Alterations in endogenous levels of cytokinins following grafting of Pinus radiata support ratio of cytokinins as an index of ageing and vigour

Markers for ageing or maturation in woody plants provides the possibility of identifying genotypes with a prolonged juvenile phase, or to evaluate reinvigoration procedures, such as grafting of adult material. This study focuses specifically on cytokinins (Cks) as markers, due to their role in the transition process from juvenile to adult states. Previous reports from our group disclosed that the ratio of isopentenyladenine-type (iP-type) to zeatin-type (Z-type) Cks decreases during tree maturation, a maturation and ageing index being likely in Pinus radiata. Based on these results, Cks were tested in terminal buds of adult and grafted adult P. radiata material to corroborate the importance of the aforementioned balance as an index in the reinvigoration process of adult trees associated with grafting procedures. Results revealed the reversion of this index pattern parallel to the reactivation of some juvenile traits, namely, increasing values of the ratio throughout the reinvigoration process. According to previous results, however, the opposite was the case during the reverse process. This fact allowed us to validate the iP-type/Z-type Ck ratio as an ageing and vigour index.     

Could plant hormones be the basis of maturation indices in Pinus radiata?

Maturation and phase change in woody plants are developmental processes at present poorly understood, especially from a physiological point of view. Maturation indices, such as phytohormones, could be used to understand more about the events that accompany the increase in the developmental state and to choose optimal conditions in order to improve forest programs and make them more profitable. In order to determine this putative index, the contents in abscisic acid-like substances (ABA-like), indole-3-acetic acid (IAA) and several cytokinins (Cks) were analysed in terminal and axillary buds of Pinus radiata trees with different developmental states to compare their phytohormonal status. An increase in the content of some zeatin-type (Z-type) Cks, and a decrease in isopentenyladenine-type (iP-type) Cks levels throughout maturation were shown both in terminal and axillary buds. This fact allowed us to consider the ratio iP-type/Z-type Cks, which decreased in parallel with the increasing developmental state of the tree, as a maturation index. According to the results, ABA-like compounds and IAA contents could not be considered as a maturation index in Pinus radiata, even though similar patterns were in fact found between the two kinds of buds.     

Abscisic acid and 3-indolacetic acid levels during the reinvigoration process of <Emphasis Type="Italic">Pinus radiata</Emphasis> D. Don adult material

The maturation and phase change processes in woody species have not been completely clarified, specially the role of growth regulators during the maturation phase. Understanding their role better will allow to comprehend the physiological aspects of the species, which would allow to chose the optimal management conditions in forestry programs and make them more useful. In the present study, the endogenous content of abscisic acid (ABA) and 3-indolacetic acid (IAA) were quantified by high performance liquid chromatography, in the reinvigoration process of elite Pinus radiata material, during the development of homomicrografts (P. radiata/P. radiata) and heteromicrografts (P. radiata/Pinus caribaea), developed under in vitro conditions. The results showed an increase in the endogenous content of ABA through time and a decrease in the content of IAA. Significant differences were found in the endogenous content of ABA, where the maximum values were detected at 120 days of micrograft culture, being similar to the values obtained in juvenile buds. As well, significant differences were also detected in the endogenous content of IAA in the micrografted buds during all the development phases, compared with adult buds. However, no significant differences were detected in the endogenous content of ABA and IAA, regarding the type of rootstock employed. Considering the endogenous content of ABA and IAA, the micrografted plant material would be equal to the juvenile buds, since statistically they were equal to these buds, considering that the values obtained in this case are independent from the type of rootstock used.     

Novel cytokinins: The predominant forms in mature buds of Pinus radiata

To elicit the roles of cytokinins in the regulation of maturation of Pinus radiata D. Don, the spectrum of endogenous cytokinins and their concentration in the mature buds were analysed using double-solvent extraction, column complex purification and separation, a novel immunoaffinity purification method, normal and reverse phase high-pressure liquid chromatography, enzymatic treatment, radioimmunoassay and electrospray MS/MS spectrometry. We have isolated two novel cytokinin glycosides whose proposed structures are isopentenyladenine-9-(glucopyranosyl riboside), dihydrozeatin-9-(glucopyranosyl riboside) and confirmed the presence of zeatin-9-(glucopyranosyl riboside). We have also found the presence of novel phosphorylated forms of these 3 cytokinin ribosyl-linked glycosides. Quantitative analyses revealed that the cytokinin ribosyl-linked glycosides predominate in P. radiata mature buds. Although cytokinin free base, riboside and nucleotide forms are also present, we could find no evidence of the traditional cytokinin O - or N-glucosides in the conifer buds. Thus, cytokinin metabolism in mature buds of P. radiata is very different from other species previously examined.     

Plant growth regulators as putative physiological markers of developmental stage in Prunus persica

Prunus persica plants of different ages and statesof maturation were analysed to compare their phytohormonal status. Endogenouslevels of indole-3-acetic acid (IAA), abscisic acid (ABA), and severalcytokinins (Cks): zeatin (Z), zeatin riboside ([9R]Z), dihydrozeatin ((diH)Z),dihydrozeatin riboside ((diH)Z[9R]), isopentenyl adenine (iP) and isopentenyladenosine ([9R]iP), were measured in order to determine their possible use asphysiological indices of phase change and maturation. A decrease in Ck levels(Z, [9R]Z, (diH)Z, (diH)[9R]Z, and iP, [9RiP) was found from the embryonic tojuvenile stage as well as a decrease in the ratio of iP-type (iP and[9R]iP)/Z-type Cks paralleling the increase in tree age. ABA levels increasedduring maturation in Prunus persica and the ratio ofCks/IAA decreased with tree age. From our results, we propose that the balancesof Cks/IAA and iP-type/Z-type Cks are good indices of different developmentalstates in Prunus persica.     

Reliable plantlet formation from embryos and seedling shoot tips of radiata pine

A method has been devised for the reliable production of plantlets from embryos and seedling shoot tips of Pinus radiata D.Don (radiata pine). Buds were induced on an agar or liquid Schenk and Hildebrandt (SH) medium containing 5.0 mg/l benzylaminopurine (BAP). Except for some abnormal buds, the buds grew into elongated shoots on an agar SH medium without cytokinin. The transfer of shoots from a SH medium to a Gresshoff and Doy (GD) medium was found to be an important pretreatment which increased the survival of the shoots when they were placed in a peat and pumice mix for root formation. Elongated shoots were induced to form roots under non-sterile conditions in a humid environment with occasional misting. An intervening 5-day treatment of shoots in an agar medium containing 2.0 mg/l indolebutyric acid (IBA) and 0.5 mg/l napthaleneacetic acid (NAA) significantly increased the percentage of shoots forming roots and the number of roots formed per shoot over control shoots placed directly in the peat:pumice mix. An enhanced level of CO₂ during root formation had no effect on the time of root formation or on the percentage of shoots forming roots. These results concerning the elongation, growth and rooting of adventitious shoots are now being applied to the development of very large numbers of plantlets starting from cotyledons from partially germinated seeds.     

Phase-change related epigenetic and physiological changes in <Emphasis Type="Italic">Pinus radiata</Emphasis> D. Don

DNA methylation and polyamine levels were analysed before and after Pinus radiata D. Don. phase change in order to identify possible molecular and physiological phase markers. Juvenile individuals (without reproductive ability) were characterised by a degree of DNA methylation of 30-35% and a ratio of free polyamines to perchloric acid-soluble polyamine conjugates greater than 1, while mature trees (with reproductive ability) had 60% 5-methylcytosine and a ratio of free polyamines to perchloric acid-soluble polyamine conjugates of less than 1. Results obtained with trees that attained reproductive capacity during the experimental period confirmed that changes in the degree of DNA methylation and polyamine concentrations found among juvenile and mature states come about immediately after the phase change. We suggest that both indicators may be associated with the loss of morphogenic ability during ageing, particularly after phase change, through a number of molecular interactions, which are subsequently discussed.     

Cytokinins and bud morphology in Pinus radiata

Cytokinins (CKs) play essential roles in the regulation of plant growth and development. In the previous paper (Zhang et al. 2001), we reported the detection and identification of a wide spectrum of CKs, including several novel forms, in the buds of Pinus radiata D. Don. In this paper we examine the relationship between the CKs and buds from juvenile and adult trees of P. radiata. During development the morphology of buds alters significantly, from buds bearing primary needles during their juvenile phase to buds sealed in scales at the adult phase. The morphology of adult buds is a very stable character, as fascicle meristems released from apical dominance, or cultured in vitro, produced only secondary needles. However, exogenous CK causes the adult buds to revert to juvenile bud development in vitro . Analyses of the endogenous CKs revealed that juvenile buds had a relatively higher level of isopentenyladenine and isopentenyladenosine, extremely low levels of phosphorylated CKs and a relatively low level of novel CK glycosides. The adult buds contained lower levels of free base and riboside CKs but very high levels of phosphorylated CKs and novel CK glycosides. Possible roles for CKs in the regulation of bud development are discussed.     

Changes in Lipids of Bean Seeds (Phaseolus vulgaris) and Corn Caryopses (Zea mays) Aged in Contrasting Environments

Seeds of French bean (Phaseolus vulgaris L. cv. The Prince)and caryopses of sweet corn (maize; Zea mays L.F1 hybrid Firstof All) were stored in environments of 79% relative humidityand 25 °C, 80% r.h. and 40 °C or 100% r.h. and 45 °C,giving ageing (loss of gcrminability and vigour) over periodsof months, weeks or days, respectively. The relationship betweenchanges in lipids and changes in germinability or vigour wasunaffected in general by the speed of ageing. In the corn caryopsesthere was no evidence of hydrolysis of phospholipids or peroxidationof fatty acids during ageing. In the bean seeds phosphatidicacid increased during the ageing period and phosphatyl cholinedeclined. The percentage of fatty acids as hnolenic acid initiallyfell during bean ageing, but in the slower ageing conditionsit rose again as germination reached zero. In bean, the presenceof phosphatidic acid could be a sensitive indictator of lossof vigour, but relative proportions of the different fatty acidswould be a misleading indicator of quality. Rapid artificialageing may be an adequate model, in some species, of ageingat moderate speeds and of ageing under some ambient conditions. French bean (Phaseolus vulgaris L. cv. The Prince), sweet corn (maize, Zea mays L., Fl hybrid First of All), seed ageing, phospholipids, fatty acids     

Loss of flower bud vigour in the Mediterranean shrub,Cistus albidus L. at advanced developmental stages

To better understand aging in perennials, age‐related changes in the physiology of leaves and flower buds of the Mediterranean shrub, Cistus albidus L. were evaluated. Two groups of different ages (5 and 10 years old), both at advanced developmental stages but of similar size, were compared. Total plant biomass, biomass produced per apical meristem and levels of cytokinins, abscisic acid and jasmonic acid in leaves and flower buds, as well as flower production, were measured. No differences in plant size, vegetative growth rates and levels of phytohormones in leaves were observed between 5‐ and 10‐year‐old plants. However, they showed significant differences in flower bud development; the older plants having reduced vigour, with 29.6% of flowers reaching anthesis compared to 52.5% in the younger plants. Furthermore, endogenous concentrations of zeatin and abscisic acid in flower buds at stage I (start of flower organ formation) were 61% and 41%, respectively, smaller in 10‐ than in 5‐year‐old plants. At stage II (with all flower organs formed), zeatin and abscisic acid concentrations decreased by ca. 90% and 80%, respectively, but differences between age groups were still evident (60% and 29% for zeatin and abscisic acid, respectively). Jasmonic acid levels in flower buds decreased by 80% from stage I to II, but did not differ between age groups. Despite reductions in flower bud vigour, total number of flowers per individual was not significantly different between age groups, so that an age‐related loss in reproductive vigour at the organ level did not lead to a decrease in flower production at the whole plant level.     

Biochemical changes in Pinus pinea seeds during storing

The changes in the germination-rate, the contents in germination-inhibitors and the biochemical differences in soluble proteins and nucleic acids in freshly harvested Pinus pinea seeds stored for various periods of time, up to 24 months, and at two different temperatures (room temperature and 4 degrees C), have been investigated. The present results show that the maturation or after-ripening process of this type of seeds might be induced during the first 6 and 12 months of storage. However, seeds stored for longer periods of time might also be thought to enter into the primary phases of the ageing process where early alterations occur, including the loss of germination-rate and germination-inhibitor contents in the seed coat, together with an incapacity for the seeds to increase their protein and nucleic acid levels during the germination process.     

高温加速老化过程中甜菜种子线粒体小分

甜菜(BetavulgarisLcv.Loke)种子的人工老化(30℃,100％RH)导致种子的干重、生活力和活力逐渐丧失,线粒体的呼吸速率、细胞色素c氧化酶和苹果酸脱氢酶的活性下降。线粒体蛋白质随着种子老化而显著变化,小分子量热休克蛋白(LMWHSP)22从老化的0d到30d增加,然后从30d到90d下降。可以认为甜菜种子人工老化过程中线粒体活性和LMWHSP22的含量变化与种子活力密切相关。     

TEMPORAL COMPETENCE OF EMBRYONIC PINUS PONDEROSA COTYLEDONS TO FORM MULTIPLE BUDS IN VITRO

To investigate temporal changes occurring during tissue maturation and loss of organogenic competence, embryos of Pinus ponderosa Laws, were excised from seed and placed horizontally on medium in sterile culture. On a hormone-free basal medium, cotyledons differentiated and failed to form multiple buds. When placed on a benzyladenine (BA)-containing medium, the lower cotyledons in contact with the medium formed multiple buds while those elevated above the medium did not. Cotyledons became incompetent to form buds when embryos were initially placed on basal medium for 2 days before being transferred to the BA-supplemented medium. In order to initiate buds, cotyledons of newly excised embryos had to be exposed to BA for at least 3 days. Exposure to BA for longer than 7 days did not significantly increase the number of cotyledonary buds. Cotyledons elevated above the BA-supplemented medium retained organogenic competence to form buds for up to 14 days but did not form buds unless the cotyledons were placed in contact with the medium. The presence of BA is required to retard cotyledon maturation and sustain tissue competence to initiate buds.     

棉花花芽分化时期茎尖内源激素的变化

实验结果表明,从子叶展平后到肉眼可花芽（现蕾）,所测几种激素（ABA、IAA、GA3、iPA、ZR）的含量均表现出明显的动态变化,而且在花芽分化临界期表现出最显著的变化（出现高峰或出现低峰）。推测所测几种激素均与花芽分化有密切关系。其中ABA、GA3和CTK（iPA、ZR）在花芽分化临界期时,其含量变化均呈现出一个高峰;而IAA则在花芽分化临界期时出现一个低峰。经比较分析得知,随着花芽分化的进行,ABA/IAA、GA3／IAA、CTK／IAA均表现一个较明显的变化规律。即从子叶展平时起,其比值开始上升,到花芽开始分化时达到一个峰值,之后逐渐下降,并维持在一个较稳定的水平。显然,ABA／IAA、GA3／IAA、CTK／IAA在棉花的花芽分化过程中起着重要的调控作用。由此推测,增加植物体内的ABA、GA3、CTK的含量或降低IAA的含量,都可以促进棉花的花芽分化;反之则抑制棉花的花芽分化。     

Molecular and biochemical characterization of the Skp2-Cks1 binding interface

SCF(Skp2) is a multisubunit E3 ubiquitin ligase responsible for ubiquitination of cell cycle inhibitor p27. Ubiquitination of p27 requires an adapter protein, Cks1, to be in direct association with Skp2. The exact interface between Skp2 and Cks1 has not been elucidated. Here we have reported the definition of the critical functional interface between Skp2 and Cks1. We have identified eight amino acid residues in two discrete regions of Skp2 that are engaged in Cks1 binding. Mutation of any of these eight residues alone or in combination results in the loss of Cks1 association and negates Skp2-dependent p27 ubiquitination. These eight amino acid residues map on the same side of the Skp2 structure and likely constitute a functional binding surface for Cks1. Four of the eight amino acid residues are located in the largely unstructured carboxyl-terminal tail region of Skp2. These results uncovered the specificity of the Skp2-Cks1 interaction and reveal a critical function for the structurally flexible carboxyl-terminal tail region of Skp2 in Cks1 recognition and substrate ubiquitination.     

Alternative and cytochrome pathway respiration during shoot bud formation in cultured Pinus radiata cotyledons

Respiration rates for excised cotyledons of Pinus radiata cultured in the presence (shoot-forming) and absence (non-shoot-forming) of N⁶-benzyladenine (BA) over a 21-day period were measured using a Clark-type oxygen electrode. The capacities and activities of cytochrome and alternative pathways of respiration were determined from titrations with KCN (1-10 m M ) and salicylhydroxamic acid (2–20 m M ) individually and in combination. Respiration accounted for by alternative (AP) and cytochrome (CP) pathways varied with both culture treatment and age in culture. Rates of total respiration, CP respiration and AP activity rose concurrent with key developmental events of shoot bud formation. The greatest AP capacity was measured at day 3 in shoot-forming tissue. In contrast, for cotyledons cultured under non-shoot-forming conditions, no AP activity was observed after day 3 despite relatively constant AP capacity throughout the culture period. Although initial increases in cotyledon respiration during the culture period may be related to wounding and introduction to a tissue culture environment, later differences in respiratory patterns between shoot-forming and non-shoot-forming cotyledons appear to be associated with the cytokinin-induced developmental changes which give rise to shoot primordia in cultured radiata pine cotyledons.     

Protein modification by Amadori and Maillard reactions during seed storage: roles of sugar hydrolysis and lipid peroxidation

The non-enzymatic modifications of proteins through Amadori and Maillard reactions play an important role in the loss of seed viability during storage. In the present study, the contribution of sugar hydrolysis and lipid peroxidation to Amadori and Maillard reactions, and to seed deterioration was investigated in mung-bean (Vigna radiata Wilczek). The contents of glucose and lipid peroxidation products in seed axes increased significantly during storage. The accumulation of Amadori products in seed axes was correlated to the lipid peroxidation, whereas the accumulation of Maillard products was closely correlated to sugar hydrolysis. The rate of accumulation of Maillard products was not well correlated to the content of Amadori products in both seed axes and protein/glucose model system, reflecting the complex nature of Amadori and Maillard reactions. The content of Amadori products in seed axes increased during the early stages of seed ageing, whereas the content of Maillard products increased steadily during the entire period of storage. The accumulation of Maillard products in seed axes was associated with the decline of seed vigour. These data suggest that, during seed ageing, sugar hydrolysis and lipid peroxidation are coupled with non-enzymatic protein modification through Amadori and Maillard reactions.     

苦瓜离体培养过程中内源激素含量的变化

针对苦瓜(Momordica charantia)离体培养中外植体易于产生愈伤组织而难以再生不定芽的问题,本文采用酶联免疫吸附法, 研究了苦瓜组织培养过程中不同发育阶段各外植体内源激素含量的变化, 以探讨不定芽分化与激素水平变化之间的关系, 以及苦瓜难以再生不定芽的内在制约因素。结果表明: (1)苦瓜外植体中IAA含量较高, 而iPAs含量过低, 是苦瓜易于产生愈伤组织而不定芽再生困难的主要原因;(2)不定芽的分化与IAA/iPAs的变化有密切的关系; (3)在离体培养过程中, 保持外源细胞分裂素类物质(如ZT)的适当浓度并及时继代, 有利于苦瓜不定芽的分化。