薇甘菊花芽分化前后内源激素及相关基因表达的研究

以云南省瑞丽市勐秀林场扦插种植的薇甘菊为试材,采用液相色谱串联质谱(LC-MS/MS)技术对花芽未分化期和花序原基分化期花芽中的生长素(IAA)、赤霉素(GA)、脱落酸(ABA)、反式玉米素(tZ)、异戊烯腺嘌呤(IP)、1-氨基环丙烷羧酸(ACC)、茉莉酸(JA)和水杨酸(SA)含量进行定量分析,同时基于转录组基因功能注释数据对内源激素合成、代谢及信号转导途径相关基因进行表达分析,以探讨不同内源激素对薇甘菊花芽形成的调控作用,以及内源激素合成和信号转导途径相关基因调控薇甘菊花芽分化的机制,为后期通过外源激素调控薇甘菊内源激素水平的方式来控制薇甘菊的有性繁殖提供理论和技术支持。结果表明：(1)薇甘菊未分化期花芽中GA₁₅、GA₁₉、GA₂₀、GA₂₄、IAA、ABA和ETH含量低于花序原基分化期,而未分化期花芽中两种细胞分裂素tZ和IP含量显著高于花序原基分化期。(2)基于RNA-seq测序结果,在薇甘菊两个花芽分化时期共获得7 116个差异表达基因(DEGs),其中上调3 907个,下调3 209个。(3)在内源激素合成方面,参与GA₁₅、GA₁₉、GA₂₀、GA₂₄、IAA、ABA和ACC合成的大量DEGs在花序原基分化期上调表达,这与它们在薇甘菊花序原基分化期的高含量趋势相一致;参与IAA合成的YUCCA基因家族和ACC合成的ACS基因在花序原基分化期的高表达也可能参与促进薇甘菊花芽分化。(4)在植物激素转导途径中,在花序原基分化期,生长素信号转导途径通过AUX/IAA(gene-E3N88_07743)的下调表达和ARF(gene-E3N88_41119)的上调表达,乙烯信号转导途径通过ERF(gene-E3N88_41547)的上调表达,赤霉素信号转导途径通过GID1(gene-E3N88_19448)基因的上调表达,细胞分裂素信号转导途径通过B-ARR(gene-E3N88_28086)和A-RRR(gene-E3N88_40764)基因的下调表达,脱落酸途径通过AREB(gene-E3N88_18558)基因的上调表达,茉莉酸信号转导途径通过JAZ(gene-E3N88_05628)的上调表达和MYC2(gene-E3N88_32405)的下调表达来调控薇甘菊花芽分化。研究发现,高水平的GA₁₅、GA₁₉、GA₂₀、GA₂₄、IAA、ABA和ACC有利于薇甘菊的花芽分化;薇甘菊在花芽分化过程中通过改变不同种类内源激素合成、代谢基因的表达来调控激素浓度,而激素又通过信号转导途径引起下游基因的表达,进而调控薇甘菊的花芽分化。     

Response of Amaranthus retroflexus L. seeds to gibberellic acid, ethylene and abscisic acid depending on duration of stratification and burial

Freshly harvested, dormant seeds of Amaranthus retroflexus were unable to germinate at 25 and 35 °C. To release their dormancy at the above temperatures, the seeds were stratified at a constant temperature (4 °C) under laboratory conditions or at fluctuating temperatures in soil or by outdoor burial in soil. Fully dormant, or seeds stratified or buried (2006/2007 and 2007/2008) for various periods were treated with exogenous gibberellic acid (GA₃), ethephon and abscisic acid (ABA). Likewise, the effects of these regulators, applied during stratification, on seed germination were determined. The results indicate that A. retroflexus seed dormancy can be released either by stratification or by autumn–winter burial. The effect of GA₃ and ethylene, liberated from ethephon, applied after various periods of stratification or during stratification, depends on dormancy level. GA₃ did not affect or only slightly stimulated the germination of non-stratified, fully dormant seeds at 25 and 35 °C respectively. Ethylene increased germination at both temperatures. Seed response to GA₃ and ethylene at 25 °C was increased when dormancy was partially removed by stratification at constant or fluctuating temperatures or autumn–winter burial. The response to GA₃ and ethylene increased with increasing time of stratification. The presence of GA₃ and ethephon during stratification may stimulate germination at 35 °C. Thus, both GA₃ and ethylene can partially substitute the requirement for stratification or autumn–winter burial. Both hormones may also stimulate germination of secondary dormant seeds, exhumed in September. The response to ABA decreased in parallel with an increasing time of stratification and burial up to May 2007 or March 2008. Endogenous GA_n, ethylene and ABA may be involved in the control of dormancy state and germination of A. retroflexus. It is possible that releasing dormancy by stratification or partial burial is associated with changes in ABA/GA and ethylene balance and/or sensitivity to these hormones.     

Changes in Parameters of the Plasmalemma ATPase during Peach Vegetative Bud Dormancy

Plant dormancy and dormancy breaking depend, at least partially, on close relationships between buds and tissues underlying bud (bud stands). In Prunus persica, the dormancy was related to high nutrient absorption in bud stands linked to high plasmalemma ATPase (EC 3.6.1.3) activity. Two plasmalemma fractions was isolated from peach vegetative buds and bud stands using aqueous phase partitioning and ultracentrifugation. Results of markers enzyme assays indicated that both plasmalemma enriched fractions obtained were highly purified. During the dormancy period plasma membrane ATPase amount and activity were higher in bud stands than in buds. Moreover, assays performed at different temperatures (4, 18, 30 °C) indicated modifications of kinetic parameters (K_m, V_m) in both tissues during dormancy release. In buds, from November to February, K_m declined at 4°C and increased at 30 °C whereas no changes was measured at 18 °C and V_m increased at all temperature. In bud stands, no changes of K_m was measured at 4 °C and 18 °C whereas an increase occurred at 30 °C and V_m decreased at all temperature. According to the results, it can be postulated that dormancy release in peach-tree could be related to modifications of plasma membrane ATPase properties, in buds and bud stands, during winter time.     

Functional diversification of the dehydrin gene family in apple and its contribution to cold acclimation during dormancy

Dehydrins (DHN) are proteins involved in plant adaptive responses to abiotic stresses, mainly dehydration. Several studies in perennial crops have linked bud dormancy progression, a process characterized by the inability to initiate growth from meristems under favorable conditions, with DHN gene expression. However, an in‐depth characterization of DHNs during bud dormancy progression is still missing. An extensive in silico characterization of the apple DHN gene family was performed. Additionally, we used five different experiments that generated samples with different dormancy status, including genotypes with contrasting dormancy traits, to analyze how DHN genes are being regulated during bud dormancy progression in apple by real‐time quantitative polymerase chain reaction (RT‐qPCR). Duplication events took place in the diversification of apple DHN family. Additionally, MdDHN genes presented tissue‐ and bud dormant‐specific expression patterns. Our results indicate that MdDHN genes are highly divergent in function, with overlapping levels, and that their expressions are fine‐tuned by the environment during the dormancy process in apple.     

Studies with Liatris spicata Willd. 1. Effect of Temperature on Sprouting, Flowering and Gibberellin Content

Dormancy was induced during storage of Liatris spicata cormsgrown in Israeli summer conditions, but plants left in soilcontinued vegetative growth. Corms of winter-grown plants sproutedfreely. Treatment with GA₃ restored both sprouting and floweringin summer-grown corms, but in winter corms GA₂ was effectiveonly after corms were stored at low temperature. All the plantsflowered after 4 weeks at 2 °C and GA³ treatment. The content of gibberellins in the main bud of freshly excavatedcorms decreased during the first 18 d of storage but increasedto the initial level after 4 months of cold storage. The numberof flowering stems increased to 2.5 per corm when corms werecold-stored up to 75 d, but decreased with a longer storage. Liatris spicata, dormancy, flowering, gibberellin, sprouting     

Variation in seed dormancy and germination among populations of Silybum marianum (Asteraceae)

Milk thistle (Silybum marianum) is a medicinal plant; however, lack of consistency in past dormancy studies has hindered propagation of this species from seeds. We tested the germination responses of freshly harvested and after-ripened (stored for 2 and 7 months; 25°C at 50% relative humidity) seeds from three populations (P1, P2 and P3) in Iran at varying constant or alternating temperatures, with or without GA₃ and in light and continuous darkness. No germination occurred in freshly harvested seeds incubated at any condition without GA₃ application, indicating that all the seeds were dormant. Seeds from P1 and P2, which developed under relatively dry, warm conditions, germinated over a wider range of temperatures after 2 months of dry storage, indicating type 6 of non-deep physiological dormancy (PD). Seeds from P3, which developed under relatively wet, cool conditions, incubated at constant temperatures (especially on GA₃), exhibited an increase in maximum temperature for germination, indicating type 1 of non-deep PD. Light improved germination of after-ripened seeds, and GA₃ application substituted for the light requirement for germination. This is the first report that environmental conditions during seed development may be correlated with differences in the type of non-deep PD. We conclude that milk thistle seeds are positively photoblastic and photodormant and the germination responses of after-ripened seeds from different populations are different under darkness. Therefore, the impacts of genetic differences and maternal effects on the induction of dormancy during seed development should be considered in attempts to domesticate this medicinal plant.     

Ethanol breaks dormancy of the potato tuber apical bud

Growing potato tubers or freshly harvested mature tubers have a dormant apical bud. Normally, this dormancy is spontaneously broken after a period of maturation of the tuber, resulting in the growth of a new sprout. Here it is shown that in in vitro-cultured growing and maturing tubers, ethanol can rapidly break this dormancy and re-induce growth of the apical bud. The in vivo promoter activity of selected genes during this secondary growth of the apical bud was monitored, using luciferase as a reporter. In response to ethanol, the expression of carbohydrate-storage, protein-storage, and cell division-related genes are rapidly down-regulated in tuber tissue. It was shown that dormancy was broken by primary but not by secondary alcohols, and the effect of ethanol on sprouting and gene expression in tuber tissue was blocked by an inhibitor of alcohol dehydrogenase. By contrast, products derived from alcohol dehydrogenase activity (acetaldehyde and acetic acid) did not induce sprouting, nor did they affect luciferase reporter gene activity in the tuber tissue. Application of an inhibitor of gibberellin biosynthesis had no effect on ethanol-induced sprouting. It is suggested that ethanol-induced sprouting may be related to an alcohol dehydrogenase-mediated increase in the catabolic redox charge [NADH/(NADH+NAD+)].     

Physiological characteristics and related gene expression of after‐ripening on seed dormancy release in rice

After‐ripening is a common method used for dormancy release in rice. In this study, the rice variety Jiucaiqing (Oryza sativa L. subsp. japonica) was used to determine dormancy release following different after‐ripening times (1, 2 and 3 months). Germination speed, germination percentage and seedling emergence increased with after‐ripening; more than 95% germination and 85% seedling emergence were observed following 1 month of after‐ripening within 10 days of imbibition, compared with <45% germination and 20% seedling emergence in freshly harvested seed. Hence, 3 months of after‐ripening could be considered a suitable treatment period for rice dormancy release. Dormancy release by after‐ripening is mainly correlated with a rapid decline in ABA content and increase in IAA content during imbibition. Subsequently, GA₁/ABA, GA₇/ABA, GA₁₂/ABA, GA₂₀/ABA and IAA/ABA ratios significantly increased, while GA₃/ABA, GA₄/ABA and GAs/IAA ratio significantly decreased in imbibed seeds following 3 months of after‐ripening, thereby altering α‐amylase activity during seed germination. Peak α‐amylase activity occurred at an earlier germination stage in after‐ripened seeds than in freshly harvested seeds. Expression of ABA, GA and IAA metabolism genes and dormancy‐related genes was regulated by after‐ripening time upon imbibition. Expression of OsCYP707A5, OsGA2ox1, OsGA2ox2, OsGA2ox3, OsILR1, OsGH3‐2, qLTG3‐1 and OsVP1 increased, while expression of Sdr4 decreased in imbibed seeds following 3 months of after‐ripening. Dormancy release through after‐ripening might be involved in weakening tissues covering the embryo via qLTG3‐1 and decreased ABA signalling and sensitivity via Sdr4 and OsVP1.     

Induction of Secondary Dormancy in Chenopodium bonus-henricus L. Seeds by Osmotic and High Temperature Treatments and Its Prevention by Light and Growth Regulators

Factors controlling the establishment and removal of secondary dormancy in Chenopodium bonus-henricus L. seeds were investigated. Unchilled seeds required light for germination. A moist-chilling treatment at 4 C for 28 to 30 days removed this primary dormancy. Chilled seeds now germinated in the dark. When chilled seeds were held in the dark in −8.6 bars polyethylene glycol 6000 solution at 15 C or in water at 29 C a secondary dormancy was induced which increased progressively with time as determined by subsequent germination. These seeds now failed to germinate under the condition (darkness) which previously allowed their germination. Continuous light or daily brief red light irradiations during prolonged imbibition in polyethylene glycol solution at 15 C or in water at 29 C prevented the establishment of the secondary dormancy and caused an advancement of subsequent germination. Far red irradiations immediately following red irradiation reestablished the secondary dormancy indicating phytochrome participation in “pregerminative” processes. The growth regulator combination, kinetin + ethephon + gibberellin A₄+A₇ (GA₄₊₇), and to a relatively lesser extent GA₄₊₇, was effective in preventing the establishment of the secondary dormancy and in advancing the germination or emergence time. Following the establishment of the secondary dormancy by osmotic or high temperature treatments the regulator combination was relatively more active than light or GA₄₊₇ in removing the dormancy. Prolonged dark treatment at 29 C seemed to induce changes that were partially independent of light or GA₄₊₇ control. The data presented here indicate that changes during germination preventing dark treatment determine whether the seed will germinate, show an advancement effect, or will become secondarily dormant. These changes appear to be modulated by light and hormones.     

Gibberellins and Subapical Cell Divisions in Relation to Bud Set and Bud Break in Salix pentandra

In young plants of Salix pentandra, a temperate zone deciduous woody species, elongation growth ceases and a terminal bud is formed at day lengths shorter than a critical length. This is the first step in dormancy development, making survival under harsh winter conditions possible. Early studies strongly indicate that gibberellin is involved in the photoperiodic control of bud set and bud break. GA₁ action was studied by application under short days to plants where cessation of shoot elongation had occurred, followed by subsequent anatomic investigations of shoot tips. Under short days the frequency of cell division decreased rapidly along with the earlier observed decrease in GA₁ levels. Application of GA₁ to short-day–induced terminal buds rapidly stimulated cell division in apices several days before visible shoot elongation in response to this treatment was observed. One day after GA₁ application a fourfold increase in cell division frequency in apices was observed, increasing to a maximum of sevenfold 2 days after application. Long-day treatment leading to induction of bud break after about 4–6 days was followed by slowly increasing frequency of cell divisions. In earlier studies of this species, short days and gibberellins had no effect on cell elongation. These data show that increased GA₁ content, by application or long-day treatment, results in increased frequency of mitosis. This strongly indicates that GA₁ affects stem elongation in connection with bud set and bud break primarily by affecting cell divisions in subapical tissues. Received February 26, 1999; accepted October 8, 1999     

桃花芽自然休眠期间水孔蛋白基因的转录表达

以10年生大田栽培及3年生盆栽曙光油桃花芽为试材,利用荧光定量PCR测定了油桃休眠及休眠解除期间(2009年9月15日-2010年1月15日)曙光油桃水孔蛋白基因δTIP1、PIP1;1的表达量,以及低温胁迫下的转录表达.结果表明:在油桃休眠及休眠解除期间,曙光油桃PIP1;1的转录水平呈现持续增高趋势,且1月的高水平表达使水分通过液泡膜和细胞质膜流出,减少了芽体水分含量,阻止细胞内冰晶的形成,从而抵御冻害;可溶性糖、可溶性蛋白、脯氨酸含量均达到最高,防止细胞的脱水伤害.低温处理2周后高水平表达说明PIP1;1为冷诱导基因.δTIP1的转录水平在休眠期间呈现波动性变化,至休眠解除时大幅度增高,这可能与休眠解除时,其上调表达被休眠解除信号及植物活性的增强所诱导有关.低温处理2周后,其表达没有升高,说明δTIP1并非冷诱导基因.     

The Cold Awakening of <Emphasis Type="Italic">Doritaenopsis</Emphasis> ‘Tinny Tender’ Orchid Flowers: The Role of Leaves in Cold-induced Bud Dormancy Release

Massive flowering of tropical Phalaenopsis orchids is coordinated by the cold-induced release of reproductive bud dormancy. Light and temperature are the two key factors integrated by the dormancy mechanism to both stop and reactivate the meristem development of many other angiosperm species, including fruit trees and ornamental plants. It is well established that leaves and roots play a major role in inducing flower development; however, currently, knowledge of molecular events associated with reproductive bud dormancy release in organs other than the bud is limited. Using differential gene expression, we have shown that the leaves of a hybrid of Phalaenopsis species, Doritaenopsis ‘Tinny Tender’, undergo major metabolic modifications. These changes result in the production of sucrose and amino acids, both of which can sustain bud outgrowth, and auxin and ethylene, which may play important roles in awaking the dormant buds. Intake of abscisic acid and synthesis of the hormone jasmonate may also explain the inhibition of vegetative growth that coincides with bud growth. Interestingly, many genes that were upregulated by cold treatment are homologous for genes involved in flower induction and vernalization in Arabidopsis, indicating that processes regulating flowering induction and those regulating reproductive bud dormancy release may use similar pathways and effector molecules.