TEMPLATE FOR PROJECT OUTLINE

Student’s name: Melissa Chen

Topic chosen: Leaf cell dedifferentiation in Bryophytes

SPECIFIC QUESTION:

What is the triggering mechanism that allows differentiated leaf cells in bryophytes (specifically, Funaria hygrometrica) to dedifferentiate and become identical to protonemal cells?

HYPOTHESIS:

The dedifferentiation of detached leaf cells is caused by the absence of certain cytoplasmic inhibitors in Funaria hygrometrica.

 

EVIDENCE ON WHICH THE HYPOTHESIS IS BASED (INCLUDE REFERENCES):

 

References are in annotated bibliography section.

 

PART 1: Regular protonemal development

Protonema is a totipotent-like state that emerges from germinating spores (Vanderpooten and Goffinet 2010). There are two stages: chloronema and caulonema. The chloronemtal stage precedes the caulonematal stage, and it has been shown that the two stages release different hormone factors into the environment (Bopp 2008, Brandes and Kende 1968). Factor H (which is secreted by caulonema) promotes bud formation, whereas Factor F (which is secreted by chloronema) inhibits bud formation. These factors are detected by neighbouring protonema in a concentration-dependant manner, and there are many cytokinin analogues that can produce similar effects.

 

PART2: Leaf dedifferentiation

It has been shown that fragments of bryophytes have the amazing ability to revert back into protonematal stages (chloronemal-identical stage) (Ishikawa et al. 2011). It seems that separating (by cutting, ripping, etc) leaves from the main stem will result in dedifferentiation, whereas attached-leaves usually do not tend form protonema (although there have been many notable exceptions) (Giles 1970). Some sources report that it is the cells near the ‘cut’ site (boundary cells) that ultimately sprout protonema (Ishikawa et al 2011), but personal observations have suggested that protonematal re-development occurs along the entire leaf lamina. It is unclear whether the dedifferentiation of boundary cells is unique to certain species (such as Physomeitralla) or situations.

 

PART3: What is known about the pathway and different hypothesis

It is known that a protein Cyclin-dependent kinase A (CDKA) is necessary for dedifferentiation of leaf cells into protonemal cells by causing leaf cells to re-enter the mitosis cycle. Since all cells in bryophytes are arrested in the S-phase of mitosis (unlike most other angiosperms, who are arrested in the G1-phase), the re-entering into the cell cycle is initiated by an asymmetrical division of leaf cells. This asymmetrical division will then give rise to an apical and basal cell—the former of which forms the first protonemal cell. Although CKDA is present in all gametophyte tissue, it must be activated by CDKD in order to promote cell division. Previous work by Ishikawa et al. (2011) has shown that boundary cells in Physomeitralla express CDKD, suggesting that injury to tissue somehow initiates cell dedifferentiation. Some sources suggest that leaf tissue in bryophytes possess an internal ‘polarity’ to them, which can be destroyed by eliminating the connection of the leaf to the stem (Giles 1970). It is also possible that gametophytic tissue excretes some type of hormone or signalling factor to repress protonemal growth in the gametophyte stage. Protonema already uses this system to signal to each other when to form gametophytic buds, so it is easy to see how such a system could be modified to perpetually supress protonemal growth. This model may also explain why leaves sometimes still dedifferentiate into protonema despite being attached to the main shoot. Lastly, it is possible that there is some ubiquitous factor within gametophytic cytoplasm that supresses the transcription of CDKD, and consequently the activity of CDKA. When tissue is damaged or cut, the loss of neighbouring cytoplasm—and consequently, repressing factors—may initiate CDKD transcription and cause dedifferentiation. This would explain by in-lab test have shown CDKD to be active only around cut sites, whereas other experiments have shown protonemal growth all over the leaf. The growth of protonema from all over the lamina could be due to small puncture wounds on the leaf when removing leaf tissue from the stem. This last model is the question I will be testing. I plan to either confirm or eliminate the possibility of cytoplasm repressive factors acting as the trigger for CDKD transcription, and ultimately cell dedifferentiation.

 

 

 

 

 

PREDICTION(S):

I predict that by removing cytoplasm from mid-lamina cells in a leaf that is still attached to the main shoot, I can induce cell dedifferentiation and resulting protonemal growth.

 

 

EXPERIMENTAL APPROACH TO TEST PREDICTION (INCLUDE ANY DETAILS THAT YOU HAVE WORKED OUT SO FAR):

 

First, obtain a transgenetic sample of Funaria hygrometrica that has a GFP reporter gene attached to the end of CDKD.

 

Next, I will attempt to induce protonemal development by removing the cytoplasm from lamina cells of leaves still attached to the main shoot. This will include 4 treatments:

 

• Whole leaves, detached from the main stem (positive control)
• Whole leaves, still attached to main stem (negative control)
• Whole leaves, still attached to main stem with mid-lamina cells punctured by a small needle (puncture control)
• Whole leaves, still attached to main stem, with mid-lamina cell cytoplasm removed with small needles.

 

All treatments will be cultured on Knop’s Agar (a common media for bryophyte cultures) and be kept under indirect sunlight at natural light cycles. I will be assessing the cultures for protonemal growth, speed of growth, and location of growth, as well as the presence of the GFP reporter gene. I expect the reporter gene will be present around the areas that protonema emerge.

 

If my hypothesis is correct, I expect to see protonemal growth at the cells directly adjacent to the cells that I choose to remove cytoplasm from. Additionally, I would expect to see no protonemal growth in the positive control or puncture control. This result would suggest that there is indeed some repressive factor in the cytoplasm that prevents spontaneous protonemal growth from occurring.

 

Some other possibilities would be if I saw the puncture controls growing protonema out of the punctured cells, but no protonema growing from the treatment with cytoplasm removed. This would instead suggest that cell injury causes some kind of protein cascade that ultimately causes CDKD expression.

 

Other interesting results may include protonema growing from cells that were neither punctured nor next to empty cells. Protonema may also grow on the positive control, which may suggest exogenous signals (hormones, gravity, nutrients, etc) at play.

 

 

LIST OF RELEVANT PRIMARY AND REVIEW ARTICLES READ, AND SUMMARY OF RELEVANT INFORMATION FROM EACH (this is the start of an annotated bibliography):

 

Brandes, H. & Kende, H. (1968). Studies on Cytokinin-Controlled Bud Formation in Moss Protonema. Plant Physiol, 43, 827-857.

This reference looks as the effect of various hormone analogues on protonema of Funaria hygrometrica. They show that some naturally produced hormones (called Factor H) induce bud formation on protonema, and that this can also be induced by adding cytokinins to the protonema. These hormones/cytokinins appear to be needed at a constant concentration in order to induce bud formation, and washing away these factors during bud development will stop development. Buds that have these factors removed will often revert back to protonema. They also show that it is possible to reverse bud formation by applying indole-3-acetic acid (IAA).

 

Szweykowska, A. (1961). Kinetin-induced formation of gametophores in dark cultures of Ceratodon purpureus. J. of Experimental Botany, 14(40), 137-141.

This reference shows that it is possible to induce bud formation in the dark by simple application of kinetin—which is crazy because one would assume light is an incredibly important energy source to have when developing gametic tissue.

 

Saunders, M.J., & Hepler, P.K. (1983). Calcium antagonists and calmodulin inhibitors block cytokinin-induced bud formation in Funaria. Developmental Biology, 99, 41-49.

This references adds onto the process of bud formation in mosses. They show that in Funaria hygrometrica, the cytokine-induced bud formation relies on the rise of intracellular calcium. It appears that calcium sources are from the outside of the cells, since calcium uptake inhibitors will prevent cell division, and consequently differentiation. This suggests that mosses are often reliant on exogenous signals to develop, and that nutrient availability may play a role in signalling to cells when to develop.

 

Bopp, M. (2008). Development of protonema and bud formation in mosses. J. Linn. Soc. (Bot.), 58(373), 305-309.

This reference shows that the hormones that protonema excrete work in a concentration-dependent manner, and that different factors are released at different stages to induce growth patterns. Protonema appear to inhibit each other, and thus promote a spread-out growth pattern. They identified two factors : Factor H (secreted from caulonema) which promotes bud formation, and Factor F (secreted from caulonema) which inhibits bud formation.

 

La Farge, C., Williams, K.H., & Engalnd, J.H. (2013). Regeneration of Little Ice Age bryophytes emerging from a polar glacier with implications of totipotency in extreme environments. PNAS, 110(24), 9839-9844.

This paper explores how mosses are able to germinate from gametophyte fragments that were previously frozen in glaciers. Most of the fragments appear completely dead—yet they are able to produce protonema!

 

Giles, K.L. (1970). Dedifferentiation and regeneration in Bryophytes: A Selective Review. New Zealand Journal of Botany, 9, 689-694.

This paper claims that leaf cells tend to de-differentiate when detached from the main plant, and that once leaves are isolated they seem to lose stability of differentiation. They report that chloroplast movement after 48 hours of isolation marks the beginning of de-differentiation. The chloroplasts will move to one side of the cell, causing elongation of the leaf cell. Isolated leaves have been reported to produce up to 100 secondary protonema.

 

Ishikawa, M., Murata, T., Sato, Yoshikatsu, Nishiyama, T, Hiwatashi, Y., Imai, A., Kimura, M., Sugimoto, Nagisa, Akita, Asaka, Oguri, Y., Friedman, W.E., Hasebe, M., & Kubo, M. (2011). Physomitrella cyclin-dependent kinase A links cell cycle reactivation to other cellular changes during reprogramming of leaf cells. The Plant Cell, 23, 2924-2938.

This paper shows the first molecular mechanism discovered in bryophytes involved in dedifferentiation of cells. First, they show that leaf cells are arrested in the S-phase by comparing the genomic content with other plants of similar genomic size. They use Physomitrella as a model organism. They show that when you cut leaves off Physomitrella, the cut edge will form cells that are indistinguishable from the apical cells of chloronemata. They found that a protein called CDKA is necessary for this cell cycle progression, and that it needs to be activated by CDKD.

 

Van der Poorten, A. & Goffinet, B. (2010). Introduction to Bryophytes.  Cambridge University Press: New York.

This textbook is a general guide to mosses and liverworts. It is helpful in the general terminology and anatomy of bryophytes.

 

 

HOW DOES THE QUESTION FIT INTO THE BROADER PICTURE, AND WHAT IS ITS IMPACT?

 

Bryophytes can revert differentiated cells into a totipotent state, and this is an ability that is not only unique, but extremely impressive. By studying the mechanisms that allow bryophytes to de-differentiate, one could apply the same concepts to other models. There is still so much to learn about the significance of the bryophyte life cycle: Why are cells arrested in the S-stage of mitosis? Is this relevant to its regenerative abilities? Can we use similar transgenic protein cascades to control the potency of human cells?—and I believe that studying this mechanism will provide insight on different approaches to stem cell research and widen our knowledge of the wonderful diversity in developmental adaptations.

 

 

 

POTENTIAL WAYS TO MAKE YOUR QUESTION KNOWN TO THE PUBLIC AT LARGE (OR TO YOUR NON-BIOLOGIST FAMILY AND FRIENDS):

 

Talk about it! Sign up for every conference I can think of and just get the information out there. I doubt most people realize the strange S-phase characteristic of mosses, and I think lots of people would be convinced mosses are cool if they are just given the chance.

 

As for my non-biologist family or friends, I would frame my work in the perspective of ‘stem cell research in plant models’. By linking this research with something they are already aware of and consider ‘important’, it would be easier to get them excited about moss research!

 

 

 

 

 

 

ANY OTHER PARTS OF THE PROJECT COMPLETED SO FAR:

 

 

Not much else—I’m planning to work on it over the break!

 

 

 

 

 

 

ANYTHING YOU WOULD LIKE SPECIFIC FEEDBACK ON:

 

Would you like me to do a more experiments in this project? I could design experiments to test some of the other hypothesis I mentioned above, but wasn’t sure if you wanted something simple or something more comprehensive.