A Transcriptome-Based Characterization of Habituation in Plant Tissue Culture

Abstract

For the last 50 years, scientists have recognized that varying ratios of the plant hormones cytokinin and auxin induce plant cells to form particular tissues: undifferentiated calli, shoot structures, root structures, or a whole plant. Proliferation of undifferentiated callus tissue, greening, and the formation of shoot structures are all cytokinin-dependent processes. Habituation refers to a naturally occurring phenomenon whereby callus cultures, upon continued passage, lose their requirement for cytokinin. Earlier studies of calli with a higher-than-normal cytokinin content indicate that overproduction of cytokinin by the culture tissues is a possible explanation for this acquired cytokinin independence. A transcriptome-based analysis of a well established habituated Arabidopsis (Arabidopsis thaliana) cell culture line was undertaken, to explore genome-wide expression changes underlying the phenomenon of habituation. Increased levels of expression of the cytokinin receptor CRE1, as well as altered levels of expression of several other genes involved in cytokinin signaling, indicated that naturally acquired deregulation of cytokinin-signaling components could play a previously unrecognized role in habituation. Up-regulation of several cytokinin oxidases, downregulation of several known cytokinin-inducible genes, and a lack of regulation of the cytokinin synthases indicated that increases in hormone concentration may not be required for habituation. In addition, up-regulation of the homeodomain transcription factor FWA, transposon-related elements, and several DNA-and chromatin-modifying enzymes indicated that epigenetic changes contribute to the acquisition of cytokinin habituation.

Signaling pathways in a Citrus EST database

“…Considering these findings, one can suggest that the homologues of the three key proteins in a His/Asp phosphorelay are expressed in Citrus, as well as the response regulators. Those results are quite in agreement with the genes that have been identified in Arabidopsis and other eukaryotes such as yeasts (Pischke et al, 2006).…”

“…CytokininIndependent 1 (CKI1) belongs to a group of putative plant histidine kinases whose members do not appear to act as ethylene receptors (Pischke et al, 2006). The regulators are named AHPs (from 1 to 5, Arabidopsis thaliana histidine phototransmitter) and ARRs classified in A-type and Btype Arabidopsis thaliana response regulators (Oka et al, 2002).…”

The Effect of a Genetically Reduced Plasma Membrane Protonmotive Force on Vegetative Growth of Arabidopsis

“…Cycle conditions were 95°C for 5 min followed by 40 cycles of 95°C for 10 s, 58°C for 7 s, and 72°C for 20s. The expression of reference genes or K + transporter genes was analyzed with gene-specific pairs of primers: ACT2 (Pischke et al, 2006), 5#-GCATGAAGATCAAGGTGGTTGCAC-3# and 5#-ATGGACCTGACTCATCGTACTCACT-3#; UBQ10 (Czechowski et al, 2005), 5#-GGCCTTGTATAATCCCTGATGAATAAG-3# and 5#-AAAGAGATAA-CAGGAACGGAAACATAGT-3#; AtHAK5 (Qi et al, 2008), 5#-CGTTTTCATT-GTTCTTCAGG-3# and 5#-ATCTTCTGGTTCTTGGTTTG-3#; AtCHX17, 5#-CCGTCCCGATATAATCTGCGTTTTC-3# and 5#-CAACGCGTGTTCCA-GAGGATTC-3#. The sequences of the primers for AtCHX17 were designed using the AtRTPrimer program (Han and Kim, 2006).…”

Epigenetics in plant tissue culture

“…A transcriptome-based analysis of habituated and non-habituated Arabidopsis thaliana plant cell cultures revealed the differential expression of more than 800 genes, including a 19-fold up-regulation of the transcript encoding the cytokinin receptor CRE1. The alterations in gene expression found among habituated and nonhabituated callus cultures do not result simply from global hypomethylation of DNA (Pischke et al 2006). Nevertheless it has been reported that treatment of in vitro cultured cells of a nontumorous Nicotiana glauca 9 N. langsdorffii hybrid with 5-azacytidine, a drug causing hypomethylation, leads to the induction of a habituated phenotype (Durante et al 1989).…”