Development in Norway Spruce, Picea abies'

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. The capacity of many somatic cells to respond to external stimuli by a developmental reorientation toward the formation of new organs or even entire new plants is unique to cells from higher plants. The mechanisms by which development is regulated in plants are largely unknown. However, endogenously produced hormones (auxins, cytokinins, gibberellins, abscisic acid, ethylene, etc.) take part in the regulation in vivo, and exogenously added growth factors are extensively used to direct morphogenesis in cells and tissue explants cultured in vitro. One growth factor-controlled event that is common to a variety of plants is the de novo formation of shoot apices mediated by cytokinin. We have initiated an analysis of the early stages of one such event, the formation of adventitious ' This work was supported by grants from the Swedish Council for Forestry and Agricultural Research and the Swedish Natural Sciences Research Council to P. E., and from the Jacob Wallenberg Foundation to T. E. buds in seed embryos of Norway spruce, Picea abies (L.) Karst. In this gymnosperm the formation ofadventitious buds can be induced in vitro from different organs by treatment with cytokinins (for review, see 23). Isolated seed embryos, when cultured in vitro, develop into plantlets in a manner similar to the development of the seedling in the seed. A pulse treatment with a cytokinin, benzyladenine, efficiently inhibits the normal development, and instead within several weeks adventitious buds develop from subepidermal cells in the hypocotyl and cotyledon parts of the embryo. The formation of adventitious buds in embryos occurs through a series of morphologically distinct, and well characterized stages. The induction is reproducible and very efficient, routinely close to 100% of the embryos form buds (22). The first distinguishable, morphological differences between bud-induced and control embryos (treated with water and allowed to germinate in vitro) occur at approximately 4 d after isolation and treatment (24). This corresponds to the time at which germination ofseed embryos is complete, e.g. the radicle protrudes through the seed coat. At this time the cells in the cortex layer, which typically divide during the germination phase, stop dividing in the control embryos, and instead become vacuolized. In contrast to this, cells in the hypocotyl of cytokinin-treated embryos continue to divide very actively. Within the following 6 to 8 d a meristematic cell-layer is formed and cells around stomata develop into meristemoids. Until this stage the process is well synchronized, both between embryos and within each embryo (24). Adventitious bud primordia and adventitious buds are observed after 3 weeks in culture and continue to form during the following weeks (25). Upon culture under appropriate conditions such buds can be regenerated to plantlets. In this report, we describe an analysis at the level of protein synthesis ofthe first phase in the development of adventitious buds. We wanted to investigate whether the alteration in development that results from the cytokinin treatment is preceded by or associated with an altered gene expression pattern in the embryo. We have monitored the changes in protein synthesis from the time of isolation and treatment of the embryos until the time, 11 d after treatment, when meristematic centers start to form. In addition we have done a similar analysis of the development of seedlings that germinate in vitro. Germination-related alterations in protein synthesis have previously been described in other plants (5, 19). A comparison of these two developmental processes has allowed us to identify a number of spruce proteins which are 1174 www.plantphysiol.org on July 15, 2017 Published by Downloaded from Copyright © 1990 American Society of Plant Biologists. All rights reserved. PROTEIN PATTERNS IN DEVELOPING SPRUCE EMBRYOS synthesized in correlation with different germination-related events. MATERIALS AND METHODS