Decrease In Abscisic Acid Content Research Articles

S-nitrosylation of the transcription factor MYB30 facilitates nitric oxide-promoted seed germination in Arabidopsis.

The gaseous signaling molecule nitric oxide (NO) plays an important role in breaking seed dormancy. NO induces a decrease in abscisic acid (ABA) content by transcriptionally activating its catabolic enzyme, the ABA 8'-hydroxylase CYP707A2. However, the underlying mechanism of this process remains unclear. Here, we report that the transcription factor MYB30 plays a critical role in NO-induced seed germination in Arabidopsis (Arabidopsis thaliana). MYB30 loss-of-function attenuates NO-mediated seed dormancy breaking. MYB30 triggers a NO-induced decrease in ABA content during germination by directly promoting CYP707A2 expression. NO induces S-nitrosylation at Cys-49 of MYB30 and enhances its transcriptional activity. Conversely, the ABA receptors PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) interact with MYB30 and repress its transcriptional activity. ABA promotes the interaction between PYL4 and MYB30, whereas S-nitrosylation releases the PYL4-mediated inhibition of MYB30 by interfering with the PYL4-MYB30 interaction. Genetic analysis showed that MYB30 functions downstream of PYLs during seed dormancy and germination in response to NO. Furthermore, MYB30 mutation significantly represses the reduced dormancy phenotype and the enhanced CYP707A2 expression of the pyr1 pyl1 pyl2 pyl4 quadruple mutant. Our findings reveal that S-nitrosylation of MYB30 precisely regulates the balance of seed dormancy and germination, providing insights into the underlying mechanism of NO-promoted seed germination.

Temporal transcriptome and metabolite analyses provide insights into the biochemical and physiological processes underlying endodormancy release in pistachio (Pistacia vera L.) flower buds.

Pistachio (Pistacia vera L.), an economically and nutritionally important tree crop, relies on winter chill for bud endodormancy break and subsequent blooming and nut production. However, insufficient winter chill poses an increasing challenge in pistachio growing regions. To gain a better understanding of the physiological and biochemical responses of endodormant pistachio buds to chilling accumulation, we investigated the global gene expression changes in flower buds of pistachio cv. Kerman that were cultivated at three different orchard locations and exposed to increasing durations of winter chill. The expression of genes encoding β-1,3-glucanase and β-amylase, enzymes responsible for breaking down callose (β-1,3-glucan) and starch (α-1,4-glucan), respectively, increased during the endodormancy break of pistachio buds. This result suggested that the breakdown of callose obstructing stomata as well as the release of glucose from starch enables symplasmic trafficking and provides energy for bud endodormancy break and growth. Interestingly, as chilling accumulation increased, there was a decrease in the expression of nine-cis-epoxycarotenoid dioxygenase (NCED), encoding an enzyme that uses carotenoids as substrates and catalyzes the rate-limiting step in abscisic acid (ABA) biosynthesis. The decrease in NCED expression suggests ABA biosynthesis is suppressed, thus reducing inhibition of endodormancy break. The higher levels of carotenoid precursors and a decrease in ABA content in buds undergoing endodormancy break supports this suggestion. Collectively, the temporal transcriptome and biochemical analyses revealed that the degradation of structural (callose) and non-structural (starch) carbohydrates, along with the attenuation of ABA biosynthesis, are critical processes driving endodormancy break in pistachio buds.

Spermidine enhances chilling tolerance of kale seeds by modulating ROS and phytohormone metabolism.

Chilling stress is an important constraint for kale seed germination and seedlings establishment. It is vital to develop an effective approach to enhance kale seed germination ability under chilling stress. The present study reported that spermidine (Spd) could improve seed chilling tolerance in two kale cultivars 'Nagoya' (MGW) and 'Pigeon' (BB) during germination. The results showed that MGW was cold tolerant with a 90.67% germination percentage (GP) under chilling stress, while BB was cold sensitive with a 70.67% GP under chilling stress. Spd content in MGW and BB seeds during seed germination were up-regulated and down-regulated by chilling stress, respectively. Besides, chilling stress apparently decreased the gibberellin (GA) and ethylene (ET) contents, while increased the levels of abscisic acid (ABA) and reactive oxygen species (ROS) in MGW and BB seeds during germination. Exogenous Spd application increased GA, ET contents and decreased ABA content through regulating the gene expressions of metabolic-related enzymes, thus effectively alleviating the low temperature damage on kale seed germination. Besides, Spd significantly increased the activities of superoxide dismutase (SOD) and peroxidase (POD), and reduced the levels of hydrogen peroxide (H2O2) and superoxide anion (O2·-). The present study demonstrated that endogenous Spd metabolism plays an important role in kale seed germination under chilling stress. The effect of exogenous Spd on the metabolism of endogenous Spd, GA, ABA, ET and antioxidant enzymes might be the important reason for promoting the kale seed vigor at low temperature.

Hydrogen-rich water treatment increased several phytohormones and prolonged the shelf life in postharvest okras.

Hydrogen-rich water (HRW) treatment has been reported to delay the softening and senescence of postharvest okras, but its regulatory mechanism remains unclear. In this paper, we investigated the effects of HRW treatment on the metabolism of several phytohormones in postharvest okras, which act as regulatory molecules in fruit ripening and senescence processes. The results showed that HRW treatment delayed okra senescence and maintained fruit quality during storage. The treatment upregulated all of the melatonin biosynthetic genes such as AeTDC, AeSNAT, AeCOMT and AeT5H, contributing to the higher melatonin content in the treated okras. Meanwhile, increased transcripts of anabolic genes but lower expression of catabolic genes involved in indoleacetic acid (IAA) and gibberellin (GA) metabolism were observed in okras when treated with HRW, which was related to the enhanced levels of IAA and GA. However, the treated okras experienced lower abscisic acid (ABA) content as compared to the non-treated fruit due to the down-regulation of its biosynthetic genes and up-regulation of the degradative gene AeCYP707A. Additionally, there was no difference in γ-aminobutyric acid between the non-treated and HRW-treated okras. Collectively, our results indicated that HRW treatment increased levels of melatonin, GA and IAA, but decreased ABA content, which ultimately delayed fruit senescence and prolonged shelf life in postharvest okras.

Phytohormone profiles and related gene expressions after endodormancy release in developing Pinus tabuliformis male strobili

Development after endo-dormancy release ensures perennial plants, such as forest trees, proper response to environmental changes and enhances their adaptability. In northern hemisphere, megasporophore and microsporophore of conifers undergo dormancy to complete their development. Here combined with transcriptome data, we used high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (ESI-HPLC-MS/MS) to quantitatively analyse the various hormones (Abscisic Acid (ABA), 3-Indoleacetic acid (IAA), Gibberellins (GAs), Cytokinin (CTK), Jasmonic acid (JA) and Salicylic acid (SA)) of Chinese pine (Pinus tabuliformis Carr.) male strobili after endo-dormancy release. More specifically, we analysed endogenous hormones and their related-genes and verified the important role of ABA in plants growth and development. We observed rapid decrease in ABA content after dormancy release, resulting in reducing the inhibitory effect on male strobili growth. Similarly, rapid drop in ABA/GA ratio was observed and was associated with the start of male strobili growth and development. Combined with transcriptome data, we found that HAB2-SnRK2.10 played a central role in the ABA pathway in the entire network of hormones regulating male strobili development. Due to external environment warming, the differentially expressed HAB2-SnRK gene led to ABA content rapid decline, thus initiating male strobili growth. We constructed a network of hormone-regulated development to understand the interactions between hormones after male strobili dormancy release of male strobili. This study provided essential foundations for studying megasporophore and microsporophore growth mechanism after endo-dormancy and offered new ideas for flower development in gymnosperms and angiosperms.

Can Bacterial Endophytes Be Used as a Promising Bio-Inoculant for the Mitigation of Salinity Stress in Crop Plants?-A Global Meta-Analysis of the Last Decade (2011-2020).

Soil salinity is a major problem affecting crop production worldwide. Lately, there have been great research efforts in increasing the salt tolerance of plants through the inoculation of plant growth-promoting endophytic bacteria. However, their ability to promote plant growth under no-stress and salinity-stress conditions remains largely uncertain. Here, we carried out a global meta-analysis to quantify the plant growth-promoting effects (improvement of morphological attributes, photosynthetic capacity, antioxidative ability, and ion homeostasis) of endophytic bacteria in plants under no-stress and salinity-stress conditions. In addition, we elucidated the underlying mechanisms of growth promotion in salt-sensitive (SS) and salt-tolerant (ST) plants derived from the interaction with endophytic bacteria under no-stress and salinity-stress conditions. Specifically, this work encompassed 42 peer-reviewed articles, a total of 77 experiments, and 24 different bacterial genera. On average, endophytic bacterial inoculation increased morphological parameters. Moreover, the effect of endophytic bacteria on the total dry biomass, number of leaves, root length, shoot length, and germination rate was generally greater under salinity-stress conditions than no-stress conditions. On a physiological level, the relative better performance of the bacterial inoculants under the salinity-stress condition was associated with the increase in total chlorophyll and chlorophyll-b, as well as with the decrease of 1-aminocylopropane-1-carboxylate concentration. Moreover, under the salinity-stress condition, bacterial inoculation conferred a significantly higher increase in root K+ concentration and decrease in leaf Na+ concentration than under the no-stress condition. In SS plants, bacterial inoculation induced a higher increase in chlorophyll-b and superoxide dismutase activity, as well as a higher decrease in abscisic acid content, than in ST plants. Under salinity-stress, endophytic bacterial inoculation increased root K+ concentration in both SS and ST plants but decreased root Na+ concentration only in ST plants. Overall, this meta-analysis suggests that endophytic bacterial inoculation is beneficial under both no salinity-stress and salinity-stress conditions, but the magnitude of benefit is definitely higher under salinity-stress conditions and varies with the salt tolerance level of plants.

CRISPR/Cas9-mediated mutagenesis of ClBG1 decreased seed size and promoted seed germination in watermelon

Abscisic acid (ABA) is a critical regulator of seed development and germination. β-glucosidases (BGs) have been suggested to be contributors to increased ABA content because they catalyze the hydrolysis of ABA-glucose ester to release free ABA. However, whether BGs are involved in seed development is unclear. In this study, a candidate gene, ClBG1, in watermelon was selected for targeted mutagenesis via the CRISPR/Cas9 system. Seed size and weight were significantly reduced in the Clbg1-mutant watermelon lines, which was mainly attributed to decreased cell number resulting from decreased ABA levels. A transcriptome analysis showed that the expression of 1015 and 1429 unique genes was changed 10 and 18 days after pollination (DAP), respectively. Cytoskeleton- and cell cycle-related genes were enriched in the differentially expressed genes of wild type and Clbg1-mutant lines during seed development. Moreover, the expression of genes in the major signaling pathways of seed size control was also changed. In addition, seed germination was promoted in the Clbg1-mutant lines due to decreased ABA content. These results indicate that ClBG1 may be critical for watermelon seed size regulation and germination mainly through the modulation of ABA content and thereby the transcriptional regulation of cytoskeleton-, cell cycle- and signaling-related genes. Our results lay a foundation for dissecting the molecular mechanisms of controlling watermelon seed size, a key agricultural trait of significant economic importance.

Salicylic acid enhances sunflower seed germination under Zn2+ stress via involvement in Zn2+ metabolic balance and phytohormone interactions

Zinc (Zn2+) stress is an important abiotic constraint affecting crop seed germination and plant growth. It is vital to exploit and utilize zinc-polluted soil by exploring technologies for promoting seed germination under Zn2+ stress. This study aims to explore the effects of salicylic acid (SA) and 2-aminoindan-2-phosphonic acid (AIP, SA synthesis inhibitor) on the Zn2+ stress resistance of sunflower during seed germination. Zn2+ treatment significantly inhibited seed germination and seedling growth of sunflower. In addition, Zn2+ treatment increased Zn2+ and abscisic acid (ABA) contents, while decreased SA and gibberellins (GA) contents of sunflower seeds. Exogenous SA effectively alleviated the inhibition of Zn2+ stress on sunflower seed germination, promoted endogenous SA anabolism, and increased the expression of zinc transporter-related genes family (MTP and HMA), which leading to decreased Zn2+accumulation. Moreover, exogenous SA increased GA content and decreased ABA content by regulating the gene expressions of metabolic related enzymes. On the contrary, AIP treatment further aggravated the inhibitory effect of Zn2+ stress on sunflower seed germination. This study demonstrated that exogenous SA could affect the Zn2+ metabolic balance in sunflower seeds by regulating the expression of Zn2+ transport-related gene (MTP and HMA), promote the biosynthesis of endogenous SA and GA, inhibit the accumulation of ABA content, and improve the germination ability of sunflower seed under zinc stress.

CO2 enrichment enhanced drought resistance by regulating growth, hydraulic conductivity and phytohormone contents in the root of cucumber seedlings

The coordinated effects of CO2 enrichment and drought stress on cucumber leaves have attracted increasing research attention, but few studies have investigated the effects of CO2 enrichment on the root system under drought stress. So we analyzed the morphological parameters, hydraulic conductivity, aquaporin-related gene expression, and endogenous phytohormone contents in roots of cucumber seedlings cultured under different CO2 concentrations (approximately 400 and 800 ± 40 μmol mol−1) and drought stresses simulated by polyethylene glycol 6000 (0%, 5%, and 10%). The results showed that under drought stress, regardless of the CO2 concentration, the root biomass and hydraulic conductivity decreased, the contents of auxin (IAA), zeatin nucleoside (ZR), and gibberellin (GA) decreased, the abscisic acid (ABA) content and the transcript levels of the aquaporin-related genes CsPIP2-4 increased, and the transcript levels of the aquaporin-related genes CsPIP2-5 and CsPIP2-7 decreased compared with no drought stress. Under moderate drought stress, CO2 enrichment decreased ABA content and the transcript level of CsPIP2-4, increased root biomass and GA content and the transcript level of CsPIP2-7, improved contribution rate of cell-to-cell water transport (mediated by aquaporins) and roots hydraulic conductivity. In summary, drought stress changed the water transport capacity of the roots and inhibited the growth of cucumber seedlings. CO2 enrichment regulated phytohormone contents and aquaporin-related gene expression, maintained the normal contribution rate of cell-to-cell water transport, and improved the root biomass and hydraulic conductivity, thereby alleviated the negative effects of drought stress on cucumber seedlings.

Relationship between cold plasma treatment-induced changes in radish seed germination and phytohormone balance

This study aimed to estimate the dependence of the effects of radish (Raphanus sativus) seed irradiation with atmospheric cold plasma (CP) on physiological seed condition or dormancy status. Seeds were treated for different durations with a scalable dielectric barrier discharge (DBD) device. The relationship between the effect of CP on germination kinetics with changes in the content of phytohormones in seeds stored for different periods of time after harvesting (1, 5, and 7 months) was estimated. CP treatment efficiently stimulated germination only in freshly harvested (not after-ripened seeds), and these changes correlated with a decrease in abscisic acid (ABA) and an increase in gibberellin (GA) content. CP treatment induced a fast decrease in ABA content and ABA/GA ratio in after-ripened seeds (5 months after harvesting) but did not stimulate germination. Primary evidence of direct ABA modification by the reactive compounds from the gaseous phase of CP was obtained.

Copper ions suppress abscisic acid biosynthesis to enhance defence against Phytophthora infestans in potato.

Copper‐based antimicrobial compounds are widely and historically used to control plant diseases, such as late blight caused by Phytophthora infestans, which seriously affects the yield and quality of potato. We previously identified that copper ion (Cu2+) acts as an extremely sensitive elicitor to induce ethylene (ET)‐dependent immunity in Arabidopsis. Here, we found that Cu2+ induces the defence response to P. infestans in potato. Cu2+ suppresses the transcription of the abscisic acid (ABA) biosynthetic genes StABA1 and StNCED1, resulting in decreased ABA content. Treatment with ABA or inhibitor fluridone made potato more susceptible or resistance to late blight, respectively. In addition, potato with knockdown of StABA1 or StNCED1 showed greater resistance to late blight, suggesting that ABA negatively regulates potato resistance to P. infestans. Cu2+ also promotes the rapid biosynthesis of ET. Potato plants treated with 1‐aminocyclopropane‐1‐carboxylate showed enhanced resistance to late blight. Repressed expression of StEIN2 or StEIN3 resulted in enhanced transcription of StABA1 and StNCED1, accumulation of ABA and susceptibility to P. infestans. Consistently, StEIN3 directly binds to the promoter regions of StABA1 and StNCED1. Overall, we concluded that Cu2+ triggers the defence response to potato late blight by activating ET biosynthesis to inhibit the biosynthesis of ABA.

γ-Irradiation of Barley Seeds and Its Effect on the Phytohormonal Status of Seedlings

We investigated the effect of γ-irradiation (4–50 Gy) of barley seeds (Hordeum vulgare L., cv. Nur) on the content of endogenous phytohormones–stimulators of plant growth and development: indol-3-acetic acid (IAA), indolyl-3-butyric acid (IBA), zeatin and abscisic acid (ABA). The ratio (IAA + IBA + zeatin)/ABA from the third to the seventh day of germination has been measured. It was shown that the changes in the content of phytohormones as a function of the radiation dose were nonlinear. In the dose range of 4–20 Gy, phytohormones balance was changed due to increased content of growth stimulators and decreased ABA content. Using a dose of 50 Gy led primarily to a decrease in the content of growth stimulators and an increase in ABA content, and the ratio (IAA + IBA + zeatin)/ABA shifted toward ABA content.

ABA Biosynthesis Genes are Down-regulated While Auxin and Cytokinin Biosynthesis Genes are Up-regulated During the Release of Grapevine Buds From Endodormancy

Auxin–cytokinin (CK) interactions have been extensively studied in the control of bud outgrowth in herbaceous plants. However, in temperate woody plants where the meristem of dormant buds can be repressed by either exogenous or endogenous factors, abscisic acid (ABA) has been suggested as a potential regulator of bud outgrowth. To investigate the involvement of ABA, CK, and auxin on bud sprouting in Vitis vinifera, single-bud cuttings were used under forced conditions. This artificial bud sprouting system mimics and hastens the natural sprouting process that occurs in spring. Our results showed that expression of the ABA biosynthesis gene VvNCED1 decreased during incubation, whereas expression of the ABA catabolism gene VvA8H3 remained unaltered. Expression of CK biosynthesis-related genes ISOPENTENYL TRANSFERASE (VvIPTs) and LONELY GUY (VvLOG1), CK catabolism-related gene CYTOKININ OXIDASE (VvCKX3), and key auxin biosynthesis gene VvYUC3 increased with incubation time. Moreover, treatment with hydrogen cyanamide (HC), a compound that breaks vine latency, increased expression of VvIPTs and VvLOG1 and reduced expression of VvCKX3 and VvNCED1. These results are consistent with previous reports indicating that HC increases CK levels and decreases ABA levels in grapevine buds. Taken together, the results suggest that in the vine, bud sprouting is preceded by a decrease in ABA content and an increase in CK and auxin levels.

Melatonin suppression of heat-induced leaf senescence involves changes in abscisic acid and cytokinin biosynthesis and signaling pathways in perennial ryegrass (Lolium perenne L.)

Leaf senescence is a typical symptom of heat damage in cool-season plant species. The objective of this study was to determine whether melatonin could suppress heat-induced leaf senescence in perennial ryegrass (Lolium perenne L.) and whether melatonin interacted with abscisic acid (ABA) and cytokinin (CK) to exert its biological function. Perennial ryegrass (cv. ‘Pinnacle’) plants were foliar sprayed with melatonin or water and exposed to heat stress at 38/33°C (day/night) and non-stress condition at 22/17°C for 28d in growth chambers. Exogenous application of melatonin alleviated growth inhibition and leaf senescence induced by heat stress, as manifested by significant higher tiller number, above-ground dry weight, plant height, turf quality, leaf chlorophyll (Chl) content, photochemical efficiency (Fv/Fm), net photosynthesis rate, and cell membrane stability in melatonin-treated plants compared to non-melatonin treatment under heat stress. The suppression of heat-induced leaf senescence by melatonin was also reflected by the reduction of transcript levels of senescence-associated genes (LpSAG12.1 and Lph36) in plants exposed to heat stress. Melatonin treatment increased the endogenous content of melatonin and CK content, whereas it decreased ABA content under heat stress. The expression of CK biosynthesis genes and its signaling response transcription factors (type B ARRs) were up-regulated, while the biosynthesis and signaling genes involved in ABA were down-regulated by melatonin treatment under heat stress. These results indicate that the suppression of heat-induced leaf senescence by exogenous melatonin could be associated with activating CK synthesis and signaling while inhibiting ABA synthesis and signaling in perennial ryegrass.

Differential hormonal and gene expression dynamics in two inbred sunflower lines with contrasting dormancy level

Seed germination and dormancy are tightly regulated by hormone metabolism and signaling pathway. We investigated the endogenous content of abscisic acid (ABA), its catabolites, and gibberellins (GAs), as well as the expression level of certain ABA and GAs metabolic and signaling genes in embryo of dry and imbibed cypselas of inbred sunflower (Helianthus annuus L., Asteraceae) lines: B123 (dormant) and B91 (non-dormant). Under our experimental conditions, the expression of RGL2 gene might be related to the ABA peak in B123 line at 3 h of imbibition. Indeed, RGL2 transcripts are absent in dry and early embedded cypselas of the non-dormant line B91. ABA increase was accompanied by a significant ABA-Glucosyl ester (ABA-GE) and phaseic acid (PA) (two ABA catabolites) decrease in B123 line (3 h) which indicates that ABA metabolism seems to be more active in this line, and that it would be involved in the imposition and maintenance of sunflower seed dormancy, as it has been reported for many species. Finally, an increase of bioactive GAs (GA1 and GA3) occurs at 12 h of imbibition in both lines after a decrease in ABA content. This study shows the first report about the RGL2 tissue-specific gene expression in sunflower inbred lines with contrasting dormancy level. Furthermore, our results provide evidence that ABA and GAs content and differential expression of metabolism and signaling genes would be interacting in seed dormancy regulation through a mechanism of action related to embryo itself.

An A20/AN1-type zinc finger protein modulates gibberellins and abscisic acid contents and increases sensitivity to abiotic stress in rice (Oryza sativa).

The plant hormones gibberellins (GA) and abscisic acid (ABA) play important roles in plant development and stress responses. Here we report a novel A20/AN1-type zinc finger protein ZFP185 involved in GA and ABA signaling in the regulation of growth and stress response. ZFP185 was constitutively expressed in various rice tissues. Overexpression of ZFP185 in rice results in a semi-dwarfism phenotype, reduced cell size, and the decrease of endogenous GA3 content. By contrast, higher GA3 content was observed in RNAi plants. The application of exogenous GA3 can fully rescue the semi-dwarfism phenotype of ZFP185 overexpressing plants, suggesting the negative role of ZFP185 in GA biosynthesis. Besides GA, overexpression of ZFP185 decreased ABA content and expression of several ABA biosynthesis-related genes. Moreover, it was found that ZFP185, unlike previously known A20/AN1-type zinc finger genes, increases sensitivity to drought, cold, and salt stresses, implying the negative role of ZFP185 in stress tolerance. ZFP185 was localized in the cytoplasm and lacked transcriptional activation potential. Our study suggests that ZFP185 regulates plant growth and stress responses by affecting GA and ABA biosynthesis in rice.

Germination induction of dormant Avena fatua caryopses by KAR1 and GA3 involving the control of reactive oxygen species (H2O2 and O2[rad]−) and enzymatic antioxidants (superoxide dismutase and catalase) both in the embryo and the aleurone layers

Avena fatua L. caryopses did not germinate at 20°C in darkness because they were dormant. However, they were able to germinate in the presence of karrikinolide (KAR1), a key bioactive compound present in smoke, and also in the presence of gibberellin A3 (GA3), a commonly known stimulator of seed germination. The aim of this study was to collect information on a possible relationship between the above regulators and abscisic acid (ABA), reactive oxygen species (ROS) and ROS scavenging antioxidants in the regulation of dormant caryopses germination. KAR1 and GA3 caused complete germination of dormant A. fatua caryopses. Hydrogen peroxide (H2O2), compounds generating the superoxide (O2−), i.e. menadione (MN), methylviologen (MV) and an inhibitor of catalase activity, aminotriazole (AT), induced germination of dormant caryopses. KAR1, GA3, H2O2 and AT decreased ABA content in embryos. Furthermore, KAR1, GA3, H2O2, MN, MV and AT increased α-amylase activity in caryopses. The effect of KAR1 and GA3 on ROS (H2O2, O2−) and activities of the superoxide dismutase (SOD) and catalase (CAT) were determined in caryopses, embryos and aleurone layers. SOD was represented by four isoforms and catalase by one. In situ localization of ROS showed that the effect of KAR1 and GA3 was associated with the localization of hydrogen peroxide mainly on the coleorhiza. However, the superoxide was mainly localized on the surface of the scutellum. Superoxide was also detected in the protruding radicle. Germination induction of dormant caryopses by KAR1 and GA3 was related to an increasing content of H2O2, O2−and activities of SOD and CAT in embryos, thus ROS homeostasis was probably required for the germination of dormant caryopses. The above regulators increased the content of ROS in aleurone layers and decreased the activities of SOD and CAT, probably leading to the programmed cell death. The presented data provide new insights into the germination induction of A. fatua dormant caryopses by KAR1 and also by GA3. In A. fatua, KAR1 or GA3 is included in the induction germination of dormant caryopses through regulation level of ABA in embryos and ROS-antioxidant status both in embryos and aleurone layers.

LEAFY COTYLEDON2-mediated control of the endogenous hormone content: implications for the induction of somatic embryogenesis in Arabidopsis

The overexpression of LEAFY COTYLEDON2 (LEC2) in Arabidopsis, results in the induction of somatic embryogenesis (SE) in an auxin-free environment and the stimulation of auxin biosynthesis was postulated as being involved in this response. To gain further insight into the hormone-related functions of LEC2 in SE, the effect of LEC2 overexpression on the hormone content in Arabidopsis plants and in vitro cultured explants was analysed. In addition to indole-3-acetic acid (IAA) and cytokinins (CKs), which are hormones that play a key role in plant development, the stress-related hormones, abscisic acid (ABA) and salicylic acid (SA), which are involved in the stress response that is related to SE-induction, were analysed. Together with the observations that LEC2 activity can compensate for the auxin treatment required for SE induction (Ledwon and Gaj in Plant Tissue Cell Org Cult 28:1677–1688, 2009) and LEC2 may control auxin biosynthesis pathway during SE induction (Wojcikowska et al. in Planta 238:425–440, 2013), a significant increase in the IAA content in response to LEC2 overexpression found in the present study supply further evidence that LEC2-controlled auxin biosynthesis may be involved in the mechanism that triggers embryogenic development in somatic cells. Moreover, LEC2-controlled SE induction was shown to be associated with a decrease in the total content of CKs and an accumulation of some specific CK types, including isopentenyl-adenin and cis-zeatin. Additionally, an increase in SA and a decrease in ABA content were also found to be related to LEC2 activity in embryogenically induced tissue. The obtained results provide further proof of the close link between LEC2 and the establishment of the hormonal environment that is required for the promotion of SE.

FLOWERING RESPONSE OF AN EARLY AND LATE FLOWERING AZALEA CULTIVAR (RHODODENDRON SIMSII) TO COLD TREATMENT

Azalea flower bud differentiation is followed by a period of dormancy which is broken by cold. In this research two cultivars with different cold requirements (the early flowering cultivar 'Nordlicht' and the late flowering cultivar 'Mw. G. Kint') were kept at 7 degrees C after flower bud differentiation and monitored weekly. To follow dormancy release, endogenous abscisic acid (ABA) concentrations in the flower buds were measured and opening of buds was assessed by an in vitro bio-assay, testing the sensitivity to exogenous ABA. Flower buds of 'Nordlicht' had a steady ABA content for 5 weeks at 7 degrees C, indicating that endogenous ABA concentrations are not directly linked to dormancy breaking. The in vitro bio-assay on the other hand showed that four weeks of 7 degrees C are required for 'Nordlicht' to lose sensitivity to exogenous ABA. For 'Mw. G. Kint' there was a decrease in ABA content until 7 weeks at 7 degrees C, and flower buds lost sensitivity to exogenous ABA after 7 weeks. Carbohydrate and starch content in leaves and buds were examined since these are important in the successive forcing period. Starch levels in the leaves decreased with increasing cold period. 'Nordlicht' flower buds responded to the cold treatment by biosynthesis of raffinose and stachyose, flower buds of 'Mw. G. Kint' maintained the initial concentration of those carbohydrates. Finally the actual flowering response was monitored by forcing weekly 10 plants from 7 degrees C in the greenhouse. The results showed that at least 4 weeks are required to break dormancy for 'Nordlicht' confirming the results of the in vitro opening of the buds, while at least 6 weeks are required for 'Mw. G. Kint'.

Chemical inhibition of potato ABA-8'-hydroxylase activity alters in vitro and in vivo ABA metabolism and endogenous ABA levels but does not affect potato microtuber dormancy duration

The effects of azole-type P450 inhibitors and two metabolism-resistant abscisic acid (ABA) analogues on in vitro ABA-8'-hydroxylase activity, in planta ABA metabolism, endogenous ABA content, and tuber meristem dormancy duration were examined in potato (Solanum tuberosum L. cv. Russet Burbank). When functionally expressed in yeast, three potato CYP707A genes were demonstrated to encode enzymatically active ABA-8'-hydroxylases with micromolar affinities for (+)-ABA. The in vitro activity of the three enzymes was inhibited by the P450 azole-type inhibitors ancymidol, paclobutrazol, diniconazole, and tetcyclasis, and by the 8'-acetylene- and 8'-methylene-ABA analogues, with diniconazole and tetcyclasis being the most potent inhibitors. The in planta metabolism of [(3)H](±)-ABA to phaseic acid and dihydrophaseic acid in tuber meristems was inhibited by diniconazole, tetcyclasis, and to a lesser extent by 8'-acetylene- and 8'-methylene-ABA. Continuous exposure of in vitro generated microtubers to diniconazole resulted in a 2-fold increase in endogenous ABA content and a decline in dihydrophaseic acid content after 9 weeks of development. Similar treatment with 8'-acetylene-ABA had no effects on the endogenous contents of ABA or phaseic acid but reduced the content of dihydrophaseic acid. Tuber meristem dormancy progression was determined ex vitro in control, diniconazole-, and 8'-acetylene-ABA-treated microtubers following harvest. Continuous exposure to diniconazole during microtuber development had no effects on subsequent sprouting at any time point. Continuous exposure to 8'-acetylene-ABA significantly increased the rate of microtuber sprouting. The results indicate that, although a decrease in ABA content is a hallmark of tuber dormancy progression, the decline in ABA levels is not a prerequisite for dormancy exit and the onset of tuber sprouting.