Effect of nucleic acid antimetabolites on the vernalization of winter wheat

Excised winter wheat embryos were cold-treated in White's mediumcontaining 2% sucrose for 60 days at 4°C. Embryos couldbe vernalized in medium containing sugar, whereas the additionof 8-azaguanine to the medium at the concentration of 10^–4M inhibited vernalization. (Received May 2, 1975; )     

Changes in nucleic acid metabolism in barley seedlings during vernalization

Changes in nucleic acid metabolism of barley seedlings duringvernalization were investigated using thymidine-³H and uridine-³H. DNA content increased in the early germination stage from the1st to 3rd week in vernalized seedlings. In unvernalized seedlings,the most rapid increase was found in the late germination stage.RNA content in the vernalized seedlings increased after 1 weekand reached maximum level after 3 weeks of vernalization treatment.The unvernalized seedlings had a comparatively high contentat 2 days' germination which then gradually increased. Much thymidine-³H was incorporated into DNA and uridine-³H intoRNA fractions in the seedlings during early vernalization. Onthe contrary, without vernalization, heavy incorporation ofthymidine-³H was delayed during the late germination stage.Incorporation of uridine-³H showed a linear increase. A more detailed distribution of thymidine-³H and uridine-³Hin the nucleic acids was examined by methylated albumin-coatedkieselguhr column chromatography. A considerable amount of theincorporated uridine-³H was found in the tenaciouslybound ribonucleicacid (TB-RNA) in the vernalized seedlings. (Received January 18, 1973; )     

Changes in hybridizable RNA in winter wheat embryos during germination and vernalization

DNA-RNA hybridization-competition experiments were performedto analyze RNA heterogeneity in wheat embryos during germinationand vernalization. A significant difference was found betweenRNA populations of 24-hr and 3-day germinated embryos, whileminor differences were detected between 3- and 5-day germinatedembryo RNAs and between 20- and 40-day cold-treated embryo RNAs.RNA populations in 30-day cold-treated embryos were significantlydifferent from those in 50-day ones. The RNA species presentin 50-day cold-treated embryos were not similar to those in3- and 5-day germinated embryos. A great portion of the RNAspecies in 3-day germinated embryos was found in 30-day cold-treatedembryos. These results suggested that some new RNA species aresynthesized in wheat embryos during 30 to 50 days of cold treatment. ¹ Present address: Department of Biochemistry, Faculty of PharmaceuticalSciences, Higashi Nippon Gakuen University, Ishikari-Tobetsu,Hokkaido 061–02, Japan. (Received February 21, 1977; )     

Changes in histories during the vernalization of wheat embryos

The effect of cold-treatment on chromatin-histone in winterwheat embryos was studied. As the embryos were vernalized, theamount of whole histone increased about two-fold, but the histonecontent in excised embryos incubated in a medium containingsugar was changed little by prolonged chilling. These resultssuggest that the increase in histone content is not necessarilyconnected with the vernalizing response. Disc electrophoretic patterns of whole histone from vernalizedembryos gave a relatively distinct band of the faster movingcomponent of Fraction F-1 histone which was very scanty in non-vernalizedembryos but distinct in spring wheat embryos. Although therewas no increase in whole histone content, the faster-movingband of F-l was more conspicuous and bands of the other fractionswere fainter in the vernalized excised embryos. These observationssuggest that this particular component of Fraction F-l histoneis connected with the vernalizing response. As vernalization proceeded, the amino acid composition of wholehistone changed and the lysine-arginine ratio became higher,in agreement with the observed increase in the lysine-rich fraction(F-l) of histones. Nuclear size increased about two-fold during the period of vernalization,supporting the view that in embryos exposed to low temperature,cell division hardly proceeds. (Received September 27, 1972; )     

Vernalization in Lolium temulentum L.: Responses of In Vitro Cultures of Mature and Immature Embryos, Shoot Apices and Callus

Response of vernalization to low temperature (2C) was studiedin a winter-annual form of Lolium temulentum L., using imbibedseeds, excised mature and immature embryos, shoot apical meristemsand callus tissue. Excised embryos, as early as 5 days afteranthesis, and excised shoot apices could be vernalized as effectivelyas imbibed seeds. Cold treatment of developing embryos withinthe ear, however, appeared to have no vernalizing effect. Plantsderived from callus by somatic embryogenesis showed varyingdegrees of vernalization response. The vernalization response in L. temulentum, as in winter annualcereals, appears to be located in the shoot apical meristemand the vernalized condition can be transmitted to new axillarymeristems formed from it. Lolium temulentum L. darnel, vernalization, embryo culture, apical meristem culture, callus culture     

INHIBITORY EFFECT OF GROWTH RETARDANTS ON THE INDUCTION OF FLOWERING IN WINTER WHEAT

1. Growth retardants, CCC, Amo-1618, Phosfon-D and B-995, appliedduring seed vernalization inhibited the ear development of winterwheat. CCC applied during green plant vernalization inhibitedflowering,but it had no appreciable effect on the final stemlength. Theinhibition by CCC was reversed by foliar applicationof gibberellin.On the other hand, CCC applied after the vernalizationperiodaffected the final stem length but not flowering.
2. Theamount of endogenous gibberellin-like substance(s) was greaterin the vernalized plant than in the non-vernalized plant. Nogibberellin-like substance was detected in the CCC-treated plant.
3. Endogenous gibberellin-like substance(s), whose biosynthesisis inhibited by some growth retardants, may play a part in thevernalization process in winter wheat.

¹Present address: National Institute of Agricultural Sciences,Nishigahara, Kitaku, Tokyo     

PROTEINS OF WHEAT EMBRYOS IN THE PERIOD OF VERNALIZATION

1. Soluble proteins prepared from non-vernalized and vernalizedwheat embryos were investigated by means of column chromatographyand disc electrophoresis. As vernalization proceeded, new proteinswere detectable as the fastest moving electrophoretic bandsor as the new peaks which were eluted with 0.025 M and 0.05M phosphate buffer (pH 7.0) from DEAE-cellulose. Plumule wasfound to be the part of embryo in which these proteins appear.After vernalization, protein pattern of winter wheat embryowas very similar to that of spring wheat embryo.
2. Histoneswere prepared from non-vernalized and vernalized wheatembryosand their chromatographic patterns were investigated.Winterwheat embryo showed more complicated pattern than didspringwheat embryo. Upon vernalization, histone pattern ofwinterwheat embryo became less complicated and similar to thatofspring wheat embryo.

(Received October 4, 1966; )     

Stimulation of generative development in partly vernalized winter wheat by animal sex hormones

The influence of selected animal steroid sex hormones, on generative development of winter wheat var. Grana was investigated. Wheat plants of this variety necessitate 63-day long vernalization. Mature, isolated embryos of wheat were cultured in vitro on media containing androsterone, androstenedione, estriol, estrone, 17β-estradiol and progesterone in concentrations 10⁻⁵ and 10⁻⁶ M. They were not vernalized or vernalized for 7, 14, 21 and 28 days (5 °C, 8 h photoperiod). Investigated steroids stimulated the generative development of winter wheat by an increasing in the percentage of heading plants and accelerating the heading. The strongest effect was observed when plants were treated with steroids during the suboptimal, 21 and 28 day, vernalization. After 28 days of vernalization, 100 % heading were observed in plants obtained on the media containing androsterone and androstenedione in concentrations 10⁻⁵ and 10⁻⁶ M or estrone, 17β-estradiol and progesterone in concentration 10⁻⁵ M. Control plants showed only 8 % heading. An erratum to this article is available at .     

Tentative determination of the molecular weight of substances stimulating the flowering of winter wheat

Murashige & Skoog nutrient was supplemented with substances of molecular weight (M_W) less than 5 kDa, which were separated from extract of winter wheat ears by means of Sephadex G-25 ultrafiltration. Isolated embryos of the same wheat cultivar (Grana) were vernalized in the nutrient for 0 and 7 days at 2 °C for 2 weeks and planted in a glass-house. After 150 days of growth (20/17 °C day/night) the development of the shoot apices was observed. It was found that substances of M_W<5 kDa strongly stimulated the generative development of the plants, enabling the earing of 30 % of non-vernalized plants (control=0%) and 100 % plants vernalized for 7 days (control=29 %). The substances present in the extract of both spring (cv. Jara) and winter (cv. Grana) varieties were fractionated by means of Sephadex chromatography into 300 fractions of M_W=1 to 5 kDa and each of them was added to the isolated embryos of cv. Grana. The embryos were vernalized at 2 °C for 7 days and then cultured as previously described. It was found that the differentiation of the shoot apices was stimulated by over 34 % by fractions of winter wheat extract and more than 50 % by fractions of the spring wheat extract. However, only a few of identical fractions of the extracts of both wheat varieties were able to induce the earing of the plants. These fractions were grouped in 4 continuous intervals of M_W equal to about 4.5–4.9, 3.2–3.3, 2.1–2.6 and 1.00–1.03 kDa. Within the three intervals was identified a small group of identical fractions, which affected the growth of the seedlings in similar mode i.e. inhibiting or stimulating. Thus it can be assumed that these intervals contained identical or similar substances capable of stimulating strongly the earing of winter wheat.     

Endogenous cytokinins in vernalized winter wheat grains

Summary The soybean callus assay was used to study the effect of vernalization on the endogenous cytokinin levels of the grains of winter wheat (Triticum vulgare L.), cv. Ferto and Fema. Vernalization of the grains was carried out at 3° C in the dark for 0, 9, 18, 27, 36 and 45 days. Cytokinin activities could be detected in both the aqueous and butanol extracts of vernalized grains of both cultivars. It is suggested that flowering of winter wheat may involve changes in the levels of endogenous cytokinins during vernalization.     

Functional analysis of the ver gene using antisense transgenic wheat

The function of ver203 , a gene related to vernalization in winter wheat, was investigated by expression of a complementary DNA as an antisense RNA in transgenic plants. A verc203:gus fusion‐expression plasmid was constructed in pBI221, which contains a CaMV (cauliflower mosaic virus) 35S‐promoter, a gus gene and a nos terminator. The construct was then introduced into the plant by the pollen‐tube pathway. The results showed that heading was strongly inhibited in 6 of 326 vernalized antisense transgenic winter wheat plants, until both the vernalized control winter wheat and sense transgenic plants ripened. The hybridization analysis of DNA, amplification of the insert DNA sequences with PCR, northern blot analysis with double‐ and single‐stranded probes, and detection of GUS activity by X‐gluc assay gave strong positive results. This suggests that the VER203 protein plays an important role in controlling heading and flower development in winter wheat.     

The Mechanism of Action of Vernalization in Lathyrus odoratus L.

In the sweet pea (Lathyrus odoratus L.) genes Dn^l andDr^h controlthe production of a graft-transmissible substance which delaysflowering and promotes outgrowth of basal laterals. Seed vernalizationpromotes flowering and reduces lateral outgrowth in intact plantsand grafted scions of genotype DnⁱDn^l, suggesting that vernalizationreduces output of the Dnⁱ system, possibly by disrupting therelationship between chronological and plastochronic age. Whenlateral outgrowth and floral abortion are used as indicatorsof inhibitor levels, it can be shown that vernalized Dnⁱ plantspossess more inhibitor but initiate flower buds at a lower nodethan unvernalized dn plants. This supports the suggestion thatin regard to floral initiation vernalization also alters thesensitivity of the shoot apex to the flowering hormone(s). InLathyrus odoratus an hormonally based vernalization responseof considerable magnitude can be shown for day-neutral (dndn)lines, supporting the suggestion that vernalization also influencesthe level of a flower promotor. Lathyrus odoratus L., sweet pea, vernalization, flowering, branching, genotype, grafting     

Effect of Vernalization and Red Light Illumination of Seedlings of Bread Wheat (Triticum aestivum L.) on the Temperature Profile of cAMP Phosphodiesterase Activity

Phenotypic manifestations of Vrn(vernalization) and Ppd (photoperiod) genes responsible for transition of bread wheat Triticum aestivumL. to generative growth (flowering) are mutually related. Since the mechanism of phytochrome-induced photoperiodism involves the enzymes of cyclic adenosine monophosphate metabolism, and phosphodiesterase in particular, we tested involvement of phosphodiesterase in the process of winter wheat vernalization and formation of flowering competence in alternate wheat requiring a long photoperiod but no vernalization for its transition to flowering. We studied temperature dependence of phosphodiesterase activity in vernalized and unvernalized winter wheat on the one hand and in etiolated and red light illuminated seedlings of alternate wheat on the other hand. Short-term experiments demonstrated that red light illumination is similar to long photoperiod by the effect on the long-day plants. Both influences induced a pronounced inversion of the temperature profile of phosphodiesterase activity in the 28–45°C range. We propose that phosphodiesterase is involved in vernalization and can serve as a receptor of low temperature in winter wheat. Changed temperature profile is a radical control mechanism of phosphodiesterase activity in response to the influences (red light and vernalizing temperatures) responsible for competence of various bread wheat forms for generative growth.     

Role of gibberellins in stem elongation and flowering in radish

The relationship among gibberellins, CCC, vernalization, and photoperiod in the flowering response of radish, Raphanus sativus L., cv. Miyashige-sofuto, was studied. The optimal condition for flowering was vernalization and a 16-hour photoperiod; GA₃ had no additional effect. Gibberellin A₃ (60 μg total) was not able to induce flowering in nonvernalized plants grown on 8-hour days, but it did increase the percentage of nonvernalized plants that flowered under long days from 60 to 100.

Gibberellin content of vernalized seedlings increased within the first 24 hours after seedlings were transferred to the greenhouse. Content reached a peak in the first 4 days after transfer and thereafter remained constant. Essentially no gibberellin was found in 2 day-old non-vernalized (control) seedlings of comparable size to the vernalized ones. Gibberellin content in the controls reached a peak on the fourth day of growth in the greenhouse; thereafter, it decreased steadily.

Bolting was inhibited slightly by CCC when applied during vernalization; it was almost completely inhibited when CCC was applied after seed vernalization. Extraction experiments revealed that CCC actually reduced the gibberellin content when applied during or after vernalization. The dwarfing agent, however, had essentially no effect on flowering. We concluded that gibberellins likely play a direct role in bolting of `Miyashige-sofuto' radish, but probably are not directly functional in initiating flowering.

     

The Role of Leaves in the Perception of Vernalizing Temperatures in Sugar Beet

Annual and biennial sugar beet varieties require long days toinduce flowering but the biennial genotypes additionally requirevernalization. Previous research has suggested that the inabilityof non-vernalized biennial plants to flower can be explainedby a lack of competence of the leaves to respond to long days.In this study defoliation experiments were used to investigatewhich leaves could perceive long daylengths and, in particular,whether leaves initiated from a non-vernalized shoot apicalmeristem could perceive vernalizing temperatures and producea floral stimulus in long days. Annual and vernalized biennialplants flowered if young leaves (i.e. those formed during orafter vernalization) were kept on the plants, but they did notflower if only older expanded leaves (including those expandedprior to vernalization) were present. No evidence was obtainedto indicate that the older leaves contained inhibitors of floweringand it seems most likely that there is a decline in responsivenessto daylength with increasing leaf age. Exposure to vernalizingtemperatures accelerated flowering of the annual and was essentialfor flowering of the biennial. The presence of a single leafinitiated, but not expanded, prior to the transfer of biennialplants to vernalizing temperatures was sufficient to induceflowering. This indicates that expanding leaves do not needto be initiated from a vernalized apical meristem to becomecompetent to produce a floral stimulus in long days. Key words: Beta vulgaris L., sugar beet, vernalization, flowering     

Vernalization-induced flowering in wheat is mediated by a lectin-like gene VER2

A vernalization-related gene VER2 was isolated from winter wheat ( Triticum aestivum L.) using a differential screening approach. The deduced VER2 is a lectin-like protein of 300 amino acids, which contains the presence of a jacalin-like GWG domain. RNA in situ hybridization results demonstrated that VER2 gene expression is restricted to the marginal meristems of immature leaves in vernalized wheat seedlings. No hybridization signal was detected in the epidermal tissue and vascular bundles. However, "devernalization" resulted in the silencing of VER2 gene activity. The gene expression pattern of VER2 induced by jasmonate was similar to that induced by vernalization. Antisense inhibition of VER2 in transgenic wheat showed that heading and maturation time were delayed up to 6 weeks compared with non-transformed wheat and the pBI121empty-vector-transformed wheat. Tissue degeneration at the top of the spike was also noticed in the antisense inhibited transgenic wheat. These results suggest that VER2 plays an important role in vernalization signaling and spike development in winter wheat.     

Nucleic acid metabolism in cold-treated wheat embryos

The incorporation of ₃₂P into nucleic acid fractions separatedon a MAK column was compared for normally germinated and cold-treatedwheat embryos. ₃₂P accumulation in DNA fraction was decreasedby cold treatment, although that in the RNA fractions was slightlypromoted. The synthesis of the fraction, probably mRNA, elutedafter the peak of heavy rRNA was enhanced in cold-treated embryosand suppressed when the embryos were cold-treated in the presenceof 8-azaguanine, an inhibitor of vernalization. (Received May 2, 1975; )     

Factors affecting plant regeneration from immature inflorescence of two winter wheat cultivars

Inflorescence explants of two winter wheat cultivars, Triticum durum cv. Kızıltan-91 and T. aestivum cv. Bezostaja-01, were used to evaluate the effects of vernalization period of donor plants, callus age and medium composition on regeneration capacity. Donor plants were grown for 7 d and they were exposed to 4 °C for 1, 2, 3, 4, and 5 weeks. The maximum inflorescence formation was observed as 79 % at 4 weeks and 73 % at 5 weeks of vernalization period for Kızıltan-91 and Bezostaja-01, respectively. Among 6 different callus induction and regeneration mediums, I₁-R₁ and I₃-R₃ have to be the best responding mediums for Kızıltan-91 and Bezostaja-01, respectively. In Kızıltan-91, calli induced from donor plants, vernalized for 3 weeks, showed a significantly lower regeneration capacity than counterparts vernalized for 4 and 5 weeks. The highest regeneration capacity of 69 % was obtained from 6-week-old calli produced from 4 weeks vernalized Kızıltan-91 donor plants. In contrast to Kızıltan-91 cultures, the effects of vernalization period and callus age on regeneration capacity were not significant in Bezostaja-01 cultures. The maximum numbers of tillers were obtained from 6-and 15-week-old calli for Bezostaja-01 and Kızıltan-91, respectively. In contrast to vernalization period of donor plants, callus age had no effect on seed number.     

Vernalization can regulate flowering time through microRNA mechanism in Brassica rapa

Vernalization is an important process that regulates the floral transition in plants. MicroRNAs (miRNAs) are endogenous non‐coding small RNA (sRNA) molecules that function in plant growth and development. Despite that miRNAs related to flowering have previously been characterized, their roles in response to vernalization in pak‐choi (Brassica rapa ssp. chinensis) has never been studied. Here, two sRNA libraries from B. rapa leaves (vernalized and non‐vernalized plants) were constructed and sequenced. Two hundred eight known and 535 novel miRNAs were obtained, of which 20 known and 66 new miRNAs were significantly differentially expressed and considered as vernalization‐related miRNAs. The corresponding targets were predicted on the basic of sequence homology search. In addition, 11 miRNAs and eight targets were selected for real‐time quantitative PCR to confirm their expression profiles. Functional annotation of targets using gene ontology and Kyoto encyclopedia of genes and genomes results suggested that most targets were significantly enriched in the hormone signaling pathway. Moreover, a decreased indole‐3‐acetic acid (IAA) and an increased GA₃ hormone were detected after vernalization, indicating that the IAA and GA₃ might response to vernalization. These results indicated that vernalization regulates flowering through microRNA mechanism by affecting endogenous hormone level in B. rapa. This study provides useful insights of promising miRNAs candidates involved in vernalization in B. rapa, and facilitates further investigation of the miRNA‐mediated molecular mechanisms of vernalization in B. rapa.