara et al (2014) found a homeobox 13-like gene (related to stem cell regulators in flowering plants) that increases when you cut cells
• My hypothesis is that there is some sort of ‘constant’ signal throughout the plant that makes it stable, and removing cytoplasm will cause dedifferentiation
  • Want to remove cytoplasm, but keep leaf cells in-tact

 

Studying stem cell regulation has always been of interest to us

• new therapeutic methods in medicine
• learn about development and important regulators that may cause genetic defects
• learn how to improve crop yields or produce clones of desirable plants

Studying in moss is interesting because it is different!

• shows more plasticity than ANY other plant
• we want to know WHY and HOW
• perhaps we can apply moss abilities to other organisms and improve culturing techniques, medical treatments, and general knowledge

 

Experimental approach:

 

1. Obtain strain of Physcomitrella patens similar to one in Ishikawa et al (2011) paper
   1. If possible, obtain the one with the CDKD-GFP-GUS transformation
   2. If not, follow the procedures in Nishiyama (2000) and Shaefer (1994) to produce a strain of Physcomitrella patens with a GFP reporter enzyme attached to the CDKD protein via homologous recombination.
   3. Raise several back-up strains—grow on BCDATG media for regular growth, and BCDAT for maintenance growth. The latter just makes protonema grow slower, which would make it easier to take care of.
   4. Pour petri plates of media covered in autoclaved cellophane; germinate with paten on top of cellophane to prevent protonema from growing into agar: makes it easy to remove whole-gametophytes and protonema
2. Treatments: For all treatments (except positive control), I will remove WHOLE gametophytes (4 weeks, Nishiyama 2000) and place horizontally on new media with cellophane. I will gently ensure some leaves are pressed down on the media. These leaves will be used for experimentation. Each gametophyte will have one of each type of treatment to ensure control of variables across treatments. Each treatment will have 5 plate replicates with 3 treatment leaves on each plate. This means each treatment will have a total of 15 leaves treated. After all treatments, plates will be incubated at 25 degrees C with constant light (Shaefer 1994).
   1. REMOVAL OF CYTOPLASM (Treatment)
      1. Use glass micropipette (stretched out vertically to produce small pore of 0.2um, as described in Mackler 1992)
         1. Mackler removed cytoplasm from neurons, but I will do it in bryophytes
      2. Remove cytoplasm from cells across the centre of the leaf cell; go all the way across the cell

• Incubate

1. PIPETTE CONTROL
   1. To control for pipette prick, repeat procedure for 1(a) except do not remove cytoplasm.
   2. Incubate
2. NEGATIVE CONTROL
   1. Do not damage or remove leaves from plant
   2. Incubate
3. POSITIVE WOUND CONTROL
   1. Using sharp knife, cut a straight line across a leaf, making sure to damage the entire line of cells
   2. Keep leaf tip ‘attached’ to leaf base
4. POSITIVE DETACHMENT CONTROL
   1. Using sharp knife, cut a straight line across a leaf
   2. Remove leaf tip and relocate 1cm away from rest of plant
5. Using a light microscope, observe plants at 1h, 6h, 12h, 48h, and 2 days and mark/observe the following traits:
   1. Chloroplast count
   2. Budding or abnormal growth
   3. Cell division
   4. At the 2 days mark, protonemal growth should have appeared already (Ishikawa et al 2011)
6. I will compare the above characteristics with CDKD-GFP expression under UV light at each time point. I will also try to characterize specific light intensities
7. Compare relative success of dedifferentiation across all treatments.

 

Possible results:

 

If my hypothesis is correct, then I expect that by removing cytoplasm