Epigenetic control on transcription of vernalization genes and whole-genome gene expression profile induced by vernalization in common wheat

Vernalization is necessary for winter wheat to flower.However,it is unclear whether vernalization is also required for spring wheat,which is frequently sown in fall,and what molecular mechanisms underlie the vernalization response in wheat varieties.In this study,we examined the molecular mechanisms that regulate vernalization response in winter and spring wheat varieties.For this purpose,we determined how major vernalization genes(VRN1,VRN2,and VRN3)respond to vernalization in these varieties and whether modifications to histones play a role in changes in gene expression.We also identified genes that are differentially regulated in response to vernalization in winter and spring wheat varieties.We found that in winter wheat,but not in spring wheat,VRN1 expression decreases when returned to warm temperature following vernalization.This finding may be associated with differences between spring and winter wheat in the levels of tri-methylation of lysine 27 on histone H3(H3K27me3)and tri-methylation of lysine 4 on histone H3(H3K4me3)at the VRN1 gene.Analysis of winter wheat transcriptomes before and after vernalization revealed that vernalization influences the expression of several genes,including those involved in leucine catabolism,cysteine biosynthesis,and flavonoid biosynthesis.These findings provide new candidates for further study on the mechanism of vernalization regulation in wheat.

Characterization and distribution of novel alleles of the vernalization gene Vrn-A1 in Chinese wheat(Triticum aestivum L.) cultivars

The ability of wheat to adapt to a wide range of environmental conditions is determined mostly by allelic diversity among genes regulating vernalization requirement.Vrn-1 is a major regulator of this requirement.In this study,two novel alleles of Vrn-A1 were discovered in Chinese cultivars:vrn-A1n was identified in two landraces,Jiunong 2 and Ganchun 16,and Vrn-A1o was detected in Duanhongmangmai.Both novel alleles showed a linked duplication in the promoter region.The common copy of these two alleles was identical to the recessive allele vrn-A1.Compared with the recessive allele vrn-A1,the other copy of vrn-A1n contained a 54-bp deletion in the promoter region and the distinct copy of Vrn-A1o contained an11-bp deletion in the promoter region.In segregating populations in the greenhouse under nonvernalizing(20–25°C)and long-day(16 h light)conditions,plants with the novel vrn-A1n allele did not head earlier than those with the recessive vrn-A1 allele.However,plants that were either homozygous or heterozygous for the novel Vrn-A1o allele headed earlier than those with the recessive vrn-A1 allele.To identify the novel allele with the small-sized product and facilitate screening,a DNA marker for the novel dominant allele Vrn-A1o was designed.Analysis of the novel-allele distribution showed that two cultivars carrying the vrn-A1n allele were dispersed in the northwestern spring wheat zone,and 12 cultivars carrying the dominant Vrn-A1o allele were widely distributed in the northwestern spring wheat zone,Xinjiang winter and spring wheat zone,Yellow and Huai River valley winter wheat zone,and QinghaiTibetan Plateau spring and winter wheat zone.Our study identifies useful germplasm and a DNA marker for wheat breeding.

Effect of vernalization and plant growth regulators on flowering of Onithogalum ‘Chesapeake Snowflake’

The effect of vernalization and two growth regulators Fascination^TM and Pro-Gibb^R on the growth, inflorescence development and flowering of Omithogalurrt Chesapeake Snowflake＇ was studied. Regardless of growth regulator treatment, chilling bulbs for 3 weeks at 10℃ before planting accelerated flowering of the first inflorescence by 5 to 6 days, elongated floral stem length by 1.5 to 2 cm and shortened leaf length by 4 to 5 cm as compared with non-chilled bulbs. When bulbs were chilled 3 weeks at 10℃ before planting, Fascination 2% and Pro-Gibb accelerated flowering of the first inflorescence by 2 to 6 days respectively as compared with the control. When bulbs were not chilled before planting, Fascination 2% and Pro-Gibb accelerated flowering of the first inflorescence by 6 to12 days respectively as compared with the control. The lengths of the leaves and the inflorescences were not affected by PGR treatment. Higher rates of Pro-Gibb （100 mg.L^-1 and 200 mg.L^-1） and more amount of Fascination 2%（100 μL and 200 μL） can cause abortion of inflorescence.

Effect of vernalization on tuberization and flowering in the Tibetan turnip is associated with changes in the expression of FLC homologues

The turnip(Brassica rapa var. rapa) is a biennial crop that is planted in late summer/early fall and forms fleshy tubers for food in temperate regions. The harvested tubers then overwinter and are planted again the next spring for flowering and seeds. FLOWERING LOCUS C(FLC) is a MADS-box transcription factor that acts as a major repressor of floral transition by suppressing the flowering promoters FT and SOC1. Here we show that vernalization effectively represses tuber formation and promotes flowering in Tibetan turnip. We functionally characterized four FLC homologues(BrrFLC1,FLC2, FLC3, and FLC5), and found that BrrFLC2 and BrrFLC1 play a major role in repressing flowering in turnip and in transgenic Arabidopsis. In contrast, tuber formation was correlated with BrrFLC1 expression in the hypocotyl and was repressed under cold treatment following the quantitative downregulation of BrrFLC1. Grafting experiments of non-vernalized and vernalized turnips revealed that vernalization independently suppressed tuberization in the tuber or hypocotyl of the rootstock or scion, which occurred in parallel with the reduction in BrrFLC1 activity. Together, our results demonstrate that the Tibetan turnip is highly responsive to cold exposure, which is associated with the expression levels of BrrFLC genes.

The Relationship Between Developmental Accumulation of Leaf Soluble Proteins and Vernalization Response of Wheat(Triticum aestivum L.em. Thell)

The relationship between vernalization requirement and quantitative and qualitative changes in total leaf soluble proteins were determined in one spring （cv. Kohdasht） and two winter （cvs. Sardari and Norstar） cultivars of wheat （Triticum aestivum L.） exposed to 4℃. Plants were sampled on days 2, 14, 21 and 35 of exposure to 4℃. The final leaf number （FLN） was determined throughout the vernalization periods （0, 7, 14, 24, and 35 d） at 4℃. The final leaf number decreased until days 24 and 35 in Sardari and Norstar eultivars, respectively, indicating the vernalization saturation at these times. No clear changes were detected in the final leaf number of Kohdash cultivar, verifying no vernalization requirement for this spring wheat cultivar. Comparing with control, clear cold-induced 2-fold increases in proteins quantity occurred after 48 h following the 4℃-treatment in the leaves of the both winter wheat cultivars but, such response was not detected in the spring cultivar. However, the electrophoretic protein patterns showed between-cultivar and between-temperature treatment differences. With increasing exposure time to 4℃, the winter cultivars tended to produce more HMW polypeptides than the spring cultivar. Similar proteins were induced in both Sardari and Norstar winter wheat cultivars, however, the long vernalization requirement in Norstar resulted in high level and longer duration of expression of cold-induced proteins compared to Sardari with a short vernalization requirement. These observations indicate that vernalization response regulates the expression of low temperature （LT） tolerance proteins and determines the duration of expression of LT- induced proteins.

MiR396 is involved in plant response to vernalization and flower development in Agrostis stolonifera

MicroRNA396(miR396)has been demonstrated to regulate flower development by targeting growth-regulating factors(GRFs)in annual species.However,its role in perennial grasses and its potential involvement in flowering time control remain unexplored.Here we report that overexpression of miR396 in a perennial species,creeping bentgrass(Agrostis stolonifera L.),alters flower development.Most significantly,transgenic(TG)plants bypass the vernalization requirement for flowering.Gene expression analysis reveals that miR396 is induced by long-day(LD)photoperiod and vernalization.Further study identifies VRN1,VRN2,and VRN3 homologs whose expression patterns in wild-type(WT)plants are similar to those observed in wheat and barley during transition from short-day(SD)to LD,and SD to cold conditions.However,compared to WT controls,TG plants overexpressing miR396 exhibit significantly enhanced VRN1 and VRN3 expression,but repressed VRN2 expression under SD to LD conditions without vernalization,which might be associated with modified expression of methyltransferase genes.Collectively,our results unveil a potentially novel mechanism by which miR396 suppresses the vernalization requirement for flowering which might be related to the epigenetic regulation of VRN genes and provide important new insight into critical roles of a miRNA in regulating vernalization-mediated transition from vegetative to reproductive growth in monocots.

Study on the Change of GAs,ABA Level and ABA/GAs Ratio in the Embryos of Spring Wheat and Winter Wheat during Vernalization

Spring wheat (cv. 7742) and winter wheat (cv. Nongda 015) were vernalized by seed chilling, and the changes of GAs, ABA content and GAs / ABA ratio in embryo were investigated. The differences between cultivars, treatments and variety-treatment interactions were significant for both GAs and ABA. The level of GAs and ABA as well as GAs / ABA ratio were hardly changed by seed chilling in spriug wheat However, in winter wheat, the GAs content decreased much less compared with ABA that reduced sharply after chilling. It showed that the GAs / ABA ratio in winter wheat was increased remarkably (4.55 times) than in spring wheat. It was suggested that the absolute GAs level was independent of the vernalization effect because there was a higher GAs level before and after seed chilling in winter wheat when compared with spring wheat, One of the vemalization effects was the decline of ABA level that led to the increase of GAs / ABA ratio.

Effects of Different Low Overwintering Temperatures on Angelica Vernalization and Premature Bolting

Angelica sinensis(Oliv.)Diels is a valuable herbal plant.In production,its premature bolting,caused by vernalization,seriously affects its root yield.In this stuty,thermal treatments of 5,0 and?5°C,as well as treatments with shifts between these temperatures,were performed in its overwintering storage to investigate the chilling effect on the early bolting of Angelica sinensis and analyze the vernalization response to different temperatures.The results indicated that the 0°C treatment rendered the highest bolting rate,and the-5°C resulted in the lowest bolting rate.The initial short-term treatment at-5°C could promote the early bolting of A.sinensis,and the treatment at 5°C prior to planting could hasten its seedling bud growth.At the appropriate temperature,the early bolting rate of Angelica was positively related to the number of treatment days.So the frozen storage at-5°C could inhibit the vernalization of Angelica seedlings and the early bolting.The vernalization of Angelica could be a continuous process related to the number of treatment days.This study would serve as reference for the overwintering storage and cultivation of Angelica.

QTL Mapping of Vernalization Requirement in Brassica rapa

An F2 population, derived from a cross between the vernalization independent genotype ＇ RcBr＇ and the vernalization dependent genotype＇08A061 ＇ , was developed to eonstruet a linkage map with 165 SSR and InDel markers. QTL analysis was performed by two phenotypie evaluation （ days to 5 em elongate stalk and flowering time） based on the difference in F2：3 families under vernalization and no vernalization. The results showed that the vernalization requirement was reeessive in Brassica rapa. Seven QTLs that controlled vernalization requirement were detected on A02 and A06 linkage groups, which explained phenotypic variation ranging from 0.08% to 22.52%. Two QTLs （ VR-DE01, VR-b391 ） were detected on the top of A02, which explained 22.52% and 14. 54% of the phenotypic variation, respectively, and closely linked with BrFLC2 gene. Two QTLs （VR-DE03, VR-DE04） were deteeted on A06 with 13.30% and 13.64% of phenotypie variation. These detected QTLs will provide useful information on understanding the genetic basis between vernalization requirement and bolting, and that will be useful for marker-assisted selection （MAS） in a breeding program for bolting resistant eultivars.

Distinct roles of H3K27me3 and H3K36me3 in vernalization response,maintenance,and resetting in winter wheat

Winter plants rely on vernalization,a crucial process for adapting to cold conditions and ensuring successful reproduction.However,understanding the role of histone modifications in guiding the vernalization process in winter wheat remains limited.In this study,we investigated the transcriptome and chromatin dynamics in the shoot apex throughout the life cycle of winter wheat in the field.Two core histone modifications,H3K27me3 and H3K36me3,exhibited opposite patterns on the key vernalization gene VERNALIZATION1(VRN1),correlating with its induction during cold exposure.Moreover,the H3K36me3 level remained high at VRN1 after cold exposure,which may maintain its active state.Mutations in FERTILIZATION-INDEPENDENT ENDOSPERM(TaFIE)and SET DOMAIN GROUP 8/EARLY FLOWERING IN SHORT DAYS(TaSDG8/TaEFS),components of the writer complex for H3K27me3 and H3K36me3,respectively,affected flowering time.Intriguingly,VRN1 lost its high expression after the cold exposure memory in the absence of H3K36me3.During embryo development,VRN1 was silenced with the removal of active histone modifications in both winter and spring wheat,with selective restoration of H3K27me3 in winter wheat.The mutant of Tafie-cr-87,a component of H3K27me3“writer”complex,did not influence the silence of VRN1during embryo development,but rather attenuated the cold exposure requirement of winter wheat.Integrating gene expression with H3K27me3 and H3K36me3 patterns identified potential regulators of flowering.This study unveils distinct roles of H3K27me3 and H3K36me3 in controlling vernalization response,maintenance,and resetting in winter wheat.

Molecular mechanism of flowering time regulation in Brassica rapa:similarities and differences with Arabidopsis

Properly regulated flowering time is pivotal for successful plant reproduction.The floral transition from vegetative growth to reproductive growth is regulated by a complex gene regulatory network that integrates environmental signals and internal conditions to ensure that flowering takes place under favorable conditions.Brassica rapa is a diploid Cruciferae species that includes several varieties that are cultivated as vegetable or oil crops.Flowering time is one of the most important agricultural traits of B.rapa crops because of its influence on yield and quality.The transition to flowering in B.rapa is regulated by several environmental and developmental cues,which are perceived by several signaling pathways,including the vernalization pathway,the autonomous pathway,the circadian clock,the thermosensory pathway,and gibberellin(GA)signaling.These signals are integrated to control the expression of floral integrators BrFTs and BrSOC1s to regulate flowering.In this review,we summarized current research advances on the molecular mechanisms that govern flowering time regulation in B.rapa and compare this to what is known in Arabidopsis.

The Characterization and Geographical Distribution of the Genes Responsible for Vernalization Requirement in Chinese Bread Wheat

The frequency and distribution of the major vernalization requirement genes and their effects on growth habits were studied. Of the 551 bread wheat genotypes tested, seven allelic combinations of the three Vrn-1 genes were found to be responsible for the spring habit, three for the facultative habit and one for the winter habit. The three Vrn-1 genes behaved additiveiy with the dominant allele of Vrn-A1 exerting the strongest effect. The allele combinations of the facultative genotypes and the discovery of spring genotypes with ＂winter＂ allele of Vrn-1 implied the presence of as yet unidentified alleles/genes for vernalization response. The dominant alleles of the three Vrn-1 genes were found in all ten ecological regions where wheat is cultivated in China, with Vrn-D1 as the most common allele in nine and Vrn-A1 in one. The combination of vrn-A1vrn-B1Vrn-D1 was the predominant genotype in seven of the regions. Compared with landraces, improved varieties contain a higher proportion of the spring type. This was attributed by a higher frequency of the dominant Vrn-A1 and Vrn-B1 alleles in the latter, Correlations between Vrn-1 allelic constitutions and heading date, spike length, plant type as well as cold tolerance were established.

The vernalization-induced long non-coding RNA VAS functions with the transcription factor TaRF2b to promote TaVRN1 expression for flowering in hexaploid wheat

Vernalization is a physiological process in which prolonged cold exposure establishes flowering competence in winter plants. In hexaploid wheat, TaVRN1 is a cold-induced key regulator that accelerates floral transition. However, the molecular mechanism underlying the gradual activation of TaVRN1 during the vernalization process remains unknown. In this study, we identified the novel transcript VAS (TaVRN1 alternative splicing) as a non-coding RNA derived from the sense strand of the TaVRN1 gene only in winter wheat, which regulates TaVRN1 transcription for flowering. VAS was induced during the early period of vernalization, and its overexpression promoted TaVRN1 expression to accelerate flowering in winter wheat. VAS physically associates with TaRF2b and facilitates docking of the TaRF2b-TaRF2a complex at the TaVRN1 promoter during the middle period of vernalization. TaRF2b recognizes the Sp1 motif within the TaVRN1 proximal promoter region, which is gradually exposed along with the disruption of a loop structure at the TaVRN1 locus during vernalization, to activate the transcription of TaVRN1. The tarf2b mutants exhibited delayed flowering, whereas transgenic wheat lines overexpressing TaRF2b showed earlier flowering. Taken together, our data reveal a distinct regulatory mechanism by which a long non-coding RNA facilitates the transcription factor targeting to regulate wheat flowering, providing novel insights into the vernalization process and a potential target for wheat genetic improvement.

Molecular cloning of a cDNA related to vernalization(verc203) in winter wheat

A cDNA clone related to the vernalization in winter wheat(verc203)was harvested from the en-riched cold-induced cDNA library of 10~4 pfu with differential screening.The insert of verc203 in λ gt10 vector wassubcloned into the sites between BamH Ⅰ and Hind Ⅲ in pUC19 plasmid after being amplified with PCR.the analysis of the Northern blotting with a probe of verc203 indicated that the verc203 has a negative signalfor the control and the devernalized mRNA and a positive signal for the vernalized winter wheat and non-vernalized spring wheat at about 2.6 kb.

Transcriptome-wide Analysis Of Vernalization Reveals Conserved and Species-specific Mechanisms in Brachypodium

Several temperate cereals need vernalization to promote flowering. Little, however, is known about the vernalization-memory-related genes, and almost no comparative analysis has been performed. Here, RNA- Seq was used for transcriptome analysis in non-vernalized, vernalized and post-vernalized Brachypodium distachyon （L.） Beauv. seedlings. In total, the expression of 1,665 genes showed significant changes （fold change 〉4） in response to vernalization. Among them, 674 putative vernalization-memory-related genes with a constant response to vernalization were significantly enriched in transcriptional regulation and monooxygenase-mediated biological processes. Comparative analysis of vernalization-memory-related genes with barley demonstrated that the oxidative-stress response was the most conserved pathway between these two plant species. Moreover, Brachypodium preferred to regulate transcription and protein phosphorylation processes, while vernalization-memory-related genes, whose products are cytoplasmic membrane-bound-vesicle-located proteins, were preferred to be regulated in barley. Correlation analysis of the vernalization-related genes with barley revealed that the vernalization mechanism was conserved between these two plant species. In summary, vernalization, including its memory mechanism, is conserved between Brachypodium and barley, although several species-specific features also exist. The data reported here will provide primary resources for subsequent functional research in vernalization.

Intron-retained alternatively spliced VIN3-LIKE 2 gene from Chimonanthus praecox promotes flowering in transgenic Arabidopsis

Vernalization is a process of acquiring or accelerating the flowering ability by prolonged cold exposure.VERNALIZATION INSENSITIVE3(VIN3)is induced by chilling and is extremely important for the vernalization response in Arabidopsis thaliana.However,the issue of the induction of the VIN3-LIKE genes in wintersweet(Chimonanthus praecox)has been largely neglected.In the present study,we explored the molecular regulation of the PHD type finger protein-encoding gene CpVIL2 in relation to the growth and development of wintersweet in Arabidopsis.In wintersweet,quantitative real-time PCR(qRT-PCR)analysis showed that the relative expression of CpVIL2-As2i(intron-retained alternatively spliced in the second intron)was extremely higher in the pistils than in the other tissues.And the relative CpVIL2-As2i expression in flower buds(FBs)treated at 8°C was higher than that of FBs in December,2016 under natural conditions,which was not detected in non-flowering FBs at 16°C.In Arabidopsis,the expression patterns of the CpVIL2-As2i gene were detected at first in CpVIL2-As2i pro::GUS(β-glucuronidase)lines,with predominantly higher expression in flowers and inflorescence.Meanwhile,the hormone-induced expression profiles of the CpVIL2-As2i promoter were confirmed using exogenous induction by abscisic acid(ABA)and indole acetic acid(IAA)phytohormones,where the GUS enzyme activity obviously decreased compared with that of control.In comparison with Arabidopsis/Col-0,early flowering was detected in ectopic 35S::CpVIL2-As2i lines.Overall,these results demonstrated the function of the CpVIL2-As2i gene,at the same time,provided us with new insights into the molecular mechanisms of early flowering and complex regulatory networks of vernalization in wintersweet.

Axenfeld-Reiger syndrome: A search for the missing links

BACKGROUND Axenfeld-Rieger syndrome(ARS)is a rare cause of congenital glaucoma and may result in loss of vision.ARS is mostly autosomal dominant in nature characterized by developmental abnormalities in the angle of anterior chamber and iris of the eye,also associated with structural abnormalities in the body.AIM To study and observe the demographics and clinical findings in a very rare ocular disease known as ARS.METHODS Case records of symptomatic patients attending Ophthalmology outpatient department and diagnosed to have ocular hypertension or glaucoma in 3 years from March 2017 to March 2020 were evaluated to search for cases diagnosed with ARS.Records of all patients diagnosed with ARS were then analysed for demographic and clinical characterization as well as management and success of therapy.RESULTS Eight out of ten patients with positive clinical signs were symptomatic and had glaucoma.One of these patients had limbal stem cell deficiency and another had vernal keratoconjunctivitis.CONCLUSION Clinical characterization of ARS is important for making a definitive diagnosis and determining prognosis.

Different Ecological FLC Gene in the Arabidopsis thaliana Sequence Analysis

[Objective]Analysis of FLC sequence that Vernalization-related genes in Arabidopsis.[Method]Advance through natural populations of Arabidopsis QTL analysis of vernalization response was found on chromosome 5 of Arabidopsis thaliana have a flowering-related QTL,this test is to use sequence analysis to determine whether it is with the FLC gene homology.[Result]Arabidopsis thaliana,Italy and Sweden in the 27th,No.461,p.501,p.638,p.738,No.884 different base.While these bases are different,but the codon encoding the first nine amino acids out of the first 167 amino acids,the first 246 amino acids,due to codon degeneracy,encode proteins that are the same.[Conclusion] Arabidopsis has a rich genetic diversity,the FLC gene is highly conserved sequence length,base variable sites rich degenerate codons encoding the same amino acids they are not affect the growth of Arabidopsis thaliana.This indicates that the Arabidopsis genome sequence will be the environment.

NaCl胁迫下秋眠型紫花苜蓿光合作用特性的研究

以中苜一号为对照,研究NaCl溶液(0,50,100,150和200 mmol.L-1)处理下秋眠型紫花苜蓿Maverick、Vernal、Pioneer5446的光响应动态,观察形态特征和盐害级别,用Li-6400光合仪测定叶片净光合速率(Pn)、气孔导度(Gs)和细胞间隙CO2浓度(Ci)。结果表明,随NaCl浓度的增加和胁迫时间的延长,Maverick、Vernal、Pioneer5446和中苜一号叶片的盐害级别均加大,但品种间和处理间盐害出现的时间早晚和最终盐害程度不同。在50和100 mmol.L-1 NaCl的较低浓度下秋眠型紫花苜蓿和中苜一号光合响应存在着预警阶段恢复阶段-耗尽阶段,在150和200 mmol.L-1 NaCl的高浓度下,紫花苜蓿光响应直接进入耗尽阶段。秋眠型紫花苜蓿的耐盐性低于中苜一号。

Expression Analysis of Genes Related to Auxin Metabolism at Different Growth Stages of Pak Choi

Pak choi is a low-temperature vernalized plant that readily undergoes premature bolting during spring, but little is known about the governing molecular regulation of vernalization. In order to enhance our understanding of mechanism about premature bolting, we discussed the relationship between auxin(indole-3-acetic acid, IAA) and flowering of pak choi. During vernalization, hormone metabolism is an important regulatory pathway, and IAA plays a specific role. IAA metabolism has been studied in Arabidopsis thaliana and other plants, but not in pak choi. In this paper, the IAA content in pak choi shoot apices during vernalization and different growth stages was compared. The IAA content decreased significantly after low-temperature treatment(4 °C) and then increased rapidly during vegetative growth. During floral bud initiation,the IAA content decreased rapidly and was the lowest. Expressions of genes encoding key IAA metabolic enzymes were analyzed, and a major synthetase-encoding gene was downregulated, while a key degrading enzyme-encoding gene was upregulated during each comparison period,resulting in decreased IAA content. Expressions of four genes(Bra034975, Bra030246, Bra012239 and Bra040296) were consistent with changes in the IAA content. The functions of differentially expressed genes(DEGs) were analyzed, and 15 DEGs were found to be related to IAA metabolism.The findings illuminated the molecular mechanism regulating IAA content during vernalization in pak choi.