Control by cytokinins of the cellular behavior in the plate meristem of zucchini cotyledons

The temporal and spatial effects of exogenous cytokinins on both cell expansion and division activity in the plate meristem of cultured zucchini cotyledons were studied. N ⁶-benzylaminopurine (1–100 μM) and N-(2-chloro-4pyridyl)-N′-phenylurea (4PU-30) (0.1–100 μM) greatly stimulated the cell growth and division. They provoked multiple cell cycles, formation of larger clusters of daughter cells and an increase of the final number of cells. Both cytokinins led to earlier achievement of final cotyledon size and shortened the cell doubling time. By contrast to the purine cytokinin, phenylurea cytokinin 4PU-30 enlarged the cotyledon predominantly in length. Zeatin and kinetin were less effective, particularly in stimulating cell expansion. In low concentrations, all cytokinins were more effective in stimulating division activity rather than expansion. The cells in the cotyledon margins displayed a higher division activity, especially when treated with exogenous cytokinins. The final cotyledon and cluster areas were not of the strict proportional dependence upon the number of their cells. These results provide a novel example where stimulated cell division fails to evoke a respective increase in the final organ size.     

Modulating zucchini cotyledon plate meristem activity by interactions between the cycline-dependent kinase inhibitor roscovitine and cytokinins

The inhibitors of cytokinin N-glucosylation are known to influence the growth of some plant objects including cotyledons. The use of the plate meristem of zucchini cotyledon as an experimental system allowed us to study for the first time the way in which the changes in the cell division are integrated in this growth reaction. Roscovitine, a potent inhibitor of cytokinin N-glucosylation and cycline-dependent kinases, did not show to have an effect on the meristem activity when applied in 100 μM to cultivated zucchini cotyledons, and acted as an inhibitor in concentrations higher than 400 μM. A 200 μM roscovitine stimulated both palisade cell division and growth. In different seed batches, 400 μM roscovitine acted as a stimulator or an inhibitor. A much stronger stimulating effect on growth and cell division was observed after application of benzyladenine (BA, 10 μM). In contrast to BA, roscovitine provoked a formation of principally flat lamina. In combined treatments, it lowered the stimulating effect of BA; 400 μM roscovitine combined with BA severely suppressed the growth and division activity. This cellular behavior and changes in cotyledon growth could be due to the roscovitine-provoked changes in endogenous cytokinin levels via the inhibition of cytokinin N-glucosylation. Roscovitine-caused stimulation of cell growth and division is stronger in the marginal meristem than that registered in central regions of the cotyledon blade. In this region it also changed the pattern of cell division and lowered the adhesion between the clusters, which enhanced the appearance of local ruptures of the cotyledon edges. The first palisade layer of the plate meristem of cultured zucchini cotyledons, the natural mono-layer of proliferating palisade cells, may be used for screening the inhibitors of cycline-dependent kinases and cytokinin N-glucosylation with regard to their effects on cell division and growth.     

The CURLY LEAF gene controls both division and elongation of cells during the expansion of the leaf blade in Arabidopsis thaliana

The CURLY LEAF (CLF ) gene in Arabidopsis thaliana (L.) Heynh. is required for stable repression of a floral homeotic gene, AGAMOUS in leaves and stems To clarify the function of CLF in organ development, we characterized clf mutants using an anatomical and genetic approach. The clf mutants had normal roots, hypocotyls, and cotyledons, but the foliage leaves and the stems had reduced dimensions. A decrease both in the extent of cell elongation and in the number of cells was evident in the clf mutant leaves, suggesting that the CLF gene might be involved in the division and elongation of cells during leaf morphogenesis. An analysis of the development of clf mutant leaves revealed that the period during which cell division or cell elongation occurred was of normal duration, while the rates of both cell production and cell elongation were lower than in the wild type. Two phases in the elongation of cells were also recognized from this analysis. From analysis of an angustifolia clf double mutant, we found that the two phases of elongation of leaf cells were regulated independently by each gene. Thus, the CLF gene appears to affect cell division at an earlier stage and cell elongation throughout the development of leaf primordia. Received: 19 February 1998 / Accepted: 24 March 1998     

Differences in cytokinin control on cellular dynamics of zucchini cotyledons cultivated in two experimental systems

The effect of endogenous cytokinins on the pattern of palisade cell division post-germination does not depend on the conditions of cotyledon development -in planta (attached to seedlings) or in vitro (isolated from dry zucchini seeds and cultured on water). In cotyledons originating from 4-day-old seedlings (experimental system 1), exogenous cytokinin temporarily (in the first 2 day of cultivation) enhanced post-mitotic cell enlargement of palisade cells, mainly due to enhanced water uptake and use of cell storage compounds, all of which lead to cotyledon senescence. Cytokinin is not able to resume the completed palisade cell division on day 5. As a result, the number of cells and the final areas of treated and control cotyledons are quite similar. By contrast, the effects of cytokinin on cotyledons isolated from dry seeds (experimental system 2) are better expressed, promoting an increase in number of palisade cells accompanied by additional cotyledon area enlargement. However, the prolonged post-mitotic cell expansion in control cotyledons compensates for the reduced speed of cell growth and division activity and decreases differences in final cotyledon area between treatments. The results define cell division as the primary target of cytokinin stimulation in cotyledon tissues competent for division, and determine the temporal patterns of palisade cell cycling related to cotyledon age. This knowledge permits a better choice of experimental system to study effects on cell proliferation and cell growth, as well as cell enlargement and senescence-related events using physiologically homogeneous material.     

Heteroblasty in the moss,Aphanoregma patens (Physcomitrella patens), results from progressive modulation of a single fundamental leaf developmental programme

Abstract

Leaves at the apex of a mature Aphanoregma patens (Hedw.) Lindb. (Physcomitrella patens (Hedw.) Bruch Schimp. in B.S.G.) gametophore differ markedly in size and form from those at its base. To determine how these differences are produced during development, we first examined qualitative and quantitative differences between successive leaves along the stem and among leaves at different developmental stages. Differences between successive leaves were slight and cumulative. Local changes in cell number and size combined to produce a regularly shaped and approximately bilaterally symmetrical leaf suggesting that cell division and cell expansion are regionally regulated and coordinated at the organ level. The midrib and marginal teeth are discrete characters, which were prefigured by changes in cell shape in leaves that lacked these characters. In leaf primordia, cell proliferation was responsible for most of the changes in leaf form and size early in development and may have continued as cell expansion took over as the primary contributor to leaf growth and morphogenesis. Thus, leaf heteroblasty in Physcomitrella probably results from modulation of a single developmental programme by external and/or internal forces, which alter progressively in intensity as a gametophore grows. We applied exogenous cytokinin and auxin separately to growing cultures to explore their effects on leaf growth. Cytokinin and auxin stimulated leaf cell division and leaf cell elongation, respectively. Also, young upper leaves of gametophores exposed to exogenous auxin closely resembled basal leaves of untreated plants. Therefore, endogenous cytokinins and auxins may be among the modulating internal forces involved in leaf morphogenesis and the establishment of leaf heteroblasty.     

Maternal genotype influences pea seed size by controlling both mitotic activity during early embryogenesis and final endoreduplication level/cotyledon cell size in mature seed

When reciprocal crosses are made between different pea genotypes, there is a strong maternal influence on mature seed size of the reciprocal hybrids, i.e. their dry weights are similar to that of seeds obtained from their maternal parents. Reciprocal crosses between pea varieties having very different mature seed sizes were used to investigate how the maternal genotype controls seed development and mature seed size. The differences in dry seed weight between genotypes and reciprocal hybrids reflected differences in both cotyledon cell number and mean cell volume, and the maternal control on the establishment of these two traits was investigated. Using flow cytometry, data relative to endoreduplication kinetics in cotyledons during the transition between the cell division phase and maturation were obtained. The appearance of nuclei having an 8C DNA content indicates the initiation of the endoreduplication phenomenon and thus the end of the cell division phase. It was shown that the duration of the cell division phase was the same in the reciprocal hybrids, its value being intermediate between those recorded for their maternal parents. This result indicates that the timing of development of the embryo is not under maternal control, but depends on its own genotype. Consequently, maternal genotype must influence the mitotic rate during the cell division phase to achieve differences in cell number found in the cotyledons of mature F1-reciprocal hybrids. The final level of endoreduplication in cotyledons of mature seeds was also investigated. This study showed that there is a close relationship (r2 = 0.919) between the endoreduplication level in mature cotyledons and seed dry weight or mean volume of cotyledon cells, suggesting that both maternal and non-maternal factors could control the number of endoreduplicating cycles in the cotyledons and, hypothetically, the cotyledon cell size.     

Photosynthesizing Tissue Development in C4 Cotyledons of Two Salsola Species (Chenopodiaceae)

The quantitative anatomy of developing cotyledons of NAD-malic enzyme species Salsola incanescens and NADP-malic enzyme species S. paulsenii (Chenopodiaceae) was studied. S. incanescens belongs to the group of species with foliar type of seedling development characterized by slowly growing cotyledons and a rosette form at juvenility. The rosette is the consequence of fast leaf formation, which was correlated with a low rate of leaf growth. S. paulsenii belongs to the group with the cotyledonous type of seedling development. A high growth rate of cotyledons, slow leaf formation, and absence of the rosette characterize this type. Slow leaf formation was correlated with a high rate of leaf growth. The Kranz–anatomy in cotyledons of S. incanescens (atriplicoid type) and S. paulsenii (salsoloid type) determines the duration of cotyledon development proceeding for 15 days after seed germination. The rate of growth changes during the developmental period was correlated with the type of seedling development. Cotyledons of a foliar species S. incanescens exhibit 2 to 5 times slower growth changes in cotyledon area, width, thickness, volume of mesophyll and bundle sheath cells, and number of chloroplasts per bundle sheath cell than the cotyledons of a cotyledonous species S. paulsenii. During cotyledon development in both species, the number of chloroplasts per mesophyll cell remained unchanged, and developmental changes in the bundle sheath occurred at higher rate than in mesophyll cells. Thus, these two indices seem to be independent of the type of Kranz–anatomy. The presence of atriplicoid type cotyledons in the species with salsoloid structure of true leaves might indicate a close genetic relationship between these two patterns of Kranz-anatomy.     

Changes in the Distribution of Magnesium, Potassium, Calcium and Phosphorus during Growth of Cucurbita Seedlings

Ockenden, I. and Lott, J. N. A. 1988. Changes in the distributionof magnesium, potassium, calcium and phosphorus during growthof Cucurbita seedlings—J. exp. Bot. 39: 973–980. Etiolated seedlings of Cucurbita maxima cv. Warted Hubbard andCucurbita andreana, grown in distilled water, showed considerablegrowth. However, only a small percentage of the minerals storedin the large cotyledons was mobilized to the root-shoot axisof the etiolated seedlings. The proportion of minerals removedfrom the cotyledons to the root-shoot axis was about two-timeshigher in C. andreana than in C. maxima. When the seedlingswere grown in distilled water in the presence of light, thepercentage of cotyledon mineral content that was mobilized waslower than the amount mobilized during growth in the dark. Cotyledonsof light-grown seedlings that were given adequate nutrients,took up minerals from the medium. These fully-expanded and photosyntheticcotyledons had slightly more P and Mg than did the cotyledonsof the dry embryos but had increased their K content 5-foldand their Ca content 50-fold. Key words: Cucurbita, minerals, seeds, seedings, NAA     

The Effects of Cell Cycle Parameters on Cell Wall Regeneration and Cell Division of Cotton Protoplasts (Gossypium hirsutum L.)

Protoplasts of cotton cotyledons were isolated and culturedto undergo cell wall regeneration and cell division. DNA contentand cell cycle parameters of nuclei from cotyledons and/or protoplastswere determined by flow cytometry. The DNA content of cotton,Gossypium hirsutum L., was estimated to be 4·34±0·12pg DNA per nucleus. There was a strong positive correlation between G₂ or Sand G₂,and cell wall regeneration and cell division and a strong negativecorrelation between G₁, and cell wall regeneration and celldivision of cotton cotyledon protoplasts. The cell cycle statusof cotyledons changes during their development; as the cotyledonsenlarge, the proportion of cells in G₀ and G₁ phases of thecell cycle increases. The implication of these results in relationto protoplast growth and development is discussed. Key words: Cell cycle parameters, cell wall regeneration, cell division, flow cytometry, Gossypium     

Deoxyribonucleic Acid and Ribonucleic Acid Synthesis during the Cell Expansion Phase of Cotyledon Development in Vicia faba L

In Vicia faba L., the tissue specific proteins, legumin and vicilin, are synthesized during the cell expansion phase of cotyledon development. During this growth period, RNA and nuclear DNA increase 8- to 10-fold. ³H-Uridine and ³H-adenosine are incorporated into ribosomal RNA, both 25S and 18S, and into transfer RNA. DNA isolated from cotyledons in the cell division phase of growth has been compared with DNA isolated from cotyledons undergoing expansion growth. Results indicate that the DNA increase involves replication of the whole genome (endoreduplication).     

The Effects of Age and Root Formation on the Life-Span of Excised Cotyledons of Cucurbita pepo

Root initiation in Cucurbita cotyledons excised at various ages was studied in nutrient solution and water. The time required for root initiation depends on the age of the cotyledon and on the rooting medium. Nutrient solution favoured root formation. Maximum number of roots, maximal total root length, and maximum life-span were found with cotyledons excised at an age of 14 days. Excision earlier or later resulted in less roots, less total root length, and shorter life-span.     

Anisocotyly and meristem initiation in an unorthodox plant, <Emphasis Type="Italic">Streptocarpus rexii</Emphasis> (Gesneriaceae)

In common with most Old World Gesneriaceae; Streptocarpus Lindl. shows anisocotylous growth, i.e., the continuous growth of one cotyledon after germination. Linked to this phenomenon is an unorthodox behaviour of the shoot apical meristem (SAM) that determines the growth pattern of acaulescent species (subgenus Streptocarpus). In contrast caulescent species develop a conventional central post-embryonic SAM (mainly subgenus Streptocarpella). We used S. rexii Lindl. as a model to investigate anisocotyly and meristem initiation in Streptocarpus by using histological techniques and analyses of the expression pattern of the meristematic marker SrSTM1 during ontogeny. In contrast to Arabidopsis thaliana (L.) Heynh., S. rexii does not establish a SAM during embryogenesis, and the first evidence of a SAM-like structure occurs during post-embryonic development on the axis (the petiolode) between the two cotyledons. The expression pattern of SrSTM1 suggests a function in maintaining cell division activity in the cotyledons before becoming localized in the basal meristem, initially at the proximal ends of both cotyledons, later at the base of the continuously growing macrocotyledon, and the groove meristem on the petiolode. The latter is equivalent to a displaced SAM seemingly originating de novo under the influence of endogenous factors. Applied cytokinin retains SrSTM1expression in the small cotyledon, thus promoting isocotyly and re-establishment of a central post-embryonic SAM. Hormone-dependent delocalization of the process of meristem development could underlie anisocotyly and the unorthodox SAM formation in Streptocarpus. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Legumin Synthesis in Developing Cotyledons of Vicia faba L

The synthesis of legumin in developing cotyledons of Vicia faba L. has been examined as a potential system for approaching the problem of differential gene expression. The pattern of legumin synthesis was determined during the growth of the cotyledon by microcomplement fixation which provided a sensitive and specific assay for legumin in the presence of vicilin. Legumin was detected even in young cotyledons. However, when the cotyledons were about 10 millimeters long, and cell division was essentially complete, there was a sharp increase in the rate of legumin accumulation.     

single cotyledon (sic) mutants of pea and their significance in understanding plant embryo development

Angiosperms are divided into two distinct classes—the dicotyledons (dicots) and monocotyledons (monocots)—based in part on the number of cotyledons in mature embryos. In this paper, we describe single‐cotyledon pea mutants, termed sic (single cotyledon), all of which show a degree of fusion between the cotyledons. The fusion in sic1 is along the margin of one cotyledon and is less complete than in sic2 embryos, but the effects of the mutations are additive in the double mutant. Occasionally sic2 mutants will show fusion of the two cotyledons into one cylindrical embryo in which the shoot apex becomes surrounded by the cotyledons. Both sic1 and sic2 mutants produce fertile plants. In the sic3 embryo, a single cotyledon is generated under the shoot apex that breaks the vascular connection between root and shoot, causing embryo lethality. The pattern of cotyledon development in all these mutants is identified by in situ mRNA hybridization and antibody labeling, using the storage protein vicilin as a cotyledon‐specific marker. These patterns indicate that the joining of the cotyledons was due to zonal growth. The results indicate that there are genes in pea that influence the positioning and the morphology of the cotyledon. A model for cotyledon development in pea is proposed that is based on the regulation of the positioning of cell clusters by the sic genes. Dev. Genet. 25:11–22, 1999. © 1999 Wiley‐Liss, Inc.     

Somatic embryogenesis and plantlet regeneration from cotyledon explants isolated from emblings and seedlings of hybrid firs

Embryogenic tissues have been initiated on cotyledon explants dissected from seedlings or emblings of hybrid firs. Cotyledons of seedling origin (Abies alba x A. cephalonica) gave a relatively low initiation frequency (1.94 percnt;). In embling-derived cotyledons (Abies alba x A. cephalonica, Abies alba x A. numidica), the initiation was cell-line dependent and reached values between 1.25 and 24.28 percnt;. The established embryogenic cell lines are being maintained in long-term cultures.The origin and development of the somatic embryos have been traced histologically. The early stages of somatic embryo development have been characterised by cell division activity (predominantly periclinal) in the epidermal and subepidermal layers of cotyledons and subsequently by development of nodular structures. Further differentiation led to the formation and emergence of somatic embryos on the surface of cotyledons.Somatic embryo development and plantlet regeneration occurred from proliferating tissues initiated from cotyledons of embling as well as seedling origin.     

The mutants compacta ?hnlich, Nitida and Grandiflora define developmental compartments and a compensation mechanism in floral development in Antirrhinum majus

In order to improve our understanding of floral size control we characterised three mutants of Antirrhinum majus with different macroscopic floral phenotypes. The recessive mutant compacta ?hnlich has smaller flowers affected mainly in petal lobe expansion, the dominant mutant Grandiflora has overall larger organs, whilst the semidominant mutation Nitida exhibits smaller flowers in a dose-dependent manner. We developed a cell map in order to establish the cellular phenotypes of the mutants. Changes in organ size were both organ- and region-specific. Nitida and compacta ?hnlich affected cell expansion in proximal and distal petal regions, respectively, suggesting differential regulation between petal lobe regions. Although petal size was smaller in compacta ?hnlich than in wild type, conical cells were significantly bigger, suggesting a compensation mechanism involved in petal development. Grandiflora had larger cells in petals and increased cell division in stamens and styles, suggesting a relationship between genes controlling organ size and organ identity. The level of ploidy in petals of Grandiflora and coan was found to be equivalent to wild type petals and leaves, ruling out an excess of growth via endoreduplication. We discuss our results in terms of current models about control of lateral organ size.     

A Comparative Study of the Role of the Cotyledon in Seedling Development

Seedling development was studied in four species in which thecotyledons had different capacities for expansion and CO²-flxation. The highest growth-rates were shown by the two species in whichthe cotyledons showed the most expansion and also the greatest¹⁴CO²-fixation capacity. Cotyledons of these species becamevery leaf-like and retained a high proportion of the radiocarbonfixation products during their growth in contrast to the hypogealrunner-bean cotyledon which had a high percentage export valuealthough total fixation was low. Seedlings with leaf-like cotyledons which had the dual roleof storage and subsequent provision of photosynthetic organshad delayed leaf development with early growth concentratedin cotyledon, hypocotyl, and root whereas species with cotyledonsless well adapted for photosynthesis produced leaves at an earlierstage. Thus, the pattern of early seedling development was closelyrelated to the degree of photosynthetic adaptation shown bythe cotyledons. The evidence suggests that in the species studied, cotyledonsadapted for the dual role appear to provide a more efficientsystem for early seedling development.     

Influence of the Embryonic Axis on Protein Hydrolysis in Cotyledons of Cucurbita maxima

Four-day time course studies of the hydrolysis of cotyledonal storage protein were conducted on intact seeds, seed cotyledons detached from their embryonic axes and on detached cotyledon pairs germinated in the presence of three excised embryonic axes of Cucurbita maxima Duch., cv. Chicago Worted Hubbard. Detached cotyledons germinated alone showed little hydrolysis of the storage protein. However, the amount of protein hydrolysis of the detached cotyledon pairs germinated in the presence of three excised embryonic axes was comparable to the amount hydrolyzed in the cotyledons of intact germinating seeds. Visual growth differences among these treatments were also evident. The size and yellow color intensity of the fourth day treatments were shown to increase in the following order: detached cotyledon pairs alone, intact seedlings, detached cotyledon pairs in the presence of three excised axes. The growth of the hypocotyl and radical was also modified by removal of the cotyledons. These findings suggest that storage protein degradation and cotyledonal growth are controled by the axis. They also indicate that the cotyledons have some influence on the growth of the axes. Time-course studies were made on the hydrolysis of storage protein in the cotyledons of squash and on the distribution of the hydrolytic products during the germination of light- and dark-grown plants. The storage protein was not hydrolyzed during the first 24 hours. It was hydrolyzed at a uniform rate from 1 to 5 days and at a slightly decreased rate from 5 to 7 days. Most of the hydrolytic products were transported to the axial tissue. Proteinase activity in the cotyledons rapidly increased during germination to a maximum level at 2 to 3 days. This was followed by a decline to about the initial value after 7 days.     

Studies of cotyledon protoplast cultures from Brassica napus,B. campestris and B. oleracea. I: Cell wall regeneration and cell division

Protoplasts isolated from cotyledons of a number of cultivars of Brassica napus, B. campestris and B. oleracea were cultured in different media to study the characteristics of cell wall regeneration and cell division at early stages of culture. Time course analysis using Calcolfluor White staining indicated that cell wall regeneration began in some protoplasts 2–4 h following isolation in all cultivars. 30–70% of cultured cotyledon protoplasts exhibited cell wall regeneration at 24 h and about 60–90% at 72 h after the initiation of culture. Results also indicated that a low percentage (0.4–5.4%) of cultured cotyledon protoplasts entered their first cell division one day after initial culture in all twelve cultivars. The percentage of dividing cells increased linearly up to 40% from 1 to 7 day, indicating that cotyledon protoplasts of Brassica had a high capacity for cell division. Factors that influence the level of cell wall regeneration and cell division during cotyledon protoplast culture have been investigated in this study. Cotyledons from seedlings germinated in a dark/dim light regime provided a satisfactory tissue source for protoplast isolation and culture for all Brassica cultivars used. The percentages of protoplasts exhibiting cell wall regeneration and division were significantly influenced by cultivar and species examined, with protoplasts from all five cultivars of B. campestris showing much lower rates of cell wall regeneration than those of B. napus and B. oleracea over 24–120 h, and with the levels of cell division in B. napus cultivars being much higher than those in B. campestris and B. oleracea over 1–9 days. The capacity of cell wall regeneration and cell division in cotyledon protoplast culture of the Brassica species appears under strong genetic control. Cell wall regeneration in protoplast culture was not affected by the culture medium used. In contrast, the composition of the culture medium played an important role in determining the level of cell division, and the interaction between medium type and cultivars was very significant.Abbreviations BA benzylaminopurine - CPW Composition of Protoplast Washing-solution - CW Calcolfluor White - EDTA ethylenediamine-tetraacetic acid - KT Kinetin - Md MS modified Murashige and Skoog medium - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - PAR photosynthetically active radiation - SDS sodium dodecyl sulfate     

Different functions of vicilin and legumin are reflected in the histopattern of globulin mobilization during germination of vetch (Vicia sativa L.)

The temporal and spatial patterns of storage-globulin mobilization were immunohistochemically pursued in the embryonic axis and cotyledons of vetch seed (Vicia sativa L.) during germination and early seedling growth. Embryonic axes as well as cotyledons of mature seeds contain protein bodies with stored globulins. Prevascular strands of axes and cotyledons, the radicle and epidermal layers of axis organs were nearly exclusively stained by vicilin antibodies whereas the cotyledonous storage mesophyll gave similar staining for vicilin and legumin. Globulin breakdown started locally where growth and differentiation commenced in the axis. There, vicilin mobilization preceded legumin mobilization. Thus vicilin represents the initial source of amino acids for early growth and differentiation processes in vetch. Legumin presumably only serves as a bulk amino acid source for subsequent seedling growth during postgerminative globulin degradation. During the first 2–3 d after the start of imbibition the axis was depleted of globulins whereas no decrease in immunostainability was detected in the cotyledons except in their vascular strands where immunostainability was almost completely lost at this time. Continuous vascular strands were established at the third day when globulin breakdown was finished in the axis but had just started in the cotyledon mesophyll. Protein mobilization proceeded in a small zone from the epidermis towards the vascular strands in the center of the cotyledons. In this zone the storage cells, which initially appeared densely packed with starch grains and protein bodies, concomitantly transformed into cells with a large central vacuole and only a thin cytoplasmic layer attached to the cell wall. These results agree well with the hypothesis that during the first 2 d after imbibition the axis is autonomous in amino acid provision. After the endogenous reserves of the axis are depleted and the conductive tissue has differentiated, globulins are mobilized in the cotyledons, suggesting that then the amino acid supply is taken over by the cotyledons. For comparison with other degradation patterns we used garden bean (Phaseolus vulgaris L) and rape (Brassica napus L.) as reference plants. Received: 3 August 1999 / Accepted: 11 December 1999