Inhibition of germination of dormant barley (Hordeum vulgare L.) grains by blue light as related to oxygen and hormonal regulation

Germination of primary dormant barley grains is promoted by darkness and temperatures below 20 °C, but is strongly inhibited by blue light. Exposure under blue light at 10 °C for periods longer than five days, results in a progressive inability to germinate in the dark, considered as secondary dormancy. We demonstrate that the inhibitory effect of blue light is reinforced in hypoxia. The inhibitory effect of blue light is associated with an increase in embryo abscisic acid (ABA) content (by 3.5‐ to 3.8‐fold) and embryo sensitivity to both ABA and hypoxia. Analysis of expression of ABA metabolism genes shows that increase in ABA mainly results in a strong increase in HvNCED1 and HvNCED2 expression, and a slight decrease in HvABA8′OH‐1. Among the gibberellins (GA) metabolism genes examined, blue light decreases the expression of HvGA3ox2, involved in GA synthesis, increases that of GA2ox3 and GA2ox5, involved in GA catabolism, and reduces the GA signalling evaluated by the HvExpA11 expression. Expression of secondary dormancy is associated with maintenance of high embryo ABA content and a low HvExpA11 expression. The partial reversion of the inhibitory effect of blue light by green light also suggests that cryptochrome might be involved in this hormonal regulation.     

Regulation of dormancy in barley by blue light and after-ripening: effects on abscisic acid and gibberellin metabolism

White light strongly promotes dormancy in freshly harvested cereal grains, whereas dark and after-ripening have the opposite effect. We have analyzed the interaction of light and after-ripening on abscisic acid (ABA) and gibberellin (GA) metabolism genes and dormancy in barley (Hordeum vulgare 'Betzes'). Analysis of gene expression in imbibed barley grains shows that different ABA metabolism genes are targeted by white light and after-ripening. Of the genes examined, white light promotes the expression of an ABA biosynthetic gene, HvNCED1, in embryos. Consistent with this result, enzyme-linked immunosorbent assays show that dormant grains imbibed under white light have higher embryo ABA content than grains imbibed in the dark. After-ripening has no effect on expression of ABA biosynthesis genes, but promotes expression of an ABA catabolism gene (HvABA8'OH1), a GA biosynthetic gene (HvGA3ox2), and a GA catabolic gene (HvGA2ox3) following imbibition. Blue light mimics the effects of white light on germination, ABA levels, and expression of GA and ABA metabolism genes. Red and far-red light have no effect on germination, ABA levels, or HvNCED1. RNA interference experiments in transgenic barley plants support a role of HvABA8'OH1 in dormancy release. Reduced HvABA8'OH1 expression in transgenic HvABA8'OH1 RNAi grains results in higher levels of ABA and increased dormancy compared to nontransgenic grains.     

Novel loci and a role for nitric oxide for seed dormancy and preharvest sprouting in barley

Barley is used for food and feed, and brewing. Nondormant seeds are required for malting, but the lack of dormancy can lead to preharvest sprouting (PHS), which is also undesired. Here, we report several new loci that modulate barley seed dormancy and PHS. Using genome‐wide association mapping of 184 spring barley genotypes, we identified four new, highly significant associations on chromosomes 1H, 3H, and 5H previously not associated with barley seed dormancy or PHS. A total of 71 responsible genes were found mostly related to flowering time and hormone signalling. A homolog of the well‐known Arabidopsis Delay of Germination 1 (DOG1) gene was annotated on the barley chromosome 3H. Unexpectedly, DOG1 appears to play only a minor role in barley seed dormancy. However, the gibberellin oxidase gene HvGA20ox1 contributed to dormancy alleviation, and another seven important loci changed significantly during after‐ripening. Furthermore, nitric oxide release correlated negatively with dormancy and shared 27 associations. Origin and growth environment affected seed dormancy and PHS more than did agronomic traits. Days to anthesis and maturity were shorter when seeds were produced under drier conditions, seeds were less dormant, and PHS increased, with a heritability of 0.57–0.80. The results are expected to be useful for crop improvement.     

AtPER1 enhances primary seed dormancy and reduces seed germination by suppressing the ABA catabolism and GA biosynthesis in Arabidopsis seeds

Seed is vital to the conservation of germplasm and plant biodiversity. Seed dormancy is an adaptive trait in numerous seed‐plant species, enabling plants to survive under stressful conditions. Seed dormancy is mainly controlled by abscisic acid (ABA) and gibberellin (GA) and can be classified as primary and secondary seed dormancy. The primary seed dormancy is induced by maternal ABA. Here we found that AtPER1, a seed‐specific peroxiredoxin, is involved in enhancing primary seed dormancy. Two loss‐of‐function atper1 mutants, atper1‐1 and atper1‐2, displayed suppressed primary seed dormancy accompanied with reduced ABA and increased GA contents in seeds. Furthermore, atper1 mutant seeds were insensitive to abiotic stresses during seed germination. The expression of several ABA catabolism genes (CYP707A1, CYP707A2, and CYP707A3) and GA biosynthesis genes (GA20ox1, GA20ox3, and KAO3) in atper1 mutant seeds was increased compared to wild‐type seeds. The suppressed primary seed dormancy of atper1‐1 was completely reduced by deletion of CYP707A genes. Furthermore, loss‐of‐function of AtPER1 cannot enhance the seed germination ratio of aba2‐1 or ga1‐t, suggesting that AtPER1‐enhanced primary seed dormancy is dependent on ABA and GA. Additionally, the level of reactive oxygen species (ROS) in atper1 mutant seeds was significantly higher than that in wild‐type seeds. Taken together, our results demonstrate that AtPER1 eliminates ROS to suppress ABA catabolism and GA biosynthesis, and thus improves the primary seed dormancy and make the seeds less sensitive to adverse environmental conditions.     

Triangular interplay between ROS,ABA and GA in dormancy alleviation of <Emphasis Type="Italic">Bunium persicum</Emphasis> seeds by cold stratification

Seeds of Bunium persicum (Boiss.) B. Fedtsch. have complex physiological dormancy that can be released by 15 weeks stratification. The present study revealed that cold stratification enhanced content of H₂O₂, O^{2 -·} and application of GA₃ and ROS donors (Fenton reagent, H₂O₂, methylviologen and menadione) did not affect or only slightly promoted the germination of non-stratified, fully dormant seeds. Dormancy was markedly decreased by ROS-generating reagents, GA₃ and fluridone (an inhibitor of ABA biosynthesis) and was enhanced by ROS-decreasing compounds (DMTU, Tiron, SB and DPI), diniconazole (Dinc, an inhibitor of ABA catabolism) and paclobutrazol (PAC, an inhibitor of GA biosynthesis) when dormancy was partially removed by cold stratification. The response to these compounds reduced with increasing time of stratification. ABA inhibited germination by repressing of NADPH oxidase activity and ROS accumulation and conversely, GA triggered germination by promoting an increase of NADPH oxidase activity and ROS levels. Data in this study, for the first time suggest releasing deep complex physiological dormancy by cold stratification is associated with interplay between ROS and ABA/GA.     

Investigating seed dormancy in cotton (Gossypium hirsutum L.): understanding the physiological changes in embryo during after‐ripening and germination

• The dormancy of seeds of upland cotton can be broken during dry after‐ripening, but the mechanism of its dormancy release remains unclear.
• Freshly harvested cotton seeds were subjected to after‐ripening for 180 days. Cotton seeds from different days of after‐ripening (DAR) were sampled for dynamic physiological determination and germination tests. The intact seeds and isolated embryos were germinated to assess effects of the seed coat on embryo germination. Content of H₂O₂ and phytohormones and activities of antioxidant enzymes and glucose‐6‐phosphate dehydrogenase were measured during after‐ripening and germination.
• Germination of intact seeds increased from 7% upon harvest to 96% at 30 DAR, while embryo germination improved from an initial rate of 82% to 100% after 14 DAR. Based on T₅₀ (time when 50% of seeds germinate) and germination index, the intact seed and isolated embryo needed 30 and 21 DAR, respectively, to acquire relatively stable germination. The content of H₂O₂ increased during after‐ripening and continued to increase within the first few hours of imbibition, along with a decrease in abscisic acid (ABA) content. A noticeable increase was observed in gibberellic acid content during germination when ABA content decreased to a lower level. Coat removal treatment accelerated embryo absorption of water, which further improved the accumulation of H₂O₂ and changed peroxidase content during germination.
• For cotton seed, the alleviation of coat‐imposed dormancy required 30 days of after‐ripening, accompanied by rapid dormancy release (within 21 DAR) in naked embryos. H₂O₂ acted as a core link between the response to environmental changes and induction of other physiological changes for breaking seed dormancy.

     

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.     

Identification of a hydrogen peroxide signalling pathway in the control of light-dependent germination in Arabidopsis

Germination is controlled by external factors, such as temperature, water, light and by hormone balance. Recently, reactive oxygen species (ROS) have been shown to act as messengers during plant development, stress responses and programmed cell death. We analyzed the role of ROS during germination and demonstrated that ROS in addition to their role as cell wall loosening factor are essential signalling molecules in this process. Indeed, we showed that ROS are released prior to endosperm rupture, that their production is required for germination, and that class III peroxidases, as ROS level regulators, colocalized with ROS production. Among ROS, H₂O₂ modifies, during germination early steps, the expression of genes encoding for enzymes regulating ROS levels. This pointing out a regulatory feedback loop for ROS production. Measurements of endogenous levels of ROS following application of GA and ABA suggested that ABA inhibits germination by repressing ROS accumulation, and that, conversely, GA triggers germination by promoting an increase of ROS levels. We followed the early visible steps of germination (testa and endosperm rupture) in Arabidopsis seeds treated by specific ROS scavengers and as the light quality perception is necessary for a regular germination, we examined the germination in presence of exogenous H₂O₂ in different light qualities. H₂O₂ either promoted germination or repressed germination depending on the light wavelengths, showing that H₂O₂ acts as a signal molecule regulating germination in a light-dependent manner. Using photoreceptors null-mutants and GA-deficient mutants, we showed that H₂O₂-dependent promotion of germination relies on phytochrome signalling, but not on cryptochrome signalling, and that ROS signalling requires GA signalling.     

Dormancy removal of apple seeds by cold stratification is associated with fluctuation in H2O2, NO production and protein carbonylation level

Reactive oxygen (ROS) and nitrogen (RNS) species play a signaling role in seed dormancy alleviation and germination. Their action may be described by the oxidative/nitrosative “window/door”. ROS accumulation in embryos could lead to oxidative modification of protein through carbonylation. Mature apple (Malus domestica Borkh.) seeds are dormant and do not germinate. Their dormancy may be overcome by 70–90 days long cold stratification. The aim of this work was to analyze the relationship between germinability of embryos isolated from cold (5 °C) or warm (25 °C) stratified apple seeds and ROS or nitric oxide (NO) production and accumulation of protein carbonyl groups. A biphasic pattern of variation in H₂O₂ concentration in the embryos during cold stratification was detected. H₂O₂ content increased markedly after 7 days of seeds imbibition at 5 °C. After an additional two months of cold stratification, the H₂O₂ concentration in embryos reached the maximum. NO production by the embryos was low during entire period of stratification, but increased significantly in germination sensu stricto (i.e. phase II of the germination process). The highest content of protein carbonyl groups was detected after 6 weeks of cold stratification treatment. Fluctuation of H₂O₂ and protein carbonylation seems to play a pivotal role in seed dormancy alleviation by cold stratification, while NO appears to be necessary for seed germination.     

New semi-dwarfing alleles with increased coleoptile length by gene editing of gibberellin 3-oxidase 1 using CRISPR-Cas9 in barley (Hordeum vulgare L.)

The green revolution was based on genetic modification of the gibberellin (GA) hormone system with “dwarfing” gene mutations that reduces GA signals, conferring shorter stature, thus enabling plant adaptation to modern farming conditions. Strong GA-related mutants with shorter stature often have reduced coleoptile length, discounting yield gain due to their unsatisfactory seedling emergence under drought conditions. Here we present gibberellin (GA) 3-oxidase1 (GA3ox1) as an alternative semi-dwarfing gene in barley that combines an optimal reduction in plant height without restricting coleoptile and seedling growth. Using large-scale field trials with an extensive collection of barley accessions, we showed that a natural GA3ox1 haplotype moderately reduced plant height by 5–10 cm. We used CRISPR/Cas9 technology, generated several novel GA3ox1 mutants and validated the function of GA3ox1. We showed that altered GA3ox1 activities changed the level of active GA isoforms and consequently increased coleoptile length by an average of 8.2 mm, which could provide essential adaptation to maintain yield under climate change. We revealed that CRISPR/Cas9-induced GA3ox1 mutations increased seed dormancy to an ideal level that could benefit the malting industry. We conclude that selecting HvGA3ox1 alleles offers a new opportunity for developing barley varieties with optimal stature, longer coleoptile and additional agronomic traits.     

Role of H2O2 dynamics in brassinosteroid‐induced stomatal closure and opening in Solanum lycopersicum

Brassinosteroids (BRs) are essential for plant growth and development; however, their roles in the regulation of stomatal opening or closure remain obscure. Here, the mechanism underlying BR‐induced stomatal movements is studied. The effects of 24‐epibrassinolide (EBR) on the stomatal apertures of tomato (Solanum lycopersicum) were measured by light microscopy using epidermal strips of wild type (WT), the abscisic acid (ABA)‐deficient notabilis (not) mutant, and plants silenced for SlBRI1, SlRBOH1 and SlGSH1. EBR induced stomatal opening within an appropriate range of concentrations, whereas high concentrations of EBR induced stomatal closure. EBR‐induced stomatal movements were closely related to dynamic changes in H₂O₂ and redox status in guard cells. The stomata of SlRBOH1‐silenced plants showed a significant loss of sensitivity to EBR. However, ABA deficiency abolished EBR‐induced stomatal closure but did not affect EBR‐induced stomatal opening. Silencing of SlGSH1, the critical gene involved in glutathione biosynthesis, disrupted glutathione redox homeostasis and abolished EBR‐induced stomatal opening. The results suggest that transient H₂O₂ production is essential for poising the cellular redox status of glutathione, which plays an important role in BR‐induced stomatal opening. However, a prolonged increase in H₂O₂ facilitated ABA signalling and stomatal closure.     

Characterizing the pathotype structure of barley powdery mildew and effectiveness of resistance genes to this pathogen in Kazakhstan

Background

Alike to Reduced height-1 (Rht-1) genes in wheat and the semi dwarfing (sd-1) gene in rice, the sdw1/denso locus involved in the metabolism of the GA, was designated as the ‘Green Revolution’ gene in barley. The recent molecular characterization of the candidate gene HvGA20ox2 for sdw1/denso locus allows to estimate the impact of the functional polymorphism of this gene on the variation of agronomically important traits in barley.

Results

We investigated the effect of the 7-bp deletion in exon 1 of HvGA20ox2 gene (sdw1.d mutation) on the variation of yield-related and malting quality traits in the population of DHLs derived from cross of medium tall barley Morex and semi-dwarf barley Barke. Segregation of plant height, flowering time, thousand grain weight, grain protein content and grain starch was evaluated in two diverse environments separated from one another by 15° of latitude. The 7-bp deletion in HvGA20ox2 gene reduced plant height by approximately 13 cm and delayed flowering time by 3–5 days in the barley segregating DHLs population independently on environmental cue. On other hand, the sdw1.d mutation did not affect significantly either grain quality traits (protein and starch content) or thousand grain weight.

Conclusions

The beneficial effect of the sdw1.d allele could be associated in barley with lodging resistance and extended period of vegetative growth allowing to accumulate additional biomass that supports higher yield in certain environments. However, no direct effect of the sdw1.d mutation on thousand grain weight or grain quality traits in barley was detected.

     

Cross-talk between salicylic acid and NaCl-generated reactive oxygen species and nitric oxide in tomato during acclimation to high salinity

Hydrogen peroxide (H₂O₂) and nitric oxide (NO) generated by salicylic acid (SA) are considered to be functional links of cross‐tolerance to various stressors. SA‐stimulated pre‐adaptation state was beneficial in the acclimation to subsequent salt stress in tomato (Solanum lycopersicum cv. Rio Fuego). At the whole‐plant level, SA‐induced massive H₂O₂ accumulation only at high concentrations (10^?3–10^?2M), which later caused the death of plants. The excess accumulation of H₂O₂ as compared with plants exposed to 100 mM NaCl was not associated with salt stress response after SA pre‐treatments. In the root tips, 10^?3–10^?2M SA triggered the production of reactive oxygen species (ROS) and NO with a concomitant decline in the cell viability. Sublethal concentrations of SA, however, decreased the effect of salt stress on ROS and NO production in the root apex. The attenuation of oxidative stress because of high salinity occurred not only in pre‐adapted plants but also at cell level. When protoplasts prepared from control leaves were exposed to SA in the presence of 100 mM NaCl, the production of NO and ROS was much lower and the viability of the cells was higher than in salt‐treated samples. This suggests that, the cross‐talk of signalling pathways induced by SA and high salinity may occur at the level of ROS and NO production. Abscisic acid (ABA), polyamines and 1‐aminocyclopropane‐1‐carboxylic acid, the compounds accumulating in pre‐treated plants, enhanced the diphenylene iodonium‐sensitive ROS and NO levels, but, in contrast to others, ABA and putrescine preserved the viability of protoplasts.     

Nitrite,sodium nitroprusside,potassium ferricyanide and hydrogen peroxide release dormancy of <Emphasis Type="Italic">Amaranthus retroflexus</Emphasis> seeds in a nitric oxide-dependent manner

Nitric oxide (NO) and reactive oxygen species (ROS) are important regulators involving various processes of plant growth and development. Amaranthus retroflexus L. seeds possess a relative dormancy property that means freshly collected seeds can only germinate over a limited, high temperature range. Here, we show that the relative dormancy of A. retroflexus seeds could be significantly released following treatments with exogenous NO/cyanide (CN) donors such as nitrite, gases evolved from acidified nitrite, sodium nitroprusside (SNP), potassium ferricyanide (Fe(III)CN) and gases evolved from SNP or Fe(III)CN solutions, as well as exogenously supplied ROS, hydrogen peroxide (H₂O₂). However, the effectiveness varied among these chemicals. Gases evolved from acidified nitrite displayed maximum effect while H₂O₂ had minimum effect. We also show that the effects of these compounds could be significantly inhibited by NO specific scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), indicating that NO signaling pathway might play a central role in the dormancy release and germination of A. retroflexus seeds, while both ROS and CN might act through NO-dependent signaling cascades.     

ARP2/3 complex‐mediated actin dynamics is required for hydrogen peroxide‐induced stomatal closure in Arabidopsis

Multiple cellular events like dynamic actin reorganization and hydrogen peroxide (H₂O₂) production were demonstrated to be involved in abscisic acid (ABA)‐induced stomatal closure. However, the relationship between them as well as the underlying mechanisms remains poorly understood. Here, we showed that H₂O₂ generation is indispensable for ABA induction of actin reorganization in guard cells of Arabidopsis that requires the presence of ARP2/3 complex. H₂O₂‐induced stomatal closure was delayed in the mutants of arpc4 and arpc5, and the rate of actin reorganization was slowed down in arpc4 and arpc5 in response to H₂O₂, suggesting that ARP2/3‐mediated actin nucleation is required for H₂O₂‐induced actin cytoskeleton remodelling. Furthermore, the expression of H₂O₂ biosynthetic related gene AtrbohD and the accumulation of H₂O₂ was delayed in response to ABA in arpc4 and arpc5, demonstrating that misregulated actin dynamics affects H₂O₂ production upon ABA treatment. These results support a possible causal relation between the production of H₂O₂ and actin dynamics in ABA‐mediated guard cell signalling: ABA triggers H₂O₂ generation that causes the reorganization of the actin cytoskeleton partially mediated by ARP2/3 complex, and ARP2/3 complex‐mediated actin dynamics may feedback regulate H₂O₂ production.     

GABA Enhances Thermotolerance of Seeds Germination by Attenuating the ROS Damage in Arabidopsis

Seeds germination is strictly controlled by environment factor such as high temperature (HT) through altering the balance between gibberellin acid (GA) and abscisic acid (ABA). Gama-aminobutyric acid (GABA) is a small molecule with four-carbon amino acid, which plays a crucial role during plant physiological process associated with pollination, wounding or abiotic stress, but its role in seeds germination under HT remains elusive. In this study we found that HT induced the overaccumulation of ROS, mainly H₂O₂ and O₂^- , to suppress seeds germination, meanwhile, HT also activated the enzyme activity of GAD for the rapid accumulation of GABA, hinting the regulatory function of GABA in controlling seeds germination against HT stress. Applying GABA directly attenuated HT-induced ROS accumulation, upregulated GA biosynthesis and downregulated ABA biosynthesis, ultimately enhanced seeds germination. Consistently, genetic analysis using the gad1/2 mutant defective in GABA biosynthesis, or pop2-5 mutant with high endogenous GABA content supported the potential function of GABA in improving seeds germination tolerance to HT through scavenging ROS overaccumulation. Based on these data, we propose that GABA acts as a novel signal to enhance thermotolerance of seeds germination through alleviating the ROS damage to seeds viability.