Proteome and metabolome profiling of cytokinin action in Arabidopsis identifying both distinct and similar responses to cytokinin down- and up-regulation

Abstract

In plants, numerous developmental processes are controlled by cytokinin (CK) levels and their ratios to levels of other hormones. While molecular mechanisms underlying the regulatory roles of CKs have been intensely researched, proteomic and metabolomic responses to CK deficiency are unknown. Transgenic Arabidopsis seedlings carrying inducible barley cytokinin oxidase/dehydrogenase (CaMV35S>GR>HvCKX2) and agrobacterial isopentenyl transferase (CaMV35S>GR>ipt) constructs were profiled to elucidate proteome- and metabolome-wide responses to down- and up-regulation of CK levels, respectively. Proteome profiling identified >1100 proteins, 155 of which responded to HvCKX2 and/or ipt activation, mostly involved in growth, development, and/or hormone and light signalling. The metabolome profiling covered 79 metabolites, 33 of which responded to HvCKX2 and/or ipt activation, mostly amino acids, carbohydrates, and organic acids. Comparison of the data sets obtained from activated CaMV35S>GR>HvCKX2 and CaMV35S>GR>ipt plants revealed unexpectedly extensive overlaps. Integration of the proteomic and metabolomic data sets revealed: (i) novel components of molecular circuits involved in CK action (e.g. ribosomal proteins); (ii) previously unrecognized links to redox regulation and stress hormone signalling networks; and (iii) CK content markers. The striking overlaps in profiles observed in CK-deficient and CK-overproducing seedlings might explain surprising previously reported similarities between plants with down- and up-regulated CK levels.

Cytokinin modulates proteomic, transcriptomic and growth responses to temperature shocks in Arabidopsis

“…In addition, to evaluate the importance of physiological levels of endogenous cytokinins in the response, we used transgenic CaMV35S > GR > HvCKX2 plants with conditionally decreased levels of endogenous cytokinins (Černý et al . ). As shown in Fig.…”

“…In parallel, seeds of transgenic CaMV35S > GR > HvCKX2 line 13, allowing conditional reductions in endogenous cytokinin levels (Černý et al . ), were sown on the same medium further supplemented with 20 μ m dexamethasone (DEX; Sigma‐Aldrich, Prague, Czech Republic, using DMSO as a carrier at the same final concentration as for the mock). The seeds were stratified on the MS plates at 4 °C for 3 d and subsequently transferred to a growth chamber (AR36LX; Percival).…”

“…Rubisco‐depleted proteomes and phosphoproteomes of the seedlings were extracted, prepared and analysed essentially as previously described (Černý et al . 2011a,b, 2013a). For proteome equalization, protein was extracted from frozen seedlings (620–650 mg, ∼900 seedlings) in 1.5 mL of isolation buffer [250 m m 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES), 10 m M ethylenediaminetetraacetic acid (EDTA), 5% gycerol, pH 7.5] supplemented with protease inhibitors and polyvinylpolypyrrolidone (PVPP) (Sigma‐Aldrich), clarified by centrifugation, concentrated to 200 μ L and processed using a Bio‐Rad ProteoMiner protein enrichment kit (for methodological principles, see, e.g.…”

The impact of heat stress targeting on the hormonal and transcriptomic response in Arabidopsis

“…Data were analyzed as described previously [19], using hierarchical clustering analysis by Gene Cluster 3.0 (http://bonsai. hgc.jp/∼mdehoon/software/cluster/), and by principal component analysis (PCA) in software R (http://www.r-project.org/).…”

Elucidation of Abiotic Stress Signaling in Plants

“…It is known that DNA repair capacity of an organism declines with age that leads to increasing accumulation of deleterious mutations promoting disease and death. This damage increases more significantly in nondividing mammalian cells causing them to malfunction that ultimately affects the whole organism (Jin 2010 ;Campisi 2000 ).…”