Proteins of cotyledons of mature horse chestnut seeds

This is the first characterization of proteins from storage parenchyma of cotyledons of mature dormant recalcitrant horse chestnut (Aesculus hippocastanum L.) seeds and evaluation the cell protein-synthesizing capacity. It was established that the content of protein in cotyledons did not exceed 0.5% of tissue fresh weight. Soluble proteins (the proteins of the postmitochondrial supernatant or cytosol) comprised the bulk (up to 90%) of total proteins. Protein of subcellular structures (20000 g-pellet) comprised 5–7% of total protein. Cotyledon proteins were heterogenous in their charges and molecular weights of subunits. Cotyledon protein was easily extracted with a salt (1 M NaCl); they comprised 90% of water-soluble albumin-like proteins. The proportion of globulins was insignificant; it did not exceed 5%. Most water-soluble proteins (more than 80%) were tolerant to heat denaturing. Among these heat-stable proteins, two major groups of polypeptides dominated: an electrophoretically homogeneous component with a mol wt of 24–25 kD and a complex group from three to five polypeptides with mol wts in the range between 6 and 12 kD. Native heat-stable proteins had disulfide bonds. Four fractions of heat-stable proteins were obtained by ammonium sulfate fractionation; three of them were alike in their polypeptide composition and contained major components with mol wts of 24–25 and 5–12 kD. It was established that the active translational machinery functioned in the cells of storage parenchyma in cotyledons of mature dormant horse chestnut seeds. During each stage of stratification, cotyledon fragments incorporated ³⁵S-methionine into TCA-insoluble material more actively than axial organs. We discuss cotyledon protein composition, their function as a storage organ, and a possible role of heat-stable proteins.     

Recalcitrant seeds of horse chestnut lack protein bodies

In recalcitrant seeds of horse chestnut (Aesculus hippocastanum L.), the bulk of protein in axial organs and cotyledons is accounted for by water-soluble proteins (albumins). In the cells of embryo, proteins are predominantly located in the cytosol, whereas the fraction of cell structures precipitate in the range from 1000 to 20000 g, accounting for only an insignificant part of total protein. Among the proteins of this fraction, there were no major components that could play a role of storage proteins. The aim of this work was to study deposition of protein in the vacuoles of cells of recalcitrant seeds of horse chestnut. Light microscopy and specific staining of protein and phytin did not detect protein bodies in the vacuoles of axial organs and cotyledons. Electron microscopy revealed traces of phytin in the vacuoles, but there were no formed globoids or considerable amount of protein therein. It is possible that precisely the absence of typical storage proteins and genetically determined desiccation in the course of maturation of recalcitrant seeds of horse chestnut stipulated preservation of the vacuoles that in mature recalcitrant seeds were not transformed into protein bodies.     

Identification and characterization of dehydrins in horse chestnut recalcitrant seeds

The fraction of heat-stable dehydrins cytosolic proteins from mature recalcitrant seeds of horse chestnut (Aesculus hippocastanum L.) was studied in the period of their dormancy and germination in order to identify and characterize stress-induced dehydrin-like polypeptides. In our experiments, in tissues of dormant seeds, dehydrin was identifies by immunoblotting as a single bright band with a mol wt of about 50 kD. Low-molecular-weight heat-stable proteins with mol wts of 25 kD and below 16 kD, which were abundant in this fraction, did not cross-react with the antibody. Dehydrin was detected in all parts of the embryo: in the cells of axial organs, cotyledon storage parenchyma, and petioles of cotyledonary leaves. This indicates the absence of tissue-specificity in distribution of these proteins in the horse chestnut seeds. Dehydrins were detected among heat-stable proteins during the entire period of stratification and also radicle emersion. During radicle emergence, not only the fraction of heat-stable proteins was reduced but also the proportion of dehydrins in it decreased. In vitro germination of axes excised at different terms of stratification also resulted in dehydrin disappearance. When growth of excised axes was retarded by treatments with ABA, cycloheximide, or α-amanitin, dehydrins did not disappeared from the fraction of heat-stable proteins. When excised axes were germinated in vitro in the presence of compounds, which did not affect their growth or stimulated it (dehydrozeatin, glucose), this resulted in dehydrin disappearance. This means that dehydrin metabolism is closely related to the process of germination. Dehydrin in the horse chestnut seeds could cross-react with the antibody against ubiquitin, which can indicate the involvement of ubiquitination in the process of dehydrin degradation during germination via the proteasome system. The analysis of total proteins of the homogenate from horse chestnut seeds revealed, along with a 50-kD heat-stable dehydrin, one more component with a mol wt of 80 kD, which was located in the fraction of heat-sensitive proteins and was named as a dehydrin-like protein. It was demonstrated that dehydrins in horse chestnut seeds represented only a very small fraction of heat-stable cytosolic proteins. The role and function of major heat-stable proteins in horse chestnut seeds are yet to be studied.     

Proteins of Axial Organs of Dormant and Germinating Horse Chestnut Seeds: 1. General Characterization

This is the first characterization of proteins from axial organs of recalcitrant horse chestnut seeds during deep dormancy, dormancy release, and germination. We demonstrated that, during the entire period of cold stratification, axial organs were enriched in easily soluble albumin-like proteins and almost devoid of globulins. About 80% of the total protein was found in the cytosol. Approximately one third of cytosolic proteins were heat-stable polypeptides, which were major components of total proteins. Heat-stable proteins comprised three groups of polypeptides with mol wts of 52–54, 24–25, and 6–12 kD with a predominance of low-molecular-weight proteins. The polypeptide patterns of heat-stable and thermolabile proteins differed strikingly. Heat-stable proteins accumulated in axes during the late seed maturation, comprising more than 30% of the total protein in axes of mature seeds. The polypeptide patterns of the total protein of axial organs and its particular fractions did not change in the course of seed dormancy and release. At early germination, the content of heat-stable proteins in axes decreased and their polypeptide pattern changed both in the cytosol and cell structures. We believe that at least some heat-stable proteins can function as storage proteins in the axes. Localization of storage proteins in the cells of axial organs and the role of heat-stable proteins in recalcitrant seeds are discussed.     

Growth Capacity of Embryo Axes Excised from Dormant and Germinating Horse Chestnut Seeds and Their Response to Exogenous Abscisic Acid

In embryo axes excised from mature horse chestnut (Aesculus hippocastanum L.) seeds, both freshly-fallen and subjected to cold stratification, the ability for growth was studied. While excised axes were kept on water at 28°C for 3 days, their fresh weight and length increased, the polypeptide composition of soluble proteins changed, the content of some heat-stable polypeptides decreased, and the capacity for protein synthesis in vivo retained. All these processes were similar to those in the axes of intact seeds during stratification until radicle protrusion. Growth of excised axes accelerated with the increasing duration of stratification. Cycloheximide (50 mg/l) and -amanitin (7 mg/l) inhibited axis growth, but an inhibitor of ABA synthesis fluridone (5 mg/l) and a natural cytokinin dihydrozeatin (10^–5 M) did not influence the growth rate. The growth capacity of axes excised from dormant and germinating horse chestnut seeds indicates the absence of dormancy in the axes of mature seeds. ABA (10^–5 M) suppressed completely the growth of axes detached from seeds experiencing cold stratification but still not germinating, although protein synthesis was not inhibited. The axes excised from the seeds after radicle emergence were insensitive to ABA and grew actively in its presence. ABA-induced growth inhibition might be related to the suppressed synthesis of minor polypeptides required for growth or to the activated synthesis of some growth-retarding proteins. The conclusion was drawn that the excised axes could be used as a model for studying the processes preceding visible germination of recalcitrant seeds.     

Thermal dissipation of light energy is regulated differently and by different mechanisms in lichens and higher plants

Modulated chlorophyll fluorescence was used to compare dissipation of light energy as heat in photosystem II of homoiohydric and poikilohydric photosynthetic organisms which were either hydrated or dehydrated. In hydrated chlorolichens with an alga as the photobiont, fluorescence quenching revealed a dominant mechanism of energy dissipation which was based on a protonation reaction when zeaxanthin was present. CO2 was effective as a weak protonating agent and actinic light was not necessary. In a hydrated cyanobacterial lichen, protonation by CO2 was ineffective to initiate energy dissipation. This was also true for leaves of higher plants. Thus, regulation of zeaxanthin-dependent energy dissipation by protonation was different in leaves and in chlorolichens. A mechanism of energy dissipation different from that based on zeaxanthin became apparent on dehydration of both lichens and leaves. Quenching of maximum or Fm fluorescence increased strongly during dehydration. In lichens, this was also true for so-called basal or Fo fluorescence. In contrast to zeaxanthin-dependent quenching, dehydration-induced quenching could not be inhibited by dithiothreitol. Both zeaxanthin-dependent and dehydration-induced quenching cooperated in chlorolichens to increase thermal dissipation of light energy if desiccation occurred in the light. In cyanolichens, which do not possess a zeaxanthin cycle, only desiccation-induced thermal energy dissipation was active in the dry state. Fluorescence emission spectra of chlorolichens revealed stronger desiccation-induced suppression of 685-nm fluorescence than of 720-nm fluorescence. In agreement with earlier reports of , fluorescence excitation data showed that desiccation reduced flow of excitation energy from chlorophyll b of the light harvesting complex II to emitting centres more than flow from chlorophyll a of core pigments. The data are discussed in relation to regulation and localization of thermal energy dissipation mechanisms. It is concluded that desiccation-induced fluorescence quenching of lichens results from the reversible conversion of energy-conserving to energy-dissipating photosystem II core complexes.