Breaking bud dormancy in apple with a plant bioregulator,thidiazuron

The effects of N-phenyl-N′-1,2,3,-thidiazol-5-ylurea (thidiazuron; Dropp; SN49537; TDZ) on metabolic changes in apple buds during dormancy break were determined. The data showed that thidiazuron has the capacity to release lateral buds from dormancy. Decreasing degree of bud break and bud growth with thidiazuron treatment occurred in a basipetal direction, suggesting a gradient of increasingly deep rest from shoot apex to base. The breaking of dormancy by thidiazuron is correlated with increase in DNA, RNA, protein, 1-aminocyclopropane-1-carboxylic acid (ACC), 1-(malonylamino) cyclopropane-1-carboxylic acid (MACC), S-adenosylmethionine (SAM) as well as with greater polyamine formation. Polyamine and ethylene biosynthesis did not seem to be competing for SAM, their common substrate, during bud break and bud development. The release of dormancy in apple bud by thidiazuron was inhibited by cordycepine, 5-fluorouracil, 6-methylpurine and cycloheximide. Inhibition of bud break and bud growth also resulted from treatment with α-difluoromethylarginine (DFMA) and α-difluoromethylornithine (DFMO). DFMO was more inhibitory than DFMA.     

Porphyrinogenesis in maize as affected by bidentate chelators of iron

Intracellular compartmentation of 1-aminocyclopropane-1-carboxylic acid (ACC) and N-malonyl-1-aminocyclopropane-1-carboxylic acid (MACC) in wheat ( Triticum aestivum L. cv. Kanzler) and barley ( Hordeum vulgare L. cv. Gerbel) leaves was studied using different methods: first, the isolation of intact vacuoles from protoplasts and, second, a non-aqueous fractionation procedure. The two methods gave similar results. ACC concentrations were similar in the extravacuolar space and in the vacuole, whereas MACC was accumulated in the vacuolar space. Transport studies revealed that no specific carrier for ACC exists at the tonoplast. MACC transfer across the tonoplast was enhanced by 120% in the presence of ATP. MACC competitively inhibited malate transport into the vacuole indicating that the same transfer system catalyzes the transfer of the two dicarboxylates.
It is concluded that malonylation of ACC is not a prerequisite for the transport of ACC through the tonoplast.     

Levels of ethylene, ACC, MACC, ABA and proline as indicators of cold hardening and frost resistance in winter wheat

Changes in ethylene production and in the contents of 1-aminocydopropane-1-carboxylic acid (ACC), 1-(malonylamin6)-cyclopropane-1-carboxylic acid (MACC), abscisic acid (ABA) and L-proline were determined after 40 days of cold hardening at 4°C in three wheat cultivars differing in frost resistance. Proline and especially ABA accumulated with hardening in all varieties in parallel with the degree of frost resistance, e.g. proline and ABA increases in the non-resistant cv. Slávia were 2x and 5x, whilst in the resistant cv. Mironovská 808 increases were 4X and 20X. Ethylene production and MACC level showed no significant changes with hardening in any of the cultivars after 40 d, but ACC levels did increase with hardening. The production of ethylene, ACC and MACC was studied during hardening. Ethylene production decreased sharply at low temperature and rose rapidly (within 1 day) on return to normal temperature, while ACC production reacted in the opposite direction. MACC levels rose rapidly during the first 4 days of cold, then more slowly for about 2 weeks, thereafter decreasing again steadily. The only varietal differences occurring at maximum levels were correlated with the degree of frost resistance.     

Metabolism of 1-aminocyclopropane-1-carboxylic acid in ripening apple fruits

In preclimacteric apple fruits ( Malus × domestica Borkh. cv. Golden Delicious) ethylene production is controlled by the rates of 1-aminocyclopropane-1-carboxylic acid (ACC) synthesis, and by its metabolism to ethylene by the ethylene-forming enzyme and to 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) by malonyl CoA-ACC transferase. The onset of the climacteric in ethylene production is associated with an increase in the activity of the ethylene-forming enzyme in the pulp and with a rise in the activity of ACC synthase. Malonyl transferase activity is very high in the skin of immature fruit, decreases sharply before the onset of the climacteric, and remains nearly constant thereafter. More than 40% of the ACC synthesized in the skin and around 5% in the flesh, are diverted to MACC at early climacteric. At the climacteric peak there are substantial gradients in ethylene production between different portions of the tissue, the inner cortical tissues producing up to twice as much as the external tissues. This increased production is associated with, and apparently due to, increased content of ACC synthase. Less than 1% of the synthesized ACC is diverted to MACC in the flesh of climacteric apples. In contrast, the skin contains high activity of malonyl transferase, and correspondingly high levels [1000 nmol (g dry weight)⁻¹] of MACC.     

Ethylene-promoted malonylation of 1-aminocyclopropane-1-carboxylic acid participates in autoinhibition of ethylene synthesis in grapefruit flavedo discs

The increase in ethylene formation and in 1-aminocyclopropane-1-carboxylic acid (ACC) content in flavedo tissue of grapefruit (Citrus paradisi Macfad. cv. Ruby Red) in response to excision was markedly inhibited by exogenous ethylene. Ethylene treatment inhibited the synthesis of ACC, but increased the tissue's capability to malonylate ACC to N-malonyl-ACC, resulting in further reduction in the endogenous ACC content. The development of extractable ACC-malonyl-transferase activity in the tissue was markedly promoted by treatment with exogenous ethylene. These results indicate that the autoinhibition of ethylene production in this tissue results not only from suppression of ACC synthesis, but also from promotion of ACC malonylation; both processes reduce the availability of ACC for ethylene synthesis.Abbreviations ACC 1-Aminocyclopropane-1-carboxylic acid - AVG aminoethyoxyvinylglycine (2-amino-4-(2-aminoexthoxy)-trans-3-butenoic acid) - MACC 1-(malonylamino)-cyclopropane-1-carboxylic acid     

Accumulation of 1-(malonylamino)cyclopropane-1-carboxylic acid in ethylene-synthesizing tobacco leaves

During the hypersensitive reaction of Samsun NN tobacco to tobacco mosaic virus (TMV) the inoculated leaves synthesize large quantities of ethylene. At the same time, 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), a conjugate of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) accumulates. Smaller amounts of MACC are formed concomitant with ethylene synthesis during the normal development of tobacco leaves. The conjugate appears neither to be hydrolysed to liberate ACC, nor to be transported to other plant parts. Its accumulation thus reflects the history of the operation of the pathway of ethylene synthesis in the leaf. In floating leaf discs exogenously applied ACC was converted only slowly to both ethylene and MACC. More ethylene and less MACC were produced in darkness than in light, suggesting that environmental conditions may influence the ratio at which ACC in converted to either ethylene or MACC.     

Changes in the level of MACC during cold hardening and dehardening in winter wheat

Changes in the level of 1-(malonylamino)-cyclopropane-1-carboxylic acid (MACC) were determined in 6 winter wheat cultivars during cold hardening at 4°C. The cultivars differed by one degree of frost resistance within the range of degree II to VII of the COMECON scale. The time-course of changes in MACC level showed a similar pattern in all 6 cultivars; i.e. increase till day 6, no changes for the next 10 days, and then a steady decrease till the end of the hardening period. There was little difference between the final and the initial levels. The increase of MACC level, expressed as per cent of the original level, was not directly correlated with either the degree of frost resistance of the actual percentage of survival. In some cultivars. mean errors exceeded the difference in MACC accumulation between cultivars closest on the resistance scale.
The fate of MACC during the second half of hardening and after transfer of plants to 25°C was studied in cultivars Bezostaya and San Pastore. During the second half of the hardening period the level of MACC decreased in the leaves of both cultivars, but increased significantly in the roots. Within two days of transfer of the hardened plants to 25°C, the MACC level in leaves increased again, while that in the roots decreased. This finding, together with the preliminary evidence of very low MACC metabolism, strongly suggest that MACC accumulates in roots during the hardening period and when transferred to 25°C, it moves from roots to leaves.     

The effect of water stress on 1-(malonylamino)cyclopropane-1-carboxylic acid concentration in plant tissues

Since the discovery of1-(malonylamino)cyclopropane-1-carboxylic acid (MACC)as a major metabolite of both endogenous andexogenously applied 1-aminocyclopropane-1-carboxylicacid (ACC), it has become evident that the formationof MACC from ACC can act to regulate ethyleneproduction in certain tissues. Hence it was suggestedthat MACC could serve as an indicator of water-stresshistory in plant tissues. The accurate quantificationof MACC in plant tissues is essential forunderstanding the role of MACC in the regulation ofethylene biosynthesis.Hoffman et al. [15] described a method for themeasurement of MACC in which MACC was hydrolysed byHCl to ACC, which was then assayed by chemicaloxidation to form ethylene. Attempts have been made byothers to raise monoclonal antibodies to MACC so thatan immunoassay could be developed in order to gain adeeper understanding of stress-induced ethyleneproduction but no further publications have beenforthcoming.Here a method employing GC-MS is compared with theindirect assay for MACC, which is based uponhydrolysis of MACC to ACC and conversion of ACC byhypochlorite reagent to ethylene which is subsequentlyquantified by GC.     

Changes in ribosome population and in nucleic acids during breaking of dormancy and development of apple flower buds

Changes in ribosome population, RNA species and DNA composition in flower buds of apples ( Malus pumila Mill. cvs Ralls and White winter pearmain) were investigated during breaking of dormancy and development. After bursting of flower buds, total ribosomes increased approximately 4-fold, and the polyribosomal fraction increased from 66% to 94% of total ribosomes. The newly synthesized ribosomes were identified by incorporation of radioactive precursor. The observed decrease in specific radioactivity of the monoribosomes is caused by the recruitment of monoribo-somes into polyribosomes after breaking of dormancy.
In both cultivars, the 25S and 18S rRNA peaks increased to a high level on April 8. The peaks of low molecular weight RNA were apparently increased after initial swelling of the flower buds. The DNA of flower buds was separated into three bands by electrophoresis. The median band is the main band of nuclear DNAs. The ahead band and the slow-moving band are satellite components of nuclear DNAs, and they obviously rose after initial swelling of the flower buds. On April 8, when the flower buds had opened, two other small DNA bands could be detected. These results suggest that the changes in level of different ribosome populations, RNA species and DNA composition are related to dormancy breaking development of apple flower buds.     

Effects of Cold Acclimation and Salicylic Acid on Changes in ACC and MACC Contents in Maize during Chilling

The effect of 0.5 mM salicylic acid (SA) pretreatment and of growing at hardening temperatures on chilling-induced changes in 1-aminocyclopropane-1-carboxylic acid (ACC) and malonyl 1-aminocyclopropane-1-carboxylic acid (MACC) was investigated in young maize (Zea mays L.) plants grown in hydroponic solution at 22/20 °C. Chilling at 5 °C caused an increase in ACC content;however, this increase was less pronounced in plants cold acclimated at 13/11 °C 4 d before the chilling treatment, and in those which were pretreated with SA for 1 d before the cold stress. Changes in MACC at low temperature showed no correlation with chilling tolerance in maize.     

Effect of paclobutrazol on water stress-induced ethylene biosynthesis and polyamine accumulation in apple seedling leaves

Ethylene biosynthesis and polyamine content were determined in [(2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol] (paclobutrazol) pre-treated and non-treated water-stressed apple seedling leaves. Paclobutrazol reduced water loss, and decreased endogenous putrescine spermidine content. Gibberellic acid (GA) counteracted the inhibitory effect of paclobutrazol on polyamine content. Paclobutrazol also prevented accumulation of water stress-induced 1-aminocyclopropane-1-carboxylic acid (ACC), 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), ethylene production and polyamines in apple leaves. α-Difluoromethylarginine (DFMA), but not α-difluoromethylornithine (DFMO), inhibited the rise of putrescine and spermidine in stressed leaves. S-Adenosylmethionine (SAM) was maintained at a steady state level even when ethylene and the polyamines were actively synthesized in stressed apple seedling leaves. The conversion of ACC to ethylene did not appear to be affected by paclobutrazol treatment.     

In vitro Protein Synthesis by Polyribosomes of Peach Flower Buds at Different Physiological Stages

The in vitro phenylalanine incorporation by polyribosomes of peach flower buds (Prunus persica Stokes) during dormancy, dormancy break and flowering was investigated. Protein synthesis was measured using as catalyst either calf liver soluble factors or the ribosomal supernatant from the peach flower buds in the presence or the absence of the synthetic mRNA, polyuridylic acid. In the presence of polyuridylic acid, the activity of protein synthesis of dormant ribosomes is the same as that of ribosomes during dormancy break and flowering. The absence of synthetic messenger did not cause a change in activity. The ribosomal supernatant of dormant buds, but not of flowering buds, reduces the phenylalanine incorporation by polyribosomes from buds harvested at dormancy break.     

Levels and Aggregation of Ribosomes during Dormancy and Dormancy Break of Peach Flower Buds

The level of ribosomes and their aggregation state (monosomes-polysomes) have been investigated in relation to dormancy in peach flower buds (Prunus persica). The ribosomes were analysed by a linear sucrose gradient. The ribosomal content hardly changes in the bud before dormancy and until dormancy breaks; on the other hand after this period there is a remarkable increase in the ribosomal level strictly related to the increase in bud weight. The ribosomal content reaches the highest value at flowering. During dormancy break the largest portion of ribosomes is localized in flower primordia, not in scales. Polysomes, which are always present in buds, remain constant during dormancy (35%), increase at dormancy breaking, reaching the highest level at flowering.     

The quantification of 1-(malonylamino)cyclopropane-1-carboxylic acid in plant tissues by quantitative mass spectrometry

A method for the quantitation of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), a conjugated form of 1-aminocyclopropane-1-carboxylic acid (ACC), in plants is described. [2,2,3,3-²H₄]MACC has been used as an internal standard for selected ion monitoring/isotope dilution quantitation of MACC in wheat seedlings and in tomato leaves. This method is compared with a widely-used two step indirect assay for MACC, which is based upon hydrolysis of MACC to ACC and conversion of ACC by hypochlorite reagent to ethylene which is subsequently quantified by gas chromatography.     

Influence of the triazole growth retardant BAS 111..W on phytohormone levels in senescing intact pods of oilseed rape

Foliar treatment of oilseed rape plants (Brassica napus L.ssp. napus cv. Linetta) with the growth retardant BAS 111..W at the 5th leaf stage delayed pod senescence during early maturation. Changes of immunoreactive cytokinin- and abscisic acid (ABA)- like substances and of the ethylene precursor 1-aminocyclo-propane-1-carboxylic acid (ACC) and its malonyl-conjugate (MACC) were determined in intact whole pods. When compared with control plants, higher levels of total chlorophyll correlated with four-fold and three-fold increases of trans-zeatin riboside- and dihydrozeatin riboside-type cytokinins, respectively, in the pods of plants treated with 0.25 mg BAS 111..W per plant. Isopentenyladenosine-type cytokinins and ACC and MACC contents remained virtually unchanged, whereas ABA levels dropped considerably below those of controls (60% reduction). However, when analysed at late pod maturity, BAS 111..W treatment no longer affected the total chlorophyll content, or the levels of cytokinins, ABA, ACC and MACC. We hypothesize that the retardant-induced changes in the hormonal status of the pods, favouring the senescence-delaying cytokinins as opposed to abscisic acid, could contribute to the developmental delay.     

Synthesis of nucleic acids and protein in tuber buds of Solanum tuberosum during dormancy and early sprouting

Autoradiographic studies have demonstrated the continuous synthesis of protein, RNA and DNA in potato ( Solanum tuberosum L. cv. King Edward) tuber buds from the time of tuber harvest throughout dormancy. Breaking of dormancy is associated with a rise in all activities. A first rise occurs just prior to (or coincident with) a general increase in cell volume. A second rise accompanies the elongation and outgrowth of the bud. The continuous metabolic activity during dormancy in the absence of cell growth is discussed.     

Changes in 1-aminocyclopropane-1-carboxylic-acid content of cut carnation flowers in relation to their senescence

The rise in ethylene production accompanying the respiration climacteric and senescence of cut carnation flowers (Dianthus caryophyllus L. cv. White Sim) was associated with a 30-fold increase in the concentration of 1-aminocyclopropane-1-carboxylic acid (ACC) in the petals (initial content 0.3 nmol/g fresh weight). Pretreatment of the flowers with silver thiosulfate (STS) retarded flower senescence and prevented the increase in ACC concentration in the petals. An increase in ACC in the remaining flower parts, which appeared to precede the increase in the petals, was only partially prevented by the STS pretreatment. Addition of aminoxyacetic acid (2 mM) to the solution in which the flowers were kept completely inhibited accumulation of ACC in all flower parts.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AOA -aminoxyacetic acid - STS silver thiosulfate complex     

休眠期间油桃花芽碳水化合物代谢及其相关基因的表达变化

以十年生大田和三年生盆栽‘曙光’油桃花芽为材料,分别测定了其休眠期碳水化合物含量、糖代谢相关基因的季节性表达及低温处理下相关基因的表达变化,旨在探讨碳水化合物及低温与休眠的关系。结果表明：休眠期间可溶性糖（主要是蔗糖）含量逐渐增加,淀粉含量则呈相反趋势。糖代谢相关基因表达明显不同：腺苷二磷酸葡萄糖焦磷酸化酶基因口GPase）无明显变化;组氨酸H3基因（HisH3）和己糖激酶I基因（胱,）在进入内休眠前有明显上升,蔗糖合酶基因（SuSy）则与之相反;尿苷二磷酸葡萄糖焦磷酸化酶（UGPase）表达总体上呈上调趋势,在进入内休眠后稍有下调。表明进入内休眠后,依赖HKl的糖信号转导途径起重要作用。在4℃处理后,与细胞分裂有关的基因HisH3含量急剧升高,而后下降,说明细胞分裂的减少并不是休眠期间抑制生长的原因;UGPase表现出与内休眠期一致的变化趋势,说明对低温有一定的适应性。     

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.