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The moss P.patens has been used as a versatile experimental model organism for the past 80 years and it falls in the division Bryophyta. Being relatively simple in morphology and it generates only few tissues that contain limited number of cell fates (Mark leech et al., 1993), it is extensively being undertaken by plant biologists on both basic and applied strategies covering major areas namely developmental biology, evolution, systems biology, biotechnology ,biodiversity etc., As the organism is predominantly haploid in nature, it is evident that it could allow to develop insights on straight forward investigation/understanding of plant systems and hence, it is amenable for genetic and molecular level studies (Cove., 2009). The availability of complete genome sequence information, genetic and physical map ease the utilization of P.patens in all most all the fields of biology (Yasuko Kamisugi., 2008). It is the only land plant with an efficient system of homologous recombination in its nuclear DNA by which specific gene targeting could be achieved and the strategy of loss of function mutants can be generated by RNAi approach. In this context, the functional genomics of P.patens helps in identifying novel genes which could be employed in metabolic engineering and stress tolerance like drought, salt and osmotic stress thus helps in improving the crop plant performance. Moreover, it serves as a valuable platform for the production of recombinant pharmaceuticals (Anna K. Beike., 2010) Thus, the transfer of novel genes from P.patens has a greater biotechnological impact and may help in better public acceptance. Hence, this presentation aims to confine the advantages of this model plant in plant molecular research by discussing its efficiency in reproduction, range of technologies applied, some interesting characteristic features behind this model and chosen case studies will establish the model behaving as a well-versed medium for wide variety of approaches
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Introduction
Established models
Emerging model system
Species fact sheet
Timescale of evolution
Life cycle
Comparative studies
Drug production
Epigenetic regulation
Moss culture
Abiotic stress tolerance
Gene silencing
Metabolic engineering
Experimental evidences
OverviewOverview OverviewOverview
Ultimate goal of modern biology- relationship between biological systems , presence and activity of genes
Model systems serve as the excellent platforms for exploring the biological relationships and functions
[Didier Schaefer.,2002]
[Cove et al., 1993]
Physcomitrella has been developed as a model system to study plant gene function
Versatile model
First established as a laboratory experimental system in the 1920s by Fritz von Wettstein (1924)
Bryophyta are the simplest and ancient lineage of land plants
It includes mosses, liverworts and hornworts
The mosses and flowering plants diverged more than 450 million years ago.
[Henrik Toft et al., 2009]
Models like Physcomitrella patens serves solving inquisitive puzzles in plant biological systems
Kingdom Plantae
Plants Division Bryophyta
Mosses Subdivision Musci
Class Bryopsida -True mosses
Subclass Bryidae
Order Funariales
Family Funariaceae
Genus Physcomitrella Bruch & Schimp.
Species Physcomitrella patens
(Hedw.) Bruch &
Schimp. [Source:USDA.gov, NRCS ]
[Knight.,2009]
[Michael Prigge et al., 2010]
Physcomitrella is well-placed phylogenetically to provide important comparisons with the flowering plants
Distinguishing features of Physcomitrella patens
P. patens is a monoecious moss- requires very simple growth conditions
It is a terrestrial non vascular plant
Relatively simple morphology, with fewer cell fates than in flowering plants.
[Mark leech et al .,1993]
Sporophyte (2n)Spore (n)
Protonema (n)
Gametophore (n)
Gametophore Colony (n)
[Sung Hyun Cho.et al.,2007]
DNA barcodingDNA barcoding
Systems biology
Applied studies
Bryotechnology
biopharmaceutical
Abiotic stress tolerance
[Tomoaki Nishiyama et al.,2003]
BryologBryology y
[Anna K. Beike et al., 2010]
The assembled P. patens genome (511 Mb) -released by the Joint Genome Institute
[Ralf Reski.,2005]
Transcriptomic analyses illustrate commonalities among plant lineages in gene content, structure, and regulation
[Ralph Quatrano.,2007]
Sequence-anchored
genetic linkage map for
the moss, P.patens has
been established
[Yasuko Kamisugi.,2008]
One-quarter genome contains genes with no known function –key to identify new and novel gene functions.
[David Cove.,2009]
More than 2,50,000 ESTs are available covering 95 % of moss transcriptome
[Rensing et al., 2002]
Phytohormones like auxin , cytokinin,ABA and photomorphogenetic pigments are found to be intact in P.patens
[Cove et al., 2009]
A remarkable feature P.patens is its ability to incorporate transforming DNA at targeted sites -Homologous recombination
[Yasuko et al.,2006]
Efficient system for reverse genetics
[Strepp et
al., 1998]
Versatility…
Genome analyses of the moss P.patens has revealed -57 families
of nuclear genes were acquired from prokaryotes, fungi or viruses
[Jipei Yue et al.,2012]
[Jipei Yue., 2012]
Horizontal gene transfer
[HK Stenoien., 2005]
[Anna Beike et al ., 2010]
Protoplasts of the moss P.patens easily regenerate into protonema and therefore provide an ideal system to explore how differentiated cells can be reprogrammed to produce stem cells.
Epigenetic regulation… Epigenetic regulation…
[Lihong Xiao et al .,2012]
[Bestor.,1988]
[Daniel Lang et al., 2008]
Culturing the mossCulturing the moss
Continuous light from fluorescent tubes at an intensity of between 5 and 20 W/m2
Either on solid and liquid culture
High capacity of regeneration
Axenic growth
Temperatures between 24°C and 26°C
[Cove.,2005]
Abiotic Stress tolerance in P.patens
[Anna Beike et al ., 2010]
Tolerates water loss of up to 92% and were able to recover successfully
Tolerates up to 350mM of NaCl and 500mM of sorbitol
P.patens is highly tolerant against drought, salt and osmotic stress
[Wolf et al., 2005]
439 genes encoding transcription –associated proteins in response to salt stress and ABA was reported by microarray expression analysis
[Sandra Richard et al., 2010]
Breakthrough technology…
miRNA important regulators of gene expression for both plants and animals
miRNA families are found to be conserved in evolution
Genome-wide expression analyses in Arabidopsis - high specificity of amiRNAs
amiRNAs can be designed to target any gene of interest- functional gene analysis
Genome-wide expression analyses in Arabidopsis - high specificity of amiRNAs
amiRNAs can be designed to target any gene of interest- functional gene analysis
Tested for amiRNA function in Physcomitrella
Gene PpFtsZ2-1, which is required for chloroplast division
PpGNT1 gene encoding an N-acetylglucosaminyl transferase
amiRNA expression in P.patens
[Basel Khraiwesh et al., 2008]
LC-PUFAs important for human diet-C22 PUFAs
Marine fish and algal oils chief source
But…
High production cost,diminishing feed stock limit the supply
Metabolic engineering of an artificial pathway that activates the production of C22-PUFAs in P.patens
Production of Docosatetraenoic acid (ADA) and n-3 docosapentaenoic acid (DPA)
Pavlova sp. Encodes D5-elongase
Transgenic P.patens with vegetable oil supplementation.
Requires a economic and sustainable source….
[Pichit et al.,2012]
I. a.Osmotic stress treatments
Experimental evidences
[Wolf et al., 2005]
I.b.Dehydration treatment
Overexpression construct
PCR screen
II. Gene silencing by amiRNA
RACE PCR for amiRNA transgenic lines RNA gel blot analysis
[Basel Khraiwesh et al., 2008]
Structure of pMDC43-PsELO5
Primers used for PCR amplification
III. Metabolic engineering
Comparison of ADA and ɷ-3DPA production
ADA-2.3% and DPA -1.1% of total fatty acids
Southern blotting of P.patens
[Pichit et al.,2012]
IMSC
www.moss-stock-center.org
To conclude…
Moss researcher consortium (left to right): Stefan Rensing, Andy Cuming, Tomoaki Nishiyama, Ralf Reski, Mitsuyasu Hasebe, Ralph Quatrano, Brent Mishler, David Cove
Source: http://www.mossgenome.org/members.php
Dr.Meena kapoor University school of biotechnology,Guru Gobind Singh Indraprastha University, New Delhi
“In the post-genomic era,……………..
to underline the most important contributions brought to science and, further, to draw attention to newcomers in the field, that are expected to fill up the gaps and answer the most specific question we face in biology,
………………………….is possible by the use of such “Classical Plant Models”
[Daniel ., 2009]
Discussion…