Abstract
Stem cells self-renew and repeatedly produce differentiated cells during development and growth. The differentiated cells can be converted into stem cells in some metazoans and land plants with appropriate treatments. After leaves of the moss Physcomitrella patens are excised, leaf cells reenter the cell cycle and commence tip growth, which is characteristic of stem cells called chloronema apical cells. To understand the underlying molecular mechanisms, a digital gene expression profiling method using mRNA 5′-end tags (5′-DGE) was established. The 5′-DGE method produced reproducible data with a dynamic range of four orders that correlated well with qRT-PCR measurements. After the excision of leaves, the expression levels of 11% of the transcripts changed significantly within 6 h. Genes involved in stress responses and proteolysis were induced and those involved in metabolism, including photosynthesis, were reduced. The later processes of reprogramming involved photosynthesis recovery and higher macromolecule biosynthesis, including of RNA and proteins. Auxin and cytokinin signaling pathways, which are activated during stem cell formation via callus in flowering plants, are also activated during reprogramming in P. patens, although no exogenous phytohormone is applied in the moss system, suggesting that an intrinsic phytohormone regulatory system may be used in the moss.
Highlights
Differentiated cells can be reprogrammed to become stem cells in some mammals and land plants with appropriate inductive treatments [reviewed in 1,2]
Double stranded cDNAs were amplified via 3 cycles of PCR and digested by EcoP15I to produce DNA fragments consisting of P2EcoP15I and 25 bp of cDNA with a two-base 59 overhang
The resulting 25 bp 59 cDNA fragments sandwiched by P1 and P2EcoP15I adaptors were amplified by 12 additional cycles of PCR for SOLiD 25 bp sequencing
Summary
Differentiated cells can be reprogrammed to become stem cells in some mammals and land plants with appropriate inductive treatments [reviewed in 1,2]. Exogenous application of the phytohormones auxin and cytokinin to the culture medium induces proliferation of explanted differentiated cells. Callus cells form shoot or root meristems depending on the ratio of exogenous auxin and cytokinin in the culture medium [4]. When a leaf of a P. patens gametophore, a haploid shoot, is dissected and incubated in culture medium without exogenous phytohormones, leaf cells facing the dissected cells start to protrude with tip growth within a few days [10]. These protruded cells have characteristics of chloronema apical cells, which are a type of stem cell in P. patens. The reprogrammed leaf cells reenter the cell cycle, self-renew, and produce cells indistinguishable from chloronema cells (Figure 1)
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