Budbreak patterns and phytohormone dynamics reveal different modes of action between hydrogen cyanamide- and defoliant-induced flower budbreak in blueberry under inadequate chilling conditions.

Under inadequate chilling conditions, hydrogen cyanamide (HC) is often used to promote budbreak and improve earliness of Southern highbush blueberry (Vaccinium corymbosum L. interspecific hybrids). However, HC is strictly regulated or even banned in some countries because of its high hazardous properties. Development of safer and effective alternatives to HC is critical to sustainable subtropical blueberry production. In this study, we examined the efficacy of HC and defoliants as bud dormancy-breaking agents for 'Emerald' blueberry. First, we compared water control, 1.0% HC (9.35 L ha-1), and three defoliants [potassium thiosulfate (KTS), urea, and zinc sulfate (ZS)] applied at 6.0% (28 kg ha-1). Model fitting analysis revealed that only HC and ZS advanced both defoliation and budbreak compared with the water control. HC-induced budbreak showed an exponential plateau function with a rapid phase occurring from 0 to 22 days after treatment (DAT), whereas ZS-induced budbreak showed a sigmoidal function with a rapid phase occurring from 15 to 44 DAT. The final budbreak percentage was similar in all treatments (71.7%-83.7%). Compared with the water control, HC and ZS increased yield by up to 171% and 41%, respectively, but the yield increase was statistically significant only for HC. Phytohormone profiling was performed for water-, HC- and ZS-treated flower buds. Both chemicals did not increase gibberellin 4 and indole-3-acetic acid production, but they caused a steady increase in jasmonic acid (JA) during budbreak. Compared with ZS, HC increased JA production to a greater extent and was the only chemical that reduced abscisic acid (ABA) concentrations during budbreak. A follow-up experiment tested ZS at six different rates (0-187 kg ha-1) but detected no significant dose-response on budbreak. These results collectively suggest that defoliants are not effective alternatives to HC, and that HC and ZS have different modes of action in budbreak induction. The high efficacy of HC as a dormancy-breaking agent could be due to its ability to reduce ABA concentrations in buds. Our results also suggest that JA accumulation is involved in budbreak induction in blueberry.

Overexpression of a wheat α-amylase type 2 impact on starch metabolism and abscisic acid sensitivity during grain germination.

Despite being of vital importance for seed establishment and grain quality, starch degradation remains poorly understood in organs such as cereal or legume seeds. In cereals, starch degradation requires the synergetic action of different isoforms of α-amylases. Ubiquitous overexpression of TaAmy2 resulted in a 2.0-437.6-fold increase of total α-amylase activity in developing leaf and harvested grains. These increases led to dramatic alterations of starch visco-properties and augmentation of soluble carbohydrate levels (mainly sucrose and α-gluco-oligosaccharide) in grain. Interestingly, the overexpression of TaAMY2 led to an absence of dormancy in ripened grain due to abscisic acid (ABA) insensitivity. Using an allosteric α-amylase inhibitor (acarbose), we demonstrated that ABA insensitivity was due to the increased soluble carbohydrate generated by the α-amylase excess. Independent from the TaAMY2 overexpression, inhibition of α-amylase during germination led to the accumulation of soluble α-gluco-oligosaccharides without affecting the first stage of germination. These findings support the hypotheses that (i) endosperm sugar may overcome ABA signalling and promote sprouting, and (ii) α-amylase may not be required for the initial stage of grain germination, an observation that questions the function of the amylolytic enzyme in the starch degradation process during germination.

miR169 and PmRGL2 synergistically regulate the NF-Y complex to activate dormancy release in Japanese apricot (Prunus mume Sieb. et Zucc.).

KEY MESSAGE: This study is the first to demonstrate that GA4-induced dormancy release is associated with the NF-Y complex, which interacts with gibberellin inhibitor RGL2 in Japanese apricot. Seasonal dormancy is not only vital for the survival in cold winter but also affects flowering of temperate fruit trees and the dormancy release depends on the accumulation of the cold temperatures (Chilling requirement-CR). To understand the mechanism of dormancy release in deciduous fruit crops, we compared miRNA sequencing data during the transition stage from paradormancy to dormancy release in the Japanese apricot and found that the miR169 family showed significant differentially up-regulated expression during dormancy induction and was down-regulated during the dormancy release periods. The 5' RACE assay and RT-qPCR validated its target gene NUCLEAR FACTOR-Y subunit A (NF-YA), which exhibited the opposite expression pattern. Further study showed that exogenous GA4 could inhibit the expression of the gibberellic acid (GA) signal transduction suppressor PmRGL2 (RGA-LIKE 2) and promote the expression of NF-Y. Moreover, the interaction between the NF-Y family and GA inhibitor PmRGL2 was verified by the yeast-two-hybrid (Y2H) system and a bimolecular fluorescence complementarity (BiFC) experiment. These results suggest that synergistic regulation of the NF-Y and PmRGL2 complex leads to the activation of dormancy release induced by GA4. These findings will help to elucidate the functional and regulatory roles of miR169 and NF-Y complex in seasonal bud dormancy induced by GA in Japanese apricot and provide new insights for the discovery of dormancy release mechanisms in woody plants.

Cover crops, hormones and herbicides: Priming an integrated weed management strategy.

Herbicide weed resistance has been a major issue of conventional global row crop agriculture for decades. Still current strategies and novel technologies available to address weed resistance are mainly herbicide-based. Thus, there is a need for innovative means of integrated weed management strategies. Our approach proposed herein integrates cover crops, plant hormones and pre-emergence (PRE) herbicides as part of weed management programs. Plant hormones such as gibberellic acid (GA3) and abscisic acid (ABA) have the potential to induce seed germination and seed dormancy, respectively. Prior to crop emergence, plant hormones are tank mixed with PRE herbicides and sprayed to cover crop residue. Two strategies are proposed (1) PRE herbicides + GA3 and (2) PRE herbicide + ABA. The hormones provide different results; GA3 is likely to stimulate a more uniform weed seed germination, thus enhancing efficacy of PRE herbicides. Conversely, ABA could promote weed seed dormancy, reducing selection pressure and weed infestations until crop canopy closure. Much research is needed to understand the impact of hormones on weed and crop species, optimize products and rates, and compatibility of hormones with herbicides and cover crops. If successful, this approach could open a new opportunity for agricultural business, enhance farming sustainability by reducing dependence on herbicides and minimizing agronomic, economic and environmental issues related to weed resistance.

Type-B cytokinin response regulators link hormonal stimuli and molecular responses during the transition from endo- to ecodormancy in apple buds.

KEY MESSAGE: Cytokinin together with MdoBRR1, MdoBRR8 and MdoBRR10 genes participate in the downregulation of MdoDAM1, contributing to the transition from endo- to ecodormancy in apple buds. The final step of cytokinin (CK) signaling pathway culminates in the activation of type-B response regulators (BRRs), important transcriptional factors in the modulation of CK-responsive genes. In this study, we performed a genome-wide analysis aiming to identify apple BRR family members and understand their involvement in bud dormancy control. The investigation identified ten MdoBRR protein-coding genes. A higher expression of three MdoBRR (MdoBRR1, MdoBRR9 and MdoBRR10) was observed in dormant buds in comparison to other developmental stages. Interestingly, in ecodormant buds these three MdoBRR genes were upregulated in a CK-dependent manner. Transcription profiles, determined during dormancy cycle under field and artificially controlled conditions, revealed that MdoBRR1 and MdoBRR8 played important roles in the transition from endo- to ecodormancy, probably mediated by endogenous CK stimuli. The expression of MdoBRR7, MdoBRR9, and MdoBRR10 was induced in ecodormant buds exposed to warm temperatures, indicating a putative role in growth resumption after chilling requirement fulfillment. Contrasting expression patternsin vivo between MdoBRRs and MdoDAM1, an essential dormancy establishment regulator, were observed during dormancy cycle and in CK-treated buds. Thereafter, in vivo transactivation assays showed that CK stimuli combined with transient overexpression of MdoBRR1, MdoBRR8, and MdoBRR10 resulted in downregulation of the reporter gene gusA driven by the MdoDAM1 promoter. These pieces of evidences point to the integration of CK-triggered responses through MdoBRRs that are able to downregulate MdoDAM1, contributing to dormancy release in apple.

Identification of potential post-ethylene events in the signaling cascade induced by stimuli of bud dormancy release in grapevine.

Ethylene signaling appears critical for grape bud dormancy release. We therefore focused on identification and characterization of potential downstream targets and events, assuming that they participate in the regulation of dormancy release. Because ethylene responding factors (ERF) are natural candidates for targets of ethylene signaling, we initially characterized the behavior of two VvERF-VIIs, which we identified within a gene set induced by dormancy release stimuli. As expected, these VvERF-VIIs are localized within the nucleus, and are stabilized upon decreases in oxygen availability within the dormant buds. Less expected, the proteins are also stabilized upon hydrogen cyanamide (HC) application under normoxic conditions, and their levels peak at deepest dormancy under vineyard conditions. We proceeded to catalog the response of all bud-expressed ERFs, and identified additional ERFs that respond similarly to ethylene, HC, azide and hypoxia. We also identified a core set of genes that are similarly affected by treatment with ethylene and with various dormancy release stimuli. Interestingly, the functional annotations of this core set center around response to energy crisis and renewal of energy resources via autophagy-mediated catabolism. Because ERF-VIIs are stabilized under energy shortage and reshape cell metabolism to allow energy regeneration, we propose that: (i) the availability of VvERF-VIIs is a consequence of an energy crisis within the bud; (ii) VvERF-VIIs function as part of an energy-regenerating mechanism, which activates anaerobic metabolism and autophagy-mediated macromolecule catabolism; and (iii) activation of catabolism serves as the mandatory switch and the driving force for activation of the growth-inhibited meristem during bud-break.

Rice OsBT1 regulates seed dormancy through the glycometabolism pathway.

Seed dormancy and germination in rice (Oryza sativa L.) are complex and important agronomic traits that involve a number of physiological processes and energy. A mutant named h470 selected from a60Co-radiated indica cultivar N22 population had weakened dormancy that was insensitive to Gibberellin (GA) and Abscisic acid (ABA). The levels of GA4 and ABA were higher in h470 than in wild-type (WT) plants. The gene controlling seed dormancy in h470 was cloned by mut-map and transgenesis and confirmed to encode an ADP-glucose transporter protein. A 1 bp deletion in Os02g0202400 (OsBT1) caused the weaker seed dormancy in h470. Metabolomics analyses showed that most sugar components were higher in h470 seeds than the wild type. The mutation in h470 affected glycometabolism.

Rice GERMIN-LIKE PROTEIN 2-1 Functions in Seed Dormancy under the Control of Abscisic Acid and Gibberellic Acid Signaling Pathways.

Seed dormancy is a natural phenomenon in plants. It ensures that seeds complete the grain-filling stage before germination and prevents germination in unsuitable ecological conditions. In this study, we determined the previously unknown function of the rice (Oryza sativa) gene GERMIN-LIKE PROTEIN 2-1 (OsGLP2-1) in seed dormancy. Using artificial microRNA and CRISPR/CAS9 approaches, suppression of OsGLP2-1 expression in rice resulted in the release of dormancy in immature seeds. Conversely, overexpression of OsGLP2-1 driven by the OsGLP2-1 native promoter led to greater seed dormancy. Seed scutellum-specific expression of OsGLP2-1 was increased by exogenous abscisic acid, but decreased with gibberellic acid treatment. We provide evidence that OsGLP2-1 is antagonistically controlled at the transcriptional level by ABA INSENSITIVE5 and GAMYB transcription factors. We conclude that OsGLP2-1 acts as a buffer, maintaining appropriate equilibrium for the regulation of primary dormancy during seed development in rice.

ABA-responsive ABRE-BINDING FACTOR3 activates DAM3 expression to promote bud dormancy in Asian pear.

Bud dormancy is indispensable for the survival of perennial plants in cold winters. Abscisic acid (ABA) has essential functions influencing the endo-dormancy status. Dormancy-associated MADS-box/SHORT VEGETATIVE PHASE-like genes function downstream of the ABA signalling pathway to regulate bud dormancy. However, the regulation of DAM/SVP expression remains largely uncharacterized. In this study, we confirmed that endo-dormancy maintenance and PpyDAM3 expression are controlled by the ABA content in pear (Pyrus pyrifolia) buds. The expression of pear ABRE-BINDING FACTOR3 (PpyABF3) was positively correlated with PpyDAM3 expression. Furthermore, PpyABF3 directly bound to the second ABRE in the PpyDAM3 promoter to activate its expression. Interestingly, both PpyABF3 and PpyDAM3 repressed the cell division and growth of transgenic pear calli. Another ABA-induced ABF protein, PpyABF2, physically interacted with PpyABF3 and disrupted the activation of the PpyDAM3 promoter by PpyABF3, indicating DAM expression was precisely controlled. Additionally, our results suggested that the differences in the PpyDAM3 promoter in two pear cultivars might be responsible for the diversity in the chilling requirements. In summary, our data clarify the finely tuned regulatory mechanism underlying the effect of ABA on DAM gene expression and provide new insights into ABA-related bud dormancy regulation.

Cold plasma treatment to release dormancy and improve growth in grape buds: a promising alternative to natural chilling and rest breaking chemicals.

Winter dormancy of temperate zone perennial plant species is commonly released by chilling temperature. If the duration of the cold weather is not adequate, plant growth becomes disorganized leading to reduced growth, spread out flowering and fruit maturation and often reduced yield. In mild-winter regions, growers commonly resort to spraying their trees with chemicals such as hydrogen cyanamide to compensate for the lack of chilling to ensure good growth and yield. Although effective, most of these chemicals are highly toxic; unfortunately, there is no effective and environmentally friendly alternative which can be used to release dormancy. In this work, we present a cold plasma treatment-based method which can effectively release the dormancy of grape buds. We have found that exposing grape buds to plasma provides improvement of several growth parameters including higher, faster and more synchronous budbreak and more vigorous vegetative growth, comparatively similar to or better than natural chilling. Biochemical analyses of bud tissue suggest that the plasma treatment triggered a marked transient oxidative stress as indicated by the increase in the concentrations of free proline, malondialdehyde (MDA) and hydrogen peroxide (H2O2). Proline appears to have played a key role; as a compatible osmolyte, it may have protected cellular structures against free radicals and as a signaling molecule, it may have induced the events leading to dormancy release. We anticipate that our work will provide a starting point for the development of novel plasma-based tools and methods to treat dormant plants. The plasma treatment method may allow higher agricultural production in several regions of the world at risk of becoming marginal for the cultivation of certain crops due to global warming.

Application of 5-azacytidine induces DNA hypomethylation and accelerates dormancy release in buds of tree peony.

Release of bud dormancy is a prerequisite for the growth resumption and production in perennial plants such as tree peony. DNA methylation plays a pivotal role in regulating gene expression. In this study, combination of morphologic observation and DNA methylation analysis indicated that 5-azacytidine (5-azaC) application for 7 d declined 5 mC quantities and promoted dormancy release. After 5-azaC treatment, total 174,341 unigenes and 1818 differentially expression genes (DEGs) were obtained by RNA-seq, of which there were 1194 DEGs after 1 d 5-azaC treatment (AD1 vs CD1), and 624 DEGs after 7 d (AD7 vs CD7), respectively. The KEGG pathway analysis identified that totally 10 DEGs annotated in DNA replication pathway were enriched when AD7 compared with CD7. Furthermore, the expression patterns of several DEGs by real-time quantitative RT-PCR were consistent with that of RNA-seq data. 5-azaC application significantly decreased the expression levels of DNA methyltransferase genes, PsCMT3, PsMET1 and PsDRM2, and increased the transcript of demethylase gene PsROS1. Simultaneously, total methyltransferases activity decreased, and demethylase activity was induced by 5-azaC. In summary, application of 5-azaC inhibited the expression of the genes related to growth and development in short-term, indicating a possible toxic effect to plant, and its long-term effect was to induce hypomethylation by increasing demethylase genes transcripts and decreasing the expressions of methyltransferase genes, and then activate cell cycle, DNA replication and glycol-metabolism processes, which subsequently accelerated dormancy release. All these would provide a new strategy to further understand the molecular mechanism of dormancy release in tree peony.

Alleviating dormancy in Brassica oleracea seeds using NO and KAR1 with ethylene biosynthetic pathway, ROS and antioxidant enzymes modifications.

BACKGROUND: Seed dormancy is a prevailing condition in which seeds are unable to germinate, even under favorable environmental conditions. Harvested Brassica oleracea (Chinese cabbage) seeds are dormant and normally germinate (poorly) at 21 °C. This study investigated the connections between ethylene, nitric oxide (NO), and karrikin 1 (KAR1) in the dormancy release of secondary dormant Brassica oleracea seeds. RESULTS: NO and KAR1 were found to induce seed germination, and stimulated the production of ethylene and 1-aminocyclopropane-1-carboxylic acid (ACC), and both ethylene biosynthesis enzyme ACC oxidase (ACO) [1] and ACC synthase (ACS) [2]. In the presence of NO and KAR1, ACS and ACO activity reached maximum levels after 36 and 48 h, respectively. The inhibitor of ethylene 2,5-norbornadiene (NBD) had an adverse effect on Brassica oleracea seed germination (inhibiting nearly 50% of germination) in the presence of NO and KAR1. The benefits from NO and KAR1 in the germination of secondary dormant Brassica oleracea seeds were also associated with a marked increase in reactive oxygen species (ROS) (H2O2 and O2˙-) and antioxidant enzyme activity at early germination stages. Catalase (CAT) and glutathione reductase (GR) activity increased 2 d and 4 d, respectively, after treatment, while no significant changes were observed in superoxide dismutase (SOD) activity under NO and KAR1 applications. An increase in H2O2 and O2˙- levels were observed during the entire incubation period, which increasing ethylene production in the presence of NO and KAR1. Abscisic acid (ABA) contents decreased and glutathione reductase (GA) contents increased in the presence of NO and KAR1. Gene expression studies were carried out with seven ethylene biosynthesis ACC synthases (ACS) genes, two ethylene receptors (ETR) genes and one ACO gene. Our results provide more evidence for the involvement of ethylene in inducing seed germination in the presence of NO and KAR1. Three out of seven ethylene biosynthesis genes (BOACS7, BOACS9 and BOACS11), two ethylene receptors (BOETR1 and BOETR2) and one ACO gene (BOACO1) were up-regulated in the presence of NO and KAR1. CONCLUSION: Consequently, ACS activity, ACO activity and the expression of different ethylene related genes increased, modified the ROS level, antioxidant enzyme activity, and ethylene biosynthesis pathway and successfully removed (nearly 98%) of the seed dormancy of secondary dormant Brassica olereace seeds after 7 days of NO and KAR1 application.

Redox metabolism and cell wall modifications as global and local targets respectively, of cyanide induced dormancy release of walnut kernels.

The HCN-induced seed dormancy release necessitates alterations in reactive oxygen species (ROS) metabolism and radicle cell wall loosening. Little is known about the interaction of ROS metabolism with cell wall hydrolytic enzymes during HCN-induced seed dormancy release. Thus dormant walnut (Juglans regia L.) kernels were exposed to HCN (4 h) and studied for redox metabolism and cell wall-modifying enzymes during 10 days of incubation (DI) i.e. before radicle emergence. HCN increased ROS especially in the embryonic axes (EAs) but decreased ROS-generating NADPH oxidase and ROS scavenging superoxide dismutase (SOD) and peroxidase (POX) with no effects on catalase (CAT), ascorbate peroxidase (APX) and cell wall-modifying enzymes activities in short term up to 2 DI. In long term roughly from 4 DI onwards, HCN-exposed EA displayed greater superoxide anions and enhanced activities of POX, APX, NADPH oxidase, cell wall peroxidase (CW-POX), ß- 1, 4-D glucanase, mannanase, polygacturonase and xylanase. Meanwhile HCN increased greater expression of POX and mannanase isoforms as revealed by in-gel activity assay. Except for higher activities of CAT, POX and APX, cotyledonary activities of CW-POX, mannanase and polygacturonase and to some extent ß- 1, 4-D glucanase remained unaffected by HCN. Thus short term ROS accumulation in HCN-treated EA is due to declined SOD and POX activities. In long term the enhanced activities of both NADPH oxidase: CW-POX couple and cell wall-modifying enzymes in EA bring about wall loosening in preparation for radicle emergence. Evidences for the simultaneous operation of both mechanisms are provided in walnut EAs during dormancy release.

ABA signaling components in Phelipanche aegyptiaca.

Obligate root holoparasite Phelipanche aegyptiaca is an agricultural pest, which infests its hosts and feeds on the sap, subsequently damaging crop yield and quality. Its notoriously viable seed bank may serve as an ideal pest control target. The phytohormone abscisic acid (ABA) was shown to regulate P. aegyptiaca seed dormancy following strigolactones germination stimulus. Transcription analysis of signaling components revealed five ABA receptors and two co-receptors (PP2C). Transcription of lower ABA-affinity subfamily III receptors was absent in all tested stages of P. aegyptiaca development and parasitism stages. P. aegyptiaca ABA receptors interacted with the PP2Cs, and inhibited their activity in an ABA-dependent manner. Moreover, sequence analysis revealed multiple alleles in two P. aegyptiaca ABA receptors, with many non-synonymous mutations. Functional analysis of selected receptor alleles identified a variant with substantially decreased inhibitory effect of PP2Cs activity in-vitro. These results provide evidence that P. aegyptiaca is capable of biochemically perceiving ABA. In light of the possible involvement of ABA in parasitic activities, the discovery of active ABA receptors and PP2Cs could provide a new biochemical target for the agricultural management of P. aegyptiaca. Furthermore, the potential genetic loss of subfamily III receptors in this species, could position P. aegyptiaca as a valuable model in the ABA perception research field.

Re-localization of hormone effectors is associated with dormancy alleviation by temperature and after-ripening in sunflower seeds.

Temperature is the primary factor that affects seed dormancy and germination. However, the molecular mechanism that underlies its effect on dormancy alleviation remained largely unknown. In this study, we investigate hormone involvement in temperature induced germination as compared to that caused by after-ripening. Dormant (D) sunflower seeds cannot germinate at 10 °C but fully germinate at 20 °C. After-ripened seeds become non-dormant (ND), i.e. able to germinate at 10 °C. Pharmacological experiments showed the importance of abscisic acid (ABA), gibberellins (GAs) and ethylene in temperature- and after-ripening-induced germination of sunflower seeds. Hormone quantification showed that after-ripening is mediated by a decline in both ABA content and sensitivity while ABA content is increased in D seeds treated at 10 or 20 °C, suggesting that ABA decrease is not a prerequisite for temperature induced dormancy alleviation. GAs and ethylene contents were in accordance with germination potential of the three conditions (GA1 was higher in D 20 °C and ND 10 °C than in D 10 °C). Transcripts analysis showed that the major change concerns ABA and GAs metabolism genes, while ABA signalling gene expression was significantly unchanged. Moreover, another level of hormonal regulation at the subcellular localization has been revealed by immunocytolocalization study. Indeed, ABA, protein Abscisic acid-Insensitive 5 (ABI5), involved in ABA-regulated gene expression and DELLA protein RGL2, a repressor of the gibberellins signalling pathway, localized mainly in the nucleus in non-germinating seeds while they localized in the cytosol in germinating seeds. Furthermore, ACC-oxidase (ACO) protein, the key ethylene biosynthesis enzyme, was detected in the meristem only in germinating seeds. Our results reveal the importance of hormone actors trafficking in the cell and their regulation in specialized tissue such as the meristem in dormancy alleviation and germination.

Nitric Oxide-Induced Dormancy Removal of Apple Embryos Is Linked to Alterations in Expression of Genes Encoding ABA and JA Biosynthetic or Transduction Pathways and RNA Nitration.

Short-term (3 h) treatment of embryos isolated from dormant apple (Malus domestica Borkh.) seeds with NO donors stimulates their transition from dormancy to germination. Seed dormancy is maintained by ABA, while germination is controlled mainly by gibberellins (GAs) and jasmonic acid (JA). NO-induced dormancy removal correlates with low ABA concentration in embryonic axes and reduced embryo sensitivity to ABA. We analyzed the expression of genes encoding key enzymes of ABA degradation (CYP707A1, CYP707A2), biosynthesis (NCED3, NCED9), and elements of the ABA transduction pathway (PYL1, PYL2, RCAR1, RCAR3, PP2CA, ABI1, ABI2, SNRK2, ABI5, AREB3, ABF). A role for JA in the regulation of germination led us to investigate the expression of genes encoding enzymes of JA biosynthesis (AOS1, JMT, JAR1) and the transduction pathway (COI1, MYC2, JAZ3, JAZ12). The expression profiles of the genes were estimated in embryonic axes isolated from dormant or NO fumigated apple embryos. The analyzed genes were differentially regulated during dormancy alleviation, the main modifications in the transcription level were detected for NCED3, NCED9, CYP707A2, RCAR1, ABF, AOS1, JMT, JAR1 and JAZ3. A regulatory role of NO in the removal of seed dormancy is associated with the stimulation of expression of genes related to ABA degradation, down-regulation of genes responsible for ABA synthesis, an increase of expression level of genes engaged in JA synthesis and modification of the expression of genes engaged in signaling pathways of the hormones. To confirm a signaling role of NO during dormancy breakage, an increased RNA nitration level in embryonic axes was demonstrated.

Developmental analysis of the early steps in strigolactone-mediated axillary bud dormancy in rice.

By contrast with rapid progress in understanding the mechanisms of biosynthesis and signaling of strigolactone (SL), mechanisms by which SL inhibits axillary bud outgrowth are less well understood. We established a rice (Oryza sativa L.) hydroponic culture system to observe axillary buds at the critical point when the buds enter the dormant state. In situ hybridization analysis indicated that cell division stops in the leaf primordia of the buds entering dormancy. We compared transcriptomes in the axillary buds isolated by laser capture microdissection before and after entering the dormant state and identified genes that are specifically upregulated or downregulated in dormant buds respectively, in SL-mediated axillary bud dormancy. Typically, cell cycle genes and ribosomal genes are included among the active genes while abscisic acid (ABA)-inducible genes are among the dormant genes. Application of ABA to the hydroponic culture suppressed the growth of axillary buds of SL mutants to the same level as wild-type (WT) buds. Tiller number was decreased in the transgenic lines overexpressing OsNCED1, the gene that encodes ABA biosynthesis enzyme. These results indicated that the main site of SL function is the leaf primordia in the axillary bud and that ABA is involved in SL-mediated axillary bud dormancy.

Involvement of ethylene biosynthesis and perception during germination of dormant Avena fatua L. caryopses induced by KAR1 or GA3.

MAIN CONCLUSION: Germination of primary dormant wild oat caused by KAR1 or GA3 is associated with ACC accumulation and increased ethylene production shortly before radicle protrusion as a result of the non-transcriptional and transcriptional activation of ACS and ACO enzymes, respectively. Response to both compounds involves the modulation of ethylene sensitivity through ethylene receptor genes. Harvested Avena fatua caryopses are primary dormant and, therefore, germinated poorly at 20 °C. Karrikin 1 (KAR1), which action probably requires endogenous gibberellins (GAs), and gibberellin A3 (GA3) was found to induce dormant caryopses to germinate. The stimulatory effects were accompanied by the activation of the ethylene biosynthesis pathway and depended on undisturbed ethylene perception. KAR1 and GA3 promoted 1-aminocyclopropane-1-carboxylic acid (ACC) accumulation during coleorhizae emergence and ethylene production shortly prior to the radicle protrusion, which resulted from the enhanced activity of two ethylene biosynthesis enzymes, ACC synthase (ACS) and ACC oxidase (ACO). The inhibitor of ACS adversely affected beneficial impacts of both KAR1 and GA3 on A. fatua caryopses germination, while the inhibitor of ACO more efficiently impeded the GA3 effect. The inhibitors of ethylene action markedly lowered germination in response to KAR1 and GA3. Gene expression studies preceded by the identification of several genes related to ethylene biosynthesis (AfACS6, AfACO1, and AfACO5) and perception (AfERS1b, AfERS1c, AfERS2, AfETR2, AfETR3, and AfETR4) provided further evidence for the engagement of ethylene in KAR1 and GA3 induced germination of A. fatua caryopses. Both AfACO1 and AfACO5 were upregulated, whereas AfACS6 remained unaffected by the treatment. This suggests the existence of different regulatory mechanisms of enzymatic activity, transcriptional for ACO and non-transcriptional for ACS. During imbibition in water, AfERS1b was stronger expressed than other receptor genes. In the presence of KAR1 or GA3, the expression of AfETR3 was substantially induced. Differential expression of ethylene receptor genes implies the modulation of caryopses sensitivity adjusted to ethylene availability and suggests the functional diversification of individual receptors.

Evaluation of dormancy breaking methods for enhanced germination in four biotypes of Brassica tournefortii.

Brassica tournefortii is an important broadleaf weed of the winter season in the northern grain region of Australia. Knowledge of germination ecology of this weed would help in implementing effective weed control programs. A series of experiments were conducted to study the germination and dormancy behavior of four biotypes of B. tournefortii seeds, biotypes A (collected from barley crop), B (barley fence lines), C (chickpea crop), and D (chickpea fence lines), collected from the St George region of Queensland. The aim of this research was to determine the effectiveness of various methods on the seed dormancy release of B. tournefortii. Water, potassium nitrate and a soil extract did not release dormancy in B. tournefortii seeds (biotype A) at 20/10 °C in the light/dark regime. Cold stratification (5 °C) also did not improve germination. However, gibberellic acid (GA3; 100-300 mg kg-1) stimulated germination (>88%). Germination also improved when seeds were immersed in sodium hypochlorite (NaOCl; 42 g L-1) for 10 minutes and the effect was more pronounced under the complete dark environment (89% germination at a day/night temperature of 20/10 °C). The NaOCl treatment makes seeds more porous and decreases sensitivity to light. Another experiment in light/dark conducted at 25/15 °C with two biotypes (A and D) showed that, without NaOCl treatment, biotype A was more sensitive to light (29% germination) as compared to biotype D (92% germination). Our results suggest that dormancy in B. tournefortii seeds can be broken by the combination of NaOCl (10 min) and a dark environment. A day/night temperature of 25/15 °C was found best for optimum germination (>87%) for all the biotypes (A-D) when incubated in dark after treating with NaOCl. This research indicated a high degree of variability in germination responses for various biotypes of B. tournefortii seeds to various sets of conditions, which may be due to metabolic changes in response to maternal environments or genetically controlled mechanisms. Information gained from this study will be important in developing a better understanding of the dormancy behavior of B. tournefortii seeds in response to tillage systems or maternal environments that could influence the weed seed bank in the soil and therefore help in designing suitable weed management programs.

Establishment of an efficient in vitro propagation system for Iris sanguinea.

Iris sanguinea is a perennial flowering plant that is typically cultivated through seeds or bulbs. However, due to limitations in conventional propagation, an alternate regeneration system using seeds was developed. The protocol included optimization of sterilization, stratification and scarification methods as iris seeds exhibit physiological dormancy. In addition to chlorine-based disinfection, alkaline or heat treatment was used to break seed dormancy and reduce contamination. When seeds were soaked in water at 80 °C overnight, and sterilized with 75% EtOH for 30 s and 4% NaOCl solution for 20 minutes, contamination was reduced to 10% and a 73.3% germination was achieved. The germinated seedlings with 2-3 leaves and radicle were used as explants to induce adventitious buds. The optimal MS medium with 0.5 mg L-1 6-benzylaminopurine, 0.2 mg L-1 NAA, and 1.0 mg L-1 kinetin resulted in 93.3% shoot induction and a proliferation coefficient of 5.30. Medium with 0.5 mg L-1 NAA achieved 96.4% rooting of the adventitious shoots. The survival rate was more than 90% after 30 days growth in the cultivated matrix. In conclusion, a successful regeneration system for propagation of I. sanguinea was developed using seeds, which could be utilized for large-scale propagation of irises of ecological and horticultural importance.