Xu, L., & Huang, H. (2014). Genetic and epigenetic controls of plant regeneration. Current topics in developmental biology. 108: 1-

 

• tissue/organ repair AND generation of new plants (Birnbaum & sanches Alvarado, 2008); sgimoto, Gordon & meyerowitz, 2011).
• Tissue colture, you can see when adding hormones (sussex 2008, gautheret 1983, Thorpe 2006, 2007)
• Animals can regenerate—but this requies movement of stem cells; plant cells can’t move
• De novo organogenesis—grows from cut pieces
• Arabidopsis thaliana—need callus-inducing medium with high auxin and low cytokinin.
• Somatic embryogenesis—single somatic cell turns into embryo of plant.
  • Yang and zhang 2010
  • Suggests totipotency of cells; verdeil, alemanno, niemenak, tranbarger 2007)
• Xu et al 2006à can regenerate root tips by cutting of cells; auxin accumulates in new ‘end’ cells and causes protein cascade which transforms QC-adjacent cells into new QC cells
• Feldman 1976—showed that root tips can be quickly regenerated into a new QC (quiescent center)[ at tip of root apical meristem (RAM) and has surrounding stem cels; hs 2-4 cells that divide very slowly; controlled by TF WOX5. (Sarkar et al 2007)
• sena et al 2009à showed stem cell niche was not needed; it cut off QC instead of lasering it off. (abalation)
  • also high levels of auxin involved
  • using auxin polar transport-inhibitor NPA (naphtylphthalamic acid)à blocked QC regeneration
  • must be within 130um; higher means less ability to regenerate
• WOUNDING
  • Basipetal auxin stream blocked at wounded positions (asahina et al 2011, reid and ross 2011)à high auxin, low auzin below. Activates high and low promoters and both needed for healing
  • Also need jasmonic acid and ethylene (asahina et al 2011)
• DENOVO ORGANOGENESIS
  • We need to induce via calluses; but in nature perhaps you produce less obvious callus
  • Callus formation is not actualy an undifferentiated totipotent group of cells; actually, it is a mass of root meristem tip cells that resembles lateral root formation (Atta et al 2009, che, lall, howell 2007, he, chen, huang and xu, 2012; sugimoto, jiao, meyerowitz 2010)
  • Callus formation initiated with divisons from xylem-pole pericycle cells (atta, che), which is also where lateral roots come from (benkova and bielach 2010; peret et al 2009)
  • Callus no longer totipotent; is pluripotent bc not dedifferentiated; its transdifferentiated (sugimoto et al 2010, 2011)
  • PcG (polycomb group) in mutants do not allow de-regulation of ‘leaf’ genes—so they thing PcG plays a role in supressing leaf genes. Done by depositing H4K27me3 histones on leaf gene loci (he et al 2012)
• De novo root organogenesis—in Arabidopsis, can see new roots originate form vascular procambium or cambium (ahkami et al 2009; correa lda et al 2012; greenwood, cui, Xu 2001)
  • – suggests that auxin needs to be involved; because blocking it prevents rooting process.
  • Possible similar to lateral formation form hyopcotyls
  • Wound signal—absent during lateral or adventitious root formation from hypocotyls; wound signal not yet defined.
  • Xylem-pole pericycle, preprocambium, procambium, cambium – all have different stem cell features
• Procambium and cambium= pluripotentn vascular peristem in primary and secondary development of vascular tissues, respectively (elo, immanene, nieminen, helariutta 2009)
• Preprocambium is progenitor of procambium (sam as above)
• Xylem-pole pericycle cells give rise to cambium *rost, barbour, stocking and murphy 1997)
  • Seems like you NEED stem cells to regenerate roots/shoots
• CARROT SOMATIC EMBRYOGENESIS
  • Steward, mapbs, mears 1958
  • Very complex; involves hormones, TF, epigenetic reg, yang and zhang 2010)
• Somatic embryogenesis
  • Balance between GA and abscisic acid (ABA)—(de castro and hilhorst, 2006; hays, mandel, pharis 2001); hu et al 2008; ogawa et al 2003; Phillips et al 1997; etc etc…
  • Embryo cells: low ratio of GA to ABA; ratio higher in somatic cells (braybrook and harada 2008)—suggested that auzin might induce/initiate somatic embryogenesis but changes in GA/ABA ratio may provide suitable environment for cells to become comptent. Also, ethylene may be involved. (bai et al 2013, piyatrakul et al 2012; zheng, zheng, perry 2013)
  • Two classes of TF important in Arabidopsis: LEAFY cotyledon (LEC) genes and agamous-like15 (AGL15)—both only expressed in embryo and overexpression shows embryo-like symptoms
  • AGL15 might inhibit GA pathway and upregulated auxin signalling gene (Zheng et al 2009)
  • LEC2 promotes auxin pathway—all these make up genetic network that promotes ABA and auxin, but inhibits GA
  • Some interplay between pathways; regulate each other
• Epigenetic regulation of somatic embryogenesis
  • pcG and PICKLE (PKL)pathways)—mutations in both can result in somatic embryogenesis
  • PcG retains somatic ell identity
  • Arabidopsis: PRC1 and PRC2 are PcG complexes.
    • PRC2—mediates H2K27me3 leaf-to-callus formation (he 2012) and also repressing embryonic genes during embryo-to-seedling phase (bouyer et al 2011)
    • PCR2= incomplete transition from embryo to seedling; mutant seedinly has disorganized cell divisions (bouyer et al 2011)
    • Lec1, lec2, fus3 extopicaly expressed in PRC2 mutant (Makarevich et al 2006)
  • Genome-wide analysis of H2K27me3 suggests embryo-sepcific genes are highly trimethylated in somatic cells (Zhang, Clarenz eta l 2007)
  • PCR1: contain 4 major proteins: LHP1 (like heterochromatin protein1, atRING1a/b, AtBMI1a/b and EMF1
    • LHP1 recognizeds H3K27me3; second two are responsible for H2A ubiquitination after PRC1 bins to targets (Bratzel et al 2010);
    • Mutations in a/aà Lecs, AGL15, etc (Bratzel et al 2010 chen et al 2010)
  • PRC1 and PRC2à may function together to control the same embryonic genes
• Epigenetic PKLà codes CHD-type ATP-dependent chromatin remodelling factor; LOF first identified as GA-deficient mutant (Ogas, cheng, sung and Somerville 1997; 1999)
  • Mutations in PKL- ectopic expression of LEC1/LEC2, FUS3
• WOUND SINGAL
  • All three types of plant regeneration triggered by wounding
  • Changes hormone biologyà gene expression
  • Molecular nature of wound singal remains unclear
  • Possible suspects: plasma transmembrane potential; Ca; reactive oxygen species; palnt hormones; metabolic processes (leon, rojo, sanches-serrano 2001; maffei, mithofer, boland 2007)—complex bc could also trigger defense
  • In arapidopsis, wound induced dedifferentiation 1 (WIND1) (iwase et al 2011; iwase, ohme-takagi, sugimoto 2011)à induced at wounded region (but don’t know how) and seems to regulate via cytokinin pathway (same as above)
• MOSS REGENERATION
  • Physcomitrella patens—regeneration possible in many tisues and cells and is easily triggered
  • Think it might be due to change sin expression of thousands of genes (xiao, zhang, yang, zhu, he 2012)
  • Ishikawa—CDKA was induced priper to transition and DNA synthesis inhibitors (aphidicolin) could NOT stop transition and did not block induction of protonema-specific genes.
    • Suggests cell-fate transition occurs before and independent of cell division

 

Sakakibara, K., Reisewitz, P., Aoyama, T., Friedrich, T., Ando, S., Sato, Y., Tamada, Y., Nishiyama, T., Hiwatashi, Y., Kurata, T., Ishikawa, M., Deguchi, H., Rensing, S.A., Werr, W., Murata, T., Hasebe, M., & Laux, T. (2014). WOX13-like genes are required for reprogramming of leaf and protoplast cells into stem cells in the moss Physcomitrella patens. Development. 141: 1660-1670.

• cells at wound margin of detached leaves become reprogrammed into stem cells
• patens WUSCHEL-related homeobox 13-like genes (PpWOX13L)—homologs of stem cell regulators in flowering plants—upregulated and required for initiation of cell growth during stem cell formation
• Deletions fail to upreg genes encoding cell wall hoosening factor homologs
• Mutant sygotes fail to expand and initate an apical stem cell to form the embryo
• Analogous to WOX stem cell functions in seed plants, but using different cellular mechanism
• Dedifferentiation occurs more readily in plants (Birnbaum and sanchez Alvarado 2008)
• NATURAL CONDITIONS? (Steeves and sussex, 1989)
• Prigge and bezanilla 2010* similar to ishikawa?
• Saw ppWOX13LA and ppWOX13LB throughout life cycle, but no ppWOX13LC at any developmental stage
  • Used GFP knock-in and saw it in all nuclei examined—but higher in apical cells of chl/caul vs subapical, and higher in egg/zyg compared to surrounding
• Did detached leaf assay—saw whole-leaf ppWOX13LA?B transcript levels transient increase after detachment
  • First 12h; all cells increased slightly—afterward, only cells facing cut site and that eventually become stem cells were bright!
• Single mutants seem normal—a little longer to grow things, but they catch up and do alright.
• Double mutants show normal protonema and gametophores; but no mRNA was detected at all. Fewer sporophytes… seemed to develop normal sexual organs…seem to fertilize okay (but don’t know for sure) but the zygote doesn’t grow and split in2 like normal WT.
  • Even under 2 months of gametangia-inducing conditions, no sporangia.
  • Some dbl mutants formed malformed porangia without normal spores… like parthenogenesis? (definition?)
• showed the gametes were OKAY by crossing with WT—92.5% success (WTxWT)vs 5.6 success (dbl mutant) vs 8.9%dblxWT) n=10
• re-grew some normal looking ones form dbl cross—one ended up having WT but 2 had all mutant alleles… so means they Can fertilize.
• Normal: divide, then grow. Some naturally fail to grow.
  • Double mutant-> less cells that can grow tip (but dividing ok)
  • Single mutant-> somewhere inbetween for a, but ok for b.
  • 48 hours later, protonema reporters RM09 and RM55 were present in all divided cells, regardless of whether they continued to grow.
    • Suggests ppWOX13L is required for cell growth, not division.
  • In normal chloronema development, they cultured WT and mutant on high osmolarity medium. Appeared delayed; dodn’t branch, but split into 2 without growing
    • But on normal medium, they look indistringuishable—suggests potential role in cell wall expansion
    • – NOT DONE

 

 

 

Kofuji, R., & Hasebe, M. (2014). Eight types of stem cells in the life cycle of the moss Physcomitrella patens. Current Opinion in Plant Biology. 17: 13-21.

 

• physcomitrella patens has 8 stem cells
• need niche cells like in metazoan
• haploid bodies of flowering plants have reduced number of cells and have no stem cells (3,4)
• chloronema has apical cell and can branch form random cells, but we don’t know how yet (9)
• chloronemaà bright green; photosynth
• caulonemaà spindle, less green; for expansion
• gametophyte has apical cellà produced primordial cells for stem and leaves (25)
• unlike other plants, no callous forms
• wounding (53)
• no exogenous hytohormones; within 48 hours!
• Intrinsic auxin, cytokinin regulatory systems, transcriptional regulators change (55, 56, 57)

 

Nishiyama, T. Digital gene expression profiling by 5’end sequencing of cDNAs during reprogramming in the moss physcomitrella patens

• how to transform P. patens
• Schaefer 20à transformation procedures
• Schaefer, D. 1994, Molecular genetic approaches to the biology of the moss Physcomitrella patens [PhD Thesis], University of Lausanne, (http://www.unil.ch/lpc/docs/ DSThesis.htm)

Busch, H. Network theory inspired analysis of time-resolved expression data reveals key players guiding P. patens stem cell development PLoS one 2013 8:

 

Chopra, RN Biology of Bryophytes

 

Xiao

(Giles, K.L. (1971). Dedifferentiation and Regeneration in Bryophytes: A Selective Review. New Zealand J of Bota. if they stay attached, they will not dedifferentated

• if they stay attached; they don’t grow
• Mnium affineà only have 10-15 bud producing cells, but can form 100s of protonema
• Splachnumà can’t tell which ones will regenerate
• FUnaria hygrometricaà
• Some kind of innate ‘stability’ in whole plant that breaks down when you take leaves off
• Perhaps some sort of polarity? (Giles, von Maltzahn 1959)
  • Suggests this because some parts of plant, like sporophyte, will better differentiate than other parts. Basal protonema tends to be better than apical (Westerdijk 1907)
  • Von Wettstein (1924)à sporophyte is better at apical and decreases at basal
  • Can also change on which direction you grow it via light
• Giles and von Maltzahn (1967; 1968)
• Different in different plants: Dawsonia don’t dedifferentiate but display chloroplast characteristics (Giles 1971)

Ishikawa

2011