Polyamines and nucleic acids during the first cell cycle of Helianthus tuberosus tissue after the dormancy break

Polyamines (spermine, spermidine, and putrescine) and nucleic acids were studied during the first cell cycle after the break of dormancy of tuber slices of Helianthus tuberosus L., cv. OB1. Immediately after the break of dormancy, a marked decrease in stored arginine and glutamine and a corresponding increase of polyamines were observed. This first synthesis of polyamines were observed. This firs synthesis of polyamines occurred very early during the G₁ phase, concomitant to the synthesis of RNAs. A RNA, probably messenger-like RNA, was synthesized very actively only during the first hours of activation in the culture medium plus 2,4-dichlorophenoxyacetic acid, or in water. At the onset of the S phase, after 12h of activation, an incorporation of [³H] thymidine was also detected. A second putrescine synthesis and polyamine accumulation began during the progression of the S phase. During the progression of mitosis, there was a decrease of polyamine synthesis and accumulation.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA₇ Gibberellin A₇ - MAK methylated albumin column     

Content and Aggregation of Ribosomes during Formation, Dormancy and Sprouting of Tubers of Helianthus tuberosus

The ribosomes and their qualitative (monosomes-polysomes) and quantitative variations over a whole vegetative period of the tuber of Helianthus tuberosus L. (cv. OB 1) were examined. Tubers in different phases of growth, dormancy and sprouting or slices of dormant tubers activated with 2 x 10^-6M indol-3-acetic acid were used. The ribosomes were analyzed by a linear sucrose gradient. During flowering, polysomes of tuber disappeared almost completely and rRNA decreased in comparison with the level present at the beginning of tuber formation. After flowering, there was a new synthesis of monosomes and polysomes until the onset of dormancy; this last period was characterized by a marked increase in polysomes and a proportional increase in monosomes. The level remained almost constant till the break of dormancy. When the tubers sprouted, ribosomes, present almost exclusively as monosomes, decreased considerably; on the contrary the non-photosynthetic sprouts contained many monosomes and polysomes. The first phases of activation (3 h) of tuber slices were characterized by a RNA synthesis, which occurred during one hour, in the subunit region of the gradient. Successively (10 h of activation) the ³²P incorporation was seen also in the polysome region and increased with time. Some possible interpretations of these last results are discussed.     

Variations in the content of free and bound amino acids during dormancy and the first cell cycle in Helianthus tuberosus tuber explants

Abstract

Qualitative and quantitative analysis of free and bound amino acids and amides during dormancy and the most important phases of the first cell cycle was carried out in tubers of Helianthus tuberosus.

In the dormant tuber arginine was confirmed to be the most abundant amino acid. A high amount of asparagine was also present; on the contrary glutamine was found in very low concentrations. During the progression of dormancy, all the free amino acids and amides declined while aspartic and glutamic acid increased.

During the G₁ phase of the first cell cycle induced by 2,4-D, all the free amino acids and amides decreased with the exception of glutamic acid.

At 18, 20, 24 h of activation with 2,4-D, corresponding to the S phase and the beginning of mitosis, bound amino acids were also determined. In these phases of the cell cycle they increased reaching a maximum at 20 h; on the other hand the free amino acid and amide content, especially aspartic acid, asparagine and arginine, decreased with the exception of glutamic acid, alanine and phenylalanine.     

Cell cycle inHelianthus tuberosus tuber tissue in relation to dormancy

Summary Observations are presented on the patterns of DNA synthesis and mitotic activity in medullary parenchyma cells excised from tubers ofHelianthus tuberosus in four different periods of dormancy. Dormancy break (activation) was induced byin vitro culture on media added with 2,4-dichlorophenoxyacetic acid. The cell cycle responsein vitro to different combinations of growth substances has also been investigated.The results show that remarkable changes in the timing of the first and second cell cycles and their phases occur with the progression of dormancy. With increasing time after tuber harvest, the following behaviours are observed: (i) a lengthening of the first cell cycle, chiefly due to a lengthening of the G₂ phase (G₂ is absent at the beginning of dormancy) and an increase in the time interval between the start of thein vitro culture and the onset of the first mitotic wave; (ii) an increased duration of the S phase; (iii) a remarkable reduction in the cell synchrony.These behaviours, as indicated also by their comparison with thein vitro response of the cell cycle to different hormonal treatments, seem to depend on the physiological status of the tubers at the time of explant. It is concluded that the analysis of the cell cycle is an useful tool for understanding some aspects of such a complex physiological situation as dormancy.Istituto di Mutagenesi e Differenziamento del C.N.R., Pisa, Italy, publication no. 321.     

Seasonal changes in the structure and function of mitochondrial membranes of artichoke tubers: acyl Fatty Acid composition and the effect of growth conditions

Changes in the temperature response, fluidity, function and the acyl fatty acid composition, were determined for a mitochondria-rich membrane fraction from Jerusalem artichoke (Helianthus tuberosus L.) tubers during dormancy for a crop which matured in midsummer. The temperature of both the upper and lower limits of the membrane lipid transition decreased during dormancy from 26 C and 1 C to 4 C and −5 C, respectively. This was similar to the changes observed with crops maturing in late autumn. The order parameter of a spin label intercalated into the membrane lipids decreased from about 0.6 to 0.5 during dormancy and returned to the original value before sprouting, showing that membrane fluidity increased during dormancy. The activation energy of succinate oxidase of tuber mitochondria was generally high at middormancy when membrane lipids were more fluid and decreased as the membranes became more rigid at the end of dormancy. The fatty acid composition of the membrane lipids did not alter significantly during dormancy. The results indicate that neither decreasing day length nor low soil temperature during tuber maturation is essential for the initiation of the membrane changes necessary for tubers to avoid low temperature injury during dormancy. The increase in membrane fluidity during dormancy could not be accounted for by an increase in the proportion of unsaturated fatty acids in the membrane lipids.     

Seasonal Changes in the Structure and Function of Mitochondrial Membranes of Artichoke Tubers: A Requisite for Surviving Low Temperatures during Dormancy

The temperature limits of the order-disorder transition, and the Arrhenius activation energy of succinate oxidase activity for mitochondria of Jerusalem artichoke (Helianthus tuberosus L.) tubers were determined from the initiation to the termination of dormancy. The temperature limits for the transition at the initiation of dormancy were 25 and 3 C. These changed to 9 and −5 C at mid-dormancy and returned to 25 and 2 C at the termination of dormancy. The Arrhenius activation energy measured in the temperature range above the transition was 35 kilojoules per mole at middormancy and decreased to 17 kilojoules per mole at the termination of dormancy when sprouting was evident. The coincidence of the changes in membrane structure and function with dormancy suggests that artichokes possess a mechanism for regulating membrane lipid structure so that cellular integrity of tuber tissue is maintained even when the tubers are exposed to low temperatures.     

Ultrastructure and autoradiography of dormant and activated parenchyma ofHelianthus tuberosus

Summary Parenchyma cells of dormant tubers ofHelianthus tuberosus L. cv. OB1 (Jerusalem artichoke) contain a very low amount of hormones, therefore they respond to 2,4-D or IAA treatment by dividing and synthesizing RNA, DNA, and polyamines.In particular the activation of the dormant tissues induces an early synthesis of DNA, which reaches the maximum at 3 hours, much before the beginning of the S phase (12 hours). By supplying [6-³H] thymidine and carrying out electron microscopic autoradiography, we were able to determine that plastids and mitochondria were the organelles responsible for this early synthesis while the DNA in the nucleus first appeared labeled at 15 hours.In addition, ultrastructural observations carried out to compare the dormant cells with activated ones, showed an increase in the nucleolar volume, a different organization of the tubular complex of the plastids and several other ultrastructural changes which indicate that at 3 hours some fundamental metabolic processes are already active; they become even more evident later on.The implications of these results in the physiology of the tuber cells during activation are discussed.     

Polyamines in Relation to Cell Division and Xylogenesis in Cultured Explants of Helianthus tuberosus: Lack of Evidence for Growth-Regulatory Action

Phillips, R., Press, M. C. and Eason, A. 1987. Polyamines inrelation to cell division and xylogenesis in cultured explantsof Helianthus tuberosus: lack of evidence for growth-regulatoryaction.—J. exp. Bot. 38: 164–172. The polyamines spermidine, diaminopropane, and cadaverine werefound to accumulate in cultured tuber explants of H. tuberosus(Jerusalem artichoke). Rapid increases in all amines occurredduring the initial 24 h corresponding to the period of activationand the onset of mitosis. Levels then declined during the followingphases of rapid cell proliferation and xylem differentiation.The type and distribution of polyamines was not markedly affectedby changes in medium or culture conditions, and the inhibitorMGBG did not alter cell division rates or polyamine contentmarkedly although xylem differentiation was substantially depressed.Exogenously supplied spermidine and putrescine did not substantiallyalter the cellular responses of explants cultured in the presenceof auxin. In the absence of supplied auxin, spermidine at 1?0mol m^–3 produced an increase in cell division, althoughthis was small in comparison with auxin-stimulated responses.The implications of these findings on the possibility that polyaminesact as growth regulators in plants is discussed. Key words: Polyamines, Jerusalem artichoke, cultured explants, cell division, xylem differentiation     

ATP-and NADH-dependent membrane potential generation in plasmalemma enriched vesicles from parenchyma of dormant and non-dormant jerusalem artichoke tubers

Using a membrane potential probe, Oxonol VI, it was possible to demonstrate generation of ATP- and NADH-dependent membrane potential across the plasmalemma, with membrane vesicles derived from parenchyma cells of Jerusalem artichoke tubers(Helianthus tuberosus L.). It was shown that ATP- and NADH-dependent membrane potential generation was higher in dormant material than in non-dormant tissue and that the effects of ATP and NADH on membrane potential generation were additive. ATP-dependent potential generation was sensitive to vanadate, an inhibitor of plasmalemma ATPase activity. The results are discussed in relation to the properties of the different enzymes bound to the plasma membrane, the morphogenetic potentialities of tuber buds and the hypothesis that tuber dormancy could be an extreme case of nutrient deficiency induced by short-distance intercellular relationships.     

Cytochemical methods for the localization of invertase activity in plant tissues

Summary Two cytochemical methods for the localization of acid and alkaline invertases are given. The first is based upon the reduction of a silver complex at two different pH ranges, whilst the second is based upon the tetrazolium raction and permits quantification of the rate of activity of alkaline invertase activity. The distribution of alkaline invertase activity throughout the root apex of Pisum sativum and the cell wall localization of acid invertase for material excised from tuber tissue of Helianthus tuberosus are both confirmed.     

Glycosylation of free trans-trans abscisic acid as a contributing factor in bud dormancy break

By means of gas-liquid chromatography determination it was found that progress of dormancy break of almond buds is a function of relative proportions of free and glycoside-bound abscisic acid. Successive stages of bud break manifest a marked increase of bound abscisic acid accompanied by a parallel decrease in endogenous levels of the free form. Of the two stereoisomers involved it was found that while the $\text{cis-trans}$ form maintains a more or less stable level throughout, changes were detected primarily in the $\text{trans-trans}$ form. It is thus postulated that the binding of free hormone as well as its total content are of major physiological importance in the process of bud dormancy break.     

Effects of Growth Regulators on H+Translocation across the Membranes of Plasma Membrane Vesicles from Potato Tuber Cells

Effects of the growth regulators epibrassinolide-694 (EB), gibberellic acid (GA), and abscisic acid (ABA) on the ATP-dependent translocation of H⁺through the membranes of plasma membrane vesicles of potato (Solanum tuberosumL.) tuber cells were studied. The ATP-dependent accumulation of H⁺in the plasma membrane vesicles from dormant tubers was inhibited by EB and ABA and stimulated by GA. After the break of dormancy, the stimulatory effect of GA increased, the inhibitory effect of ABA decreased, and EB stimulated the accumulation of H⁺in the vesicles. The data suggest that the plasma membrane H⁺ATPase is a target of phytohormones that regulate the dormancy of potato tubers.     

In Vitro Study of Mitochondrial Protein Synthesis during Mitochondrial Biogenesis in Excised Plant Storage Tissue

Mitochondrial biogenesis was induced in Jerusalem artichoke (Helianthus tuberosus) tuber by aging tissue discs in distilled water for up to 26 hours. Changes in the purified mitochondrial fraction during aging included an increase in both protein content and specific respiratory activity. Using intact isolated mitochondria, conditions were optimized for incorporation of radioactive amino acid into protein. Incorporation was dependent upon the supply of an oxidizable substrate or an external ATP-generating system and showed characteristic sensitivity to inhibitors of protein synthesis. Aging of the tissue resulted in a 3-fold increase in the rate of in vitro incorporation of [³⁵S]methionine into mitochondrial protein. An analysis of the free amino acid pool in the mitochondrial fraction showed that the decrease in methionine level during aging of intact tissue was sufficient to account for the increased rate of protein labeling. The activation of mitochondrial biogenesis which occurs after slicing is not dependent on an increase in the capacity of mitochondria to synthesize protein as assayed in vitro.     

Polyamines bound to nucleic acids during dormancy and activation of tuber cells of Helianthus tuberosus

Tuber tissue of Helianthus tuberosus L. (cv. OB1) contains a low amount of polyamines during dormancy but they are rapidly synthesized when tuber cells are activated in a growth medium and enter a new cell cycle. It was assumed that one of the reasons for this synthesis is that polyamines are necessary for the active conformation and correct functioning of nucleic acids. Complexes were found between spermine, spermidine and putrescine and rRNA, tRNA and an RNA fraction which contains poly(A) RNA and proteins. The amount of RNA-bound polyamines in the parenchyma cells of dormant tubers is dependent on the stage of dormancy and clearly increases (especially putrescine) when cells are activated. There are both tightly-bound and non-tightlybound polyamines. The significance of these bound polyamines is discussed in relation to their stabilizing role on nucleic acids.     

Correlation between Changes in Mitochondrial Membranes of Artichoke Tubers and Their Hardening and Dormancy

Spin labeling studies using mitochondrial membranes of Jerusalem artichoke (Helianthus tuberosus L.) showed that the decrease during winter in the temperatures of the upper and lower lipid transitions correlated with the development of freezing hardiness of the tubers. The killing temperature for tuber tissue reached a minimum of −12 C, about 5 C degrees lower than the lower transition. Freeze-hardiness decreased when the lower transition increased at the time of sprouting.     

Diamine Oxidase Activity in Different Physiological Stages of Helianthus tuberosus Tuber

Diamine oxidase (DAO, EC 1.4.3.6) activity was examined in relation to polyamine content in Helianthus tuberosus L. during the first synchronous cell cycle induced in vitro by 2,4,-dichloro-phenoxyacetic acid in tuber slices and during the in vivo formation of the tuber. The optimal pH, buffer and dithiothreitol concentrations for the enzyme extraction and assay were determined. When added in the assay mixture, catalase enhanced DAO activity, while polyvinylpyrrolidone had no effect; both aminoguanidine and hydrazine inhibited enzyme activity. The time course of the reaction, based on the recovery of Δ¹-pyrroline from labeled putrescine in lipophilic solvents, showed that it was linear up to 30 minutes; the K_m of the enzyme for putrescine was of the order of 10⁻⁴ molar. During the first cell cycle, DAO activity exhibited a peak at 15 hours of activation while putrescine content gave a peak at 12 hours. During tuber formation (from August till October) DAO activity was relatively high during the first phase of growth (cell division), decreased until flowering (end of September-early October), and then newly increased during the cell enlargement phase preceding the entry into dormancy (November). Maximum putrescine content was observed at the end of October. The increase in DAO activity paralleled the accumulation of putrescine. This indicates a direct correlation between the biosynthesis and oxidation of putrescine which, as already demonstrated in animal systems, occur simultaneously in physiological stages of intense metabolism such as cell division or organ formation.     

Identification of abscisic acid in flower buds of Coffea arabica (L.)

Summary Extracts of flower buds of Coffea arabica (L.) collected before and after bud break contain abscisic acid. This was demonstrated using thin layer chromatography and gas chromatography combined with mass spectroscopy. Abscisic acid accounts for about 75% of the inhibitory activity in the acidic extract. The possible role of abscisic acid in the dormancy of coffee flower buds is discussed.     

Brassinolide Effect on Growth of Apical Meristems, Ethylene Production, and Abscisic Acid Content in Potato Tubers

Treatment of intact potato (Solanum tuberosum L., cv. Nevskii) tubers with 24-epibrassinolide (EB) resulted in prolonged deep dormancy, increased production of ethylene and higher contents of free and bound abscisic acid (ABA) in buds. EB at the most efficient concentration 0.021 mg dm^–3, applied immediately after tuber harvest, inhibited sprouting by 36 – 38 d, increased ethylene formation after 1 and 7 d of storage by almost 300 and 150%, respectively, and increased the content of both free and bound ABA during the whole period of storage (on average by about 80%). Electron microscopic and morphometric studies showed that EB brings about a decrease in cell volume in tunica and all types of meristems and an increase in the number of vacuoles, accompanied by a decrease in their volume.     

Changes of the Protein Synthesis System during the Maturation of Potato Tubers

Respiratory activity, RNA contents and protein synthesis capacity of isolated ribosomes of tubers of Solanum tuberosum, cv. Tonda di Berlino, at progressive stages of development have been determined. In the immature, still growing tubers, respiration steadily decreases with the increase of fresh weight. The entering into dormancy of the tubers collected when immature corresponds to a rapid drop of respiration to the values characteristic of mature tubers. The RNA contents per tuber increase progressively during maturation. A decrease of the RNA contents per tuber (ca. 50 %) is observed in the period between the moment of harvesting and the end of dormancy. The endogenous capacity of isolated ribosomes to carry on amino acid polymerization strongly decreases during tuber development, while the activity supported by polyuridilic acid remains almost unchanged when measured at optimum Mg²⁺ concentrations, and becomes larger for the more mature tubers at superoptimum Mg²⁺ concentrations. These changes of the protein synthesis system during tuber development are compared with those occurring during seed maturation.