Changes in Protein Synthesis upon Cytokinin-Mediated Adventitious Bud Induction and during Seedling Development in Norway Spruce, Picea abies

Abstract

A pulse-treatment of embryos of Picea abies (L.) Karst with cytokinin efficiently and reproducibly induces the coordinate de novo formation of bud primordia from subepidermal cells. The cytokinin treatment also affects the germinative development of the embryo; chloroplast maturation is delayed, and cell elongation is completely suppressed. We have analyzed the protein patterns in developing spruce embryos with the aim of identifying proteins which are differentially synthesized during early bud-differentiation and germination. In addition to a set of major seed storage proteins and a large set of constitutively synthesized proteins, we distinguish two sets of proteins that showed different patterns of synthesis in relation to germination. One was synthesized at high rates during germination, and the second set during postgerminative seedling development. Twenty-two proteins were differentially synthesized in the bud-induced versus the germinating embryos. Interestingly, all 22 belonged to either the germination phase-abundant or the seedling protein sets, whereas the constitutively synthesized proteins were unaffected by the treatment. Proteins synthesized exclusively in bud-induced embryos were not found. In total, the bud-induction treatment caused a maintenance of a protein synthesis pattem typical for the germination phase in the nontreated embryos, and the de novo formation of buds was not preceded by a major change in gene expression in the tissue.

Differential gene expression during germination and after the induction of adventitious bud formation in Norway spruce embryos

“…1C). Day 3 corresponds to late germination and the time at which synthesis of post germinationabundant proteins is initiated [30]. The changes in poly(A) + content in the bud-induced embryos during the time period studied was similar to those in the in vitro germinating embryos.…”

“…With the aim of improving our understanding of the changes in gene expression that underly, and possibly control, the development of the seedling and the adventitious bud formation induced by the cytokinin treatment, we have previously identified a number of proteins that were differentially synthesized in embryos treated to induce the formation of adventitious buds as compared to in vitro germinating embryos [30]. Interestingly, all these proteins belonged to the minority of proteins in the embryos which was also differentially synthesized during different stages of germination in vitro, in contrast to the majority of proteins constitutively synthesized during germination, and unaffected by the cytokinin treatment.…”

ALBINO3, an Arabidopsis nuclear gene essential for chloroplast differentiation, encodes a chloroplast protein that shows homology to proteins present in bacterial membranes and yeast mitochondria.

“…Half of the chloroplast pellet was used for protein isolation, and the other half was used for isolation of chloroplast membranes, according to a lowsalt method (Steinback et al, 1982), with subsequent isolation of proteins. Protein was extracted from membranes, chloroplasts, dark-treated 3-week-old seedlings, and different parts of 4-week-old wild-type and alb3 plants, according to Stabel et al (1990), loaded on denaturing 12% SDS-polyacrylamide gels (Sambrook et al, 1989), and transferred to lmmobilon polyvinylidene difluoride transfer membranes (Millipore AB, Sundbyberg, Sweden) by electroblotting, according to the manufacturer's instructions. The membrane was incubated with anti-ALB3 antibodies, washed with PBS, incubated with horseradish peroxidase-conjugated antibodies from the horseradish peroxidase-conjugate substrate kit (Bio-Rad), and subjected to the horseradish peroxidase substrate 4-chloro-1 -naphthol, according to the manufacturer's instructions.…”

Induction of Anthocyanin Accumulation by Cytokinins in Arabidopsis thaliana

“…A partia1 list of genes that are up-regulated by cytokinin includes nitrate reductase, rbcS, cab, hydroxypyruvate reductase in excised pumpkin cotyledons, a "multiple stimulus response" gene in Nicotiana plumbaginifolia cells, the early nodulin gene SrEnod2 from Sesbania rostrata, and a number of unidentified cDNAs from cultured soybean cells (Flores and Tobin, 1986;Chen, 1989;Crowell et al, 1990;Stabel et al, 1990;Dehio and de Bruijn, 1992;Dominov et al, 1992). Expression of phytochrome and a cucumber gene encoding a cDNA called CR9 are repressed by cytokinin (Cotton et al, 1990;Teramoto et al, 1994).…”