Identification of P-type plasma membrane H^(+)-ATPases in common wheat and characterization of TaHA7 associated with seed dormancy and germination

The P-type plasma membrane(PM)H^(+)-ATPases(HAs)are crucial for plant development,growth,and defense.The HAs have been thoroughly characterized in many different plants.However,despite their importance,the functions of HAs in germination and seed dormancy(SD)have not been validated in wheat.Here,we identified 28 TaHA genes(TaHA1-28)in common wheat,which were divided into five subfamilies.An examination of gene expression in strong-and weak-SD wheat varieties led to the discovery of six candidate genes(TaHA7/-12/-14/-16/-18/-20).Based on a single nucleotide polymorphism(SNP)mutation(C/T)in the TaHA7 coding region,a CAPS marker(HA7)was developed and validated in 168 wheat varieties and 171 Chinese mini-core collections that exhibit diverse germination and SD phenotypes.We further verified the roles of the two allelic variations of TaHA7 in germination and SD using wheat mutants mutagenized with ethyl methane sulphonate(EMS)in‘Jimai 22’and‘Jing 411’backgrounds,and in transgenic Arabidopsis lines.TaHA7 appears to regulate germination and SD by mediating gibberellic acid(GA)and abscisic acid(ABA)signaling,metabolism,and biosynthesis.The results presented here will enable future research regarding the TaHAs in wheat.

Research Progress of Molecular Regulation Mechanism of Seed Dormancy

Dormancy is the biological adaptation behavior for seed, and it is regulated by many factors. A lot of studies have been done on various aspects of seed dormancy, and the breadth and width of these studies are in constant expanding and deepening. With the wide application of means of quantitative genetics method and mutant in the study on seed dormancy, a large number of quantitative trait loci（QTL） and mutant genes related with dormancy have been identified, which is of great significance for the further revelation of the detail mechanisms of plant seed dormancy and germination. This paper briefly introduced the genetic variation of seed dormancy in recent years, described the inductive formation of plant seed dormancy from the four aspects of dormancy specific gene, chromatin, seed growth development and hormone, and stated the action mechanism of environmental factors on seed dormancy from genetic and molecular levels, as well as the regulation and control mechanism of dormancy release, with the aim to provide certain references for the study on seed dormancy.

OsVP1 activates Sdr4 expression to control rice seed dormancy via the ABA signaling pathway

Pre-harvest sprouting(PHS)is a disadvantageous trait in cereal production worldwide,causing large economic losses each year.Its regulation mechanism is still unclear.We generated the Oryza sativa Viviparous1(OsVP1)mutant using gene editing technique,which shows increased PHS compared with that of the wild type Nipponbare.OsVP1 is localized mainly in the nucleus and expressed in various tissues and organs.Expression of Seed dormancy 4(Sdr4),a key gene controlling PHS,was sharply reduced in OsVP1 mutants.OsVP1 bound to the specific motif CACCTG in the promoter of Sdr4 and activated its expression in rice protoplasts.Overexpression of Sdr4 reduced the high seed germination rate of OsVP1 mutant cr-osvp1-1,showing that Sdr4 acts as a downstream target of OsVP1.Both OsVP1 and Sdr4 loss-of-function mutants were insensitive to exogenous ABA and employed the ABA signaling pathway in regulating seed dormancy.These findings shed light on the control of seed dormancy aimed at preventing PHS in rice.

Breaking seed dormancy in Cupressus atlantica Gaussen,an endemicand threatened coniferous tree in Morocco

Cupressus atlantica Gaussen （Cupressaceae） is an endemic and endangered coniferous tree geographically restricted to the N＇Fis valley in South-Western Morocco. Like many forest species, C. atlantica exhibits dormancy which delays and reduces germination. To improve seed germination, different pre-treatments were conducted on C. atlantica seeds after storage for different periods （one, two and five years） including： scarification with sandy paper; soaking seeds in hot distilled water at 60℃ and 80℃ for 15 min and soaking seeds for 48 h in a gibberellic acid （GA3） at 1,000 and 2,000 mg·L-1. Results showed that scarification with sandy paper increased the germination rate of Atlas cypress by up to 67%, indicating that the species possess essentially an exogenous dormancy （physical dormancy） due to the hard seed coat （hardseededness）. Exogenous application of gibberellic acid （GA3） at 1,000 mg.L-1 was also effective in breaking seed dormancy and germination induction. These two treatments induced faster speed germination expressed by low number of days to first germination （8-10 days） and low values of mean germination times （MGT）. However, germination rate, under any treatment, is greatly dependent on the year of seed collection. Seeds collected in year 2004 gave the highest value, suggesting that even after five years of storage, the germination capacity of C. atlantica seeds could remain high. This observation is very interesting in the ex-situ conservation of such endemic and endangered species where the production of seeds is irregular over the years.

Effects of different treatments on seed germination and breaking seed dormancy of Prosopis koelziana and Prosopis Juliflora

For improving seed germination of Prosopis koelziana and Prosopis juliflora, different treatments of seeds were conducted, including scarification with sulfuric acid 98% for 10 and 15 min, sandy paper, hot water for 5 and 10 min, potasium nitrate 0.1%, gibberellic acid at 250 mg·L^-1 and 500 mg·L^-1 and combinational treatment of scarification with gibberellic acid of 250 mg·L^-1 and 500 mg·L^-1. The results show that scarifications with sandy paper and sulfuric acids 98% were the most effective treatments on breaking seed dormancy and seed germination induction. Scarification with sulfuric acid 98% for 15 min was the best treatment. According to the positive effect of scarification and lack of reaction of seeds against KNO3 and gibberellic acid, the kind of seed dormancy was determined as exogenous.

Correlation Between Parents and F_1 and Combining Ability of Parents on Seed Dormancy in indica Rice (Oryza sativa)

Dormancy indices of hulled and dehulled seeds were investigated by using 19 cytoplasmic male sterile （CMS） lines, 9 restorer lines and their 109 F1 hybrids of indica hybrid rice. The seeds of each F1 and the parents were harvested on 35 days after flowering. Combining ability was analyzed in 25 combinations made by 5 CMS lines and 5 restorer lines （North Carolina II mating design）. The seed dormancy index of F1 was positively and highly significantly correlated with those of their parents and mid-parent value. Out of the 109 combinations, 82 combinations showed mid-parent heterosis, and 43 heterobeltiosis. Seed dormancy indices of F1s and their parents declined dramatically in dehulled seeds compared with hulled seeds, indicating that the hull played an important role in seed dormancy. However, the trends were similar in hulled seeds and dehulled seeds in terms of relationships between the seed dormancy indicices in F1 and their parents. The influence of hull on seed dormancy mainly depended on F1 genotype, not on the hull from maternal parent. The variances of general combining ability （GCA） in female and male parents occupied 59.2% and 31.1% of total variance, respectively. The variance of specific combining ability （SCA） in combinations occupied 9.7% of total variance, indicating that gene additive effects were principal. Among the 5 CMS lines, II112A had the highest GCA effect for seed dormancy, followed by D62A. Among the 5 restorer lines, IRl12 had the highest GCA effect for seed dormancy, followed by 2786. These lines are elite parental materials for breeding F1 hybrid rice with stronger seed dormancy.

Seed dormancy,germination and storage behavior of Magnolia sinica,a plant species with extremely small populations of Magnoliaceae

Magnolia sinica is one of the most endangered Magnoliaceae species in China.Seed biology information concerning its long-term ex situ conservation and utilization is insufficient.This study investigated dormancy status,germination requirements and storage behavior of M.sinica.Freshly matured seeds germinated to ca.86.5%at 25/15℃but poorly at 30℃;GA3 and moist chilling promoted germination significantly at 20℃.Embryos grew at temperatures(alternating or constant)between 20℃and 25℃,but not at 5℃or 30℃.Our results indicate that M.sinica seeds possibly have non-deep simple mor-phophysiological dormancy(MPD).Seeds survived desiccation to 9.27%and 4.85%moisture content(MC)as well as a further 6-month storage at-20℃and in liquid nitrogen,including recovery in vitro as excised embryos.The established protocol ensured that at least 58%of seedlings were obtained after both cold storage and cryopreservation.These results indicate that both conventional seed banking and cryopreservation have potential as long-term ex situ conservation methods,although further optimized approaches are recommended for this critically endangered magnolia species.

Analysis of QTL for Seed Dormancy and Their Response to Dry Heat Treatment in Rice (Oryza sativa L.)

Quantitative trait loci (QTL) controlling seed dormancy in rice were identified usingrecombinant inbred lines (RILs) population derived from the cross between a japonicavariety Kinmaze and an indica variety DV85. Seeds of two parental cultivars and each RILwere harvested in 35d after heading. The germination percentage of these seeds at 30℃for 7 days were measured as the degree of seed dormancy. QTL analysis was performed withWindows QTL Cartographer 1.13a program by composite interval mapping. A total of four QTLfor seed dormancy were detected on chromosome 2 (two regions), 5 and 11, respectively.Phenotypic variation explained by each QTL ranged from 8.37 to 17.40%. Responses of suchloci to a dormancy-breaking treatment with dry heat were further detected. The resultsshowed that two alleles of qDOR-2-1 and qDOR-5 from DV85 as well as the allele of qDOR-11 from Kinmaze increased the seed dormancy, which seemed to be easily broken by dry heattreatment. Such loci of seed dormancy may be applied to rice genetic improvement. Theallele of qDOR-2-2 from DV85 increased the seed dormancy, which could not be broken bydry heat treatment.

Mapping and Analysis of Quantitative Trait Loci Controlling Seed Dormancy in Rice

Seed dormancy is one of the most important traits related to the rice grain quality and seeds application, because it is associated with pre-harvest sprouting, resulting in a downgrading of quality and severe limitations in end-use application. The recent development of DNA markers and linkage maps of rice has made possible mapping of individual genes associated with complex seed dormancy traits, analyzing the genetics effects of individual genes and genotype-by- environment interactions. Up to now, numerous quantitative trait loci (QTLs) associated with seed dormancy in rice have been identified and mapped in the molecular genetic map by different populations. In this review, we focus on the genetic base of seed dormancy in rice, especially compare QTLs controlling seed dormancy reported up to now, analyze the expression and stability of QTLs controlling seed dormancy, and discuss the present problems. Finally we show a new pathway to further research on seed dormancy.

Seed Dormancy and Seedlings Physiological Response to Topramezone in Green Foxtail(Setaria viridis)

Green foxtail(Setaria viridis)is a notorious weed in corn fields in Heilongjiang Province.To investigate the best method to break the seed dormancy of green foxtail and its physiological response to topramezone,this study selected newly harvested and one-year stored green foxtail seeds as research subjects.The seeds were treated with HCl,Na OH,gibberellic acid(GA),different water temperatures and polyethylene glycol(PEG)to study the seed dormancy and drought resistance of green foxtail.The results showed that newly harvested seeds exhibited dormancy,and treatments with HCl,NaOH and different water temperatures were unable to break the dormancy.Soaking the seeds in GA could overcome dormancy,but the seeds failed to germinate when exposed to 25%PEG concentration.When topramezone was applied at rates of 22.5 and 45.0 g a.i.·hm^(-2)at the 3-leaf and 5-leaf stages,respectively,the chlorophyll content reached the lowest value at 28 days after treatment(DAT).At the 7-leaf stage,the chlorophyll content reached the lowest value at 7 DAT.The activity of 4-hydroxyphenylpy-ruvate dioxygenase(HPPD)enzyme after topramezone application reached the maximum value at 7 DAT for different leaf ages,and the higher the leaf age,the higher the HPPD activity,which was an important factor contributing to the resistance of green foxtail to topramezone.

TaSRO1 interacts with TaVP1 to modulate seed dormancy and pre-harvest sprouting resistance in wheat

Dormancy is an adaptive trait which prevents seeds from germinating under unfavorable environmental conditions.Seeds with weak dormancy undergo pre-harvest sprouting(PHS)which decreases grain yield and quality.Understanding the genetic mechanisms that regulate seed dormancy and resistance to PHS is crucial for ensuring global food security.In this study,we illustrated the function and molecular mechanism of TaSRO1 in the regulation of seed dormancy and PHS resistance by suppressing TaVP1.The tasro1 mutants exhibited strong seed dormancy and enhanced resistance to PHS,whereas the mutants of tavp1 displayed weak dormancy.Genetic evidence has shown that TaVP1 is epistatic to TaSRO1.Biochemical evidence has shown that TaSRO1 interacts with TaVP1 and represses the transcriptional activation of the PHS resistance genes TaPHS1 and TaSdr.Furthermore,TaSRO1 undermines the synergistic activation of TaVP1 and TaABI5 in PHS resistance genes.Finally,we highlight the great potential of tasro1 alleles for breeding elite wheat cultivars that are resistant to PHS.

PIF4 interacts with ABI4 to serve as a transcriptional activator complex to promote seed dormancy by enhancing ABA biosynthesis and signaling

Transcriptional regulation plays a key role in the control of seed dormancy,and many transcription factors(TFs)have been documented.However,the mechanisms underlying the interactions between different TFs within a transcriptional complex regulating seed dormancy remain largely unknown.Here,we showed that TF PHYTOCHROME-INTERACTING FACTOR4(PIF4)physically interacted with the abscisic acid(ABA)signaling responsive TF ABSCISIC ACID INSENSITIVE4(ABI4)to act as a transcriptional complex to promote ABA biosynthesis and signaling,finally deepening primary seed dormancy.Both pif4 and abi4 single mutants exhibited a decreased primary seed dormancy phenotype,with a synergistic effect in the pif4/abi4 double mutant.PIF4 binds to ABI4 to form a heterodimer,and ABI4 stabilizes PIF4 at the protein level,whereas PIF4 does not affect the protein stabilization of ABI4.Subsequently,both TFs independently and synergistically promoted the expression of ABI4 and NCED6,a key gene for ABA anabolism.The genetic evidence is also consistent with the phenotypic,physiological and biochemical analysis results.Altogether,this study revealed a transcriptional regulatory cascade in which the PIF4–ABI4 transcriptional activator complex synergistically enhanced seed dormancy by facilitating ABA biosynthesis and signaling.

Dormancy of Amaranthus retroflexus L. Seeds and Physiological Response Seedlings to Acifluorfen Sodium

Amaranthus retroflexus L. is a serious and widespread malignant weed in soybean fields in Heilongjiang Province. Exploring the dormancy characteristics of A. retroflexus L. seeds and the physiological response of its seedlings to acifluorfen sodium can provide a basis for further researches on its resistance mechanism. Using newly harvested and stored A. retroflexus L. seeds for one year as experimental materials, the effects of different concentrations of HCl, NaOH, water temperature, gibberellic acid(GA) and polyethylene glycol(PEG) on the dormancy and germination of A. retroflexus L. seeds were studied. The sensitivity of A. retroflexus L.to acifluorfen sodium was determined using bioassay. The effects on leaf chlorophyll content and target enzyme activity were studied at a normal dosage of 360 g a.i. hm^(-2) and a doubling dosage of 720 g a.i. hm^(-2) of acifluorfen sodium. Newly harvested seeds exhibiting dormancy were soaked in water of various temperatures and in different concentrations of NaOH and HCl, which were ineffective in breaking the seed dormancy. GA could break seed dormancy, and the highest seed germination rate reached 93.33% when they were soaked at 3 000 mg·L^(-1) for 72 h and 4 000 mg·L^(-1) for 48 h. The drought stress was simulated with a 15%-25% polyethylene glycol solution, which had no significant effect on the seed germination rate. The GR_(50) value of acifluorfen sodium for A. retroflexus L. was 705.7 g a.i. hm^(-2), which was 1.96 times the recommended dose in the field. After the application of different doses of acifluorfen sodium, the chlorophyll content of A. retroflexus L. reached its minimum value 3 days after treatment(DAT), and then gradually increased. The activity of the target enzyme protoporphyrinogen oxidase(PPO) reached the highest value at 7 DAT under different dosages, and gradually returned to normal levels thereafter. Soaking with gibberellin was an effective method to break seed dormancy. A. retroflexus L. seeds had certain drought resistance during the germination process. A. retroflexus L. was not sensitive to acifluorfen sodium and acifluorfen sodium ether, and could not effectively inhibit the PPO activity, indicating that A. retroflexus L. had target resistance to acifluorfen sodium.

Changes in the biochemical composition and enzyme activity during dormancy release of Cyclocarya paliurus seeds

Cyclocarya paliurus is only propagated from seeds which have pronounced dormancy. Overcoming seed dormancy is an important component of efficient and cost-effective seedling production of Cyclocarya paliurus. Changes in biochemical composition and enzyme activity were investigated during dormancy release. The activities of all the studied enzymes in the stratified seeds increased significantly, compared to those in the control samples. Of the enzymes examined, the activities of protease increased the most （413.8%）, followed by peroxidase （278.7%）, lipase （161.0%）, glucose-6-phosphate dehydrognase （149.1%） and amylase （60.6%） after 8 months of stratification. Crude fat and protein constituted the bulk of the storage reserves in mature seeds of C. pal# urus. Compared with the seeds before stratification, about 45% of the starch, 46% of the protein and 11% of the crude fat were depleted during dormancy release of C. paliurus seeds, while the soluble sugar content was enhanced by 101.5% in the germinating seeds. Correlation analysis showed, during dormancy release of C. paliurus seeds, a close positive relationship between POD and G6PDH activity as well as soluble sugar content and amylase activity, while there was a significant negative relationship between storage substances and their related enzyme activities.

Effect of different seed treatments on dormancy breaking and germination in three species in arid and semi-arid lands

Seeds of many woody plant species have one of several types of dormancy. They do not germinate unless ,specific environmental signals are in place or events occur. This study was conducted to evaluate the effects of scarification treatments on seed dormancy and germination of Acacia nilotica （L.） Willd. ex Del., Prosopis juliflora （Sw.） DC. ：and Dodonaea viscosa （L.） Jacq. The following treatments were applied： T1, untreated seed （control）; T2, sulphuric acid （97%） for 45 min; T3, boiling water for 5 min. The seeds were cultured on a Murashige and Skoog （MS） medium after sterilization. The responses of seeds to treatments were compared with each other and with the control treatment. Germination was observed daily for a 30-day period. Results indicated positive responses to treatments, while impermeable Seed coats may be responsible for low germination rates in intact seeds as seen experimentally in the untreated control. The highest germination was obtained for P. juliflora and D. viscosa acid-scarified seeds （80.8%-90.8%） and for scarified seeds of A. nilotica （50.2%） boiled in water. The germination indices, i.e., final germination percentage （FG）, mean daily germination （MDG） and germination rate （GR）, were significantly affected by treatments and species （p 〈 0.01）.

The GATA transcription factor TaGATA1 recruits demethylase TaELF6-A1 and enhances seed dormancy in wheat by directly regulating TaABI5

Seed dormancy is an important agronomic trait in crops, and plants with low dormancy are prone to preharvest sprouting(PHS) under high-temperature and humid conditions. In this study,we report that the GATA transcription factor TaGATA1 is a positive regulator of seed dormancy by regulating TaABI5 expression in wheat.Our results demonstrate that TaGATA1 overexpression significantly enhances seed dormancy and increases resistance to PHS in wheat. Gene expression patterns, abscisic acid(ABA) response assay, and transcriptome analysis all indicate that TaGATA1 functions through the ABA signaling pathway. The transcript abundance of TaABI5, an essential regulator in the ABA signaling pathway,is significantly elevated in plants overexpressing TaGATA1. Chromatin immunoprecipitation assay(ChIP) and transient expression analysis showed that TaGATA1 binds to the GATA motifs at the promoter of TaABI5 and induces its expression.We also demonstrate that TaGATA1 physically interacts with the putative demethylase TaELF6-A1, the wheat orthologue of Arabidopsis ELF6.ChIP–qPCR analysis showed that H3K27me3 levels significantly decline at the TaABI5 promoter in the TaGATA1-overexpression wheat line and that transient expression of TaELF6-A1 reduces methylation levels at the TaABI5 promoter, increasing TaABI5 expression. These findings reveal a new transcription module, including TaGATA1–TaELF6-A1–TaABI5, which contributes to seed dormancy through the ABA signaling pathway and epigenetic reprogramming at the target site. TaGATA1 could be a candidate gene for improving PHS resistance.

The Evening Complex and the Chromatin-Remodeling Factor PICKLE Coordinately Control Seed Dormancy by Directly Repressing DOG1 in Arabidopsis

Primary seed dormancy is acquired during seed development and maturation,which is important for plant fitness and survival.DELAY OF GERMINATION1(DOG1)plays a critical role in inducing seed dormancy.DOG1 expression increases rapidly during seed development,but the precise mechanism underlying this process remains elusive.In this study,we showed that mutants with a loss or reduced function of the chromatin-remodeling factor PICKLE(PKL)exhibit increased seed dormancy.PKL associates with DOG1 chromatin and inhibits its transcription.We found that PKL physically interacts with LUX ARRHYTHMO(LUX),a member of the evening complex(EC)of the circadian clock.Furthermore,LUX directly binds to a specific coding sequence of DOG1,and DOG1 acts genetically downstream of PKL and LUX.Mutations in either LUX or EARLY FLOWERING3(ELF3)encoding another member of the EC led to increased DOG1 expression and enhanced seed dormancy.Surprisingly,these phenotypes were abolished when the parent plants were grown under continuous light.In addition,we observed that loss of function of either PKL or LUX decreased H3K27me3 levels at the DOG1 locus.Taken together,our study reveals a regulatory mechanism in which EC proteins coordinate with PKL to transmit circadian signals for directly regulating DOG1 expression and seed dormancy during seed development.

The MKK3–MPK7 cascade phosphorylates ERF4 and promotes its rapid degradation to release seed dormancy in Arabidopsis

Seeds establish dormancy to delay germination until the arrival of a favorable growing season.In this study,we identify a fate switch comprised of the MKK3–MPK7 kinase cascade and the ethylene response factor ERF4 that is responsible for the seed state transition from dormancy to germination.We show that dormancy-breaking factors activate the MKK3–MPK7 module,which affects the expression of some a-EXPANSIN(EXPA)genes to control seed dormancy.Furthermore,we identify a direct downstream substrate of this module,ERF4,which suppresses the expression of these EXPAs by directly binding to the GCC boxes in their exon regions.The activated MKK3–MPK7 module phosphorylates ERF4,leading to its rapid degradation and thereby releasing its inhibitory effect on the expression of these EXPAs.Collectively,our work identifies a signaling chain consisting of protein phosphorylation,degradation,and gene transcription,by which the germination promoters within the embryo sense and are activated by germination signals from ambient conditions.

Differences in seed dormancy and germination in amphicarpic legumes:manifold bet-hedging in space and time

Aims Dispersal through space or time via dormancy is one of the primary processes whereby organisms can influence the environment they experience.In plants,strong evolutionary correlations are expected between the two kinds of dispersal because both are performed by the seeds and play comparable adaptive roles.In this paper,we investigated these evolutionary correlations using amphicarpic plants,which simultaneously produce aerial seeds with high spatial dispersal propensity and subterranean seeds that do not disperse.Methods We investigated the variation in dormancy and germination in aerial and subterranean seeds of two amphicarpic legumes(Vicia amphicarpa L.and Lathyrus amphicarpos L.)and two closely related homocarpic taxa(Vicia sativa L.and Lathyrus cicera L.)by estimating germination percentages following different combinations of dormancy breaking treatments(i.e.dry after-ripening,cold stratification and physical scarification).Important Findings Our results showed complex interactions between spatial and temporal dispersal.Right after dispersal,aerial seeds were more dormant than their subterranean counterparts,but this trend reversed with after-ripening,as seeds developed physical dormancy.Seeds of homocarpic plants germinated at higher percentages than those of their amphicarpic congeners and lost dormancy homogeneously with after-ripening.Conversely,amphicarpic seeds exhibited varied dormancy strategies modulated by both physiological and physical dormancy.These are expected to increase variation in emergence timing,providing multiple levels of diversifying bet-hedging.This strategy might be adaptive under highly unpredictable conditions by enabling plants to rely on historically favorable sites in good seasons without preventing spatial and temporal migration.

Gene editing applications to modulate crop flowering time and seed dormancy

Gene editing technologies such as CRISPR/Cas9 have been used to improve many agricultural traits,from disease resistance to grain quality.Now,emerging research has used CRISPR/Cas9 and other gene editing technologies to target plant reproduction,including major areas such as flowering time and seed dormancy.Traits related to these areas have important implications for agriculture,as manipulation of flowering time has multiple applications,including tailoring crops for regional adaptation and improving yield.Moreover,understanding seed dormancy will enable approaches to improve germination upon planting and prevent pre-harvest sprouting.Here,we summarize trends and recent advances in using gene editing to gain a better understanding of plant reproduction and apply the resulting information for crop improvement.