Isolation and Characterization of a Cytokinin Up-Regulated Gene from Tobacco Mesophyll Protoplasts

Abstract

We have isolated a cytokinin up-regulated cDNA clone, H13, from an early stage of cultured tobacco mesophyll protoplasts by a differential display method. The expression of this gene was specifically induced by natural and synthetic cytokinins including N-(2-chloro-4-pyridyl)-N'-phenylurea (4PU30), a diphenylurea-type cytokinin, although the simultaneous presence of auxin was also required. It seems that the preceding treatment of the tobacco mesophyll protoplasts by auxin is necessary for the gene to respond to cytokinin. The addition of a cytokinin antagonist, compound 182, which suppressed the induction of cell division in tobacco mesophyll protoplasts, completely abolished the expression of this gene. Though the predicted gene product of H13 did not suggest us any sequences of defined functions, two domains of the predicted sequence had significant homology to several reported sequences in the data base. The gene product of H13 is proposed to have a role in regenerating cell wall in cultured protoplasts, since a cDNA clone E6, from cotton fiber cells, which has the most closely related structure to H13, has been isolated from cells which showed active cellulose synthesis. This supposition is supported by the evidence that in the absence of cytokinin, cell wall regeneration was significantly suppressed, resulting in failure of the induction of cell division. Thus, the gene product of H13 is supposed to have a role in regenerating cell walls and facilitating the progression of the cell cycle, resulting in the sustained cell division of tobacco mesophyll protoplasts.

Use of genetically ablated stigmas for the isolation of genes expressed specifically in the stigma epidermis

“…However, this domain is not characteristic of arabinogalactan proteins (AGPs), and the Pis63 protein is not a typical AGP. In addition, Pis 63 and related genes share an amino acid motif, [(Q/E)(Q/K/R/)QG(M/L/)SDTRFLXNGKX-(Y/X)YD] as described in Iwahara et al (1998), that is present in several reported sequences including AGPNa2, AGPPc2 (Mau et al 1995), E6 (John and Crow 1992), and a cytokinin-upregulated gene (Iwahara et al 1998). …”

A magnetic affinity approach to identify plant GABA-binding proteins

“…Since the probe was feasible for labeling GABA B receptors, we further tested whether the probe could bind to similar GABA receptors on the surface of plant protoplasts. Mature mesophyll protoplasts were isolated according to a previously published protocol (Iwahara et al, 1998); mature pollen protoplasts were also isolated using a germination-enzymatic treatment protocol established in our lab (Yu et al, 2006). Both types of living protoplasts were isolated, collected, and incubated with the probes.…”

Dicamba-responsive genes in herbicide-resistant and susceptible biotypes of kochia (Kochia scoparia)

“…Differential display is a polymerase chain reaction (PCR)based procedure for amplifying and comparing messenger RNA (mRNA) populations between two contrasting physiological states, i.e., tissue types, developmental stages, or treated and untreated tissues (Liang and Pardee 1992). The technique has been used to isolate genes whose expression patterns are affected by the phytohormones ethylene (Hajouj et al 2000;Zegzouti et al 1999), abscisic acid (Yoshida et al 2001), cytokinin (Iwahara et al 1998), and auxin (Dargeviciute et al 1998;Hutchison et al 1999). We previously used the procedure to isolate genes associated with early events of seed dormancy maintenance and release (Johnson et al 1995).…”