Effect of Photoperiod on the Regulation of Wheat Vernalization Genes VRN1 and VRN2

Wheat is usually classified as a long day (LD) plant because most varieties flower earlier when exposed to longer days. In addition to LD, winter wheats require a long exposure to low temperatures (vernalization) to become competent for flowering. Here we show that in some genotypes this vernalization requirement can be replaced by interrupting the LD treatment by 6 weeks of short day (SD), and that this replacement is associated with the SD down-regulation of the VRN2 flowering repressor. In addition, we found that SD down-regulation of VRN2 at room temperature is not followed by the up-regulation of the meristem identity gene VRN1 until plants are transferred to LD. This result contrasts with the VRN1 up-regulation observed after the VRN2 down-regulation by vernalization, suggesting the existence of a second VRN1 repressor. Analysis of natural VRN1 mutants indicated that a CArG-box located in the VRN1 promoter is the most likely regulatory site for the interaction with this second repressor. Up-regulation of VRN1 under SD in accessions carrying mutations in the CArG-box resulted in an earlier initiation of spike development, compared to other genotypes. However, even the genotypes with CArG box mutations required LD for a normal and timely spike development. The SD acceleration of flowering was observed in photoperiod sensitive winter varieties. Since vernalization requirement and photoperiod sensitivity are ancestral traits in Triticeae species we suggest that wheat was initially a SD–LD plant and that strong selection pressures during domestication and breeding resulted in the modification of this dual regulation. The down-regulation of the VRN2 repressor by SD is likely part of the mechanism associated with the SD–LD regulation of flowering in photoperiod sensitive winter wheat. These authors contributed equally to this work     

CONSTANS delays Arabidopsis flowering under short days

Long days (LD) promote flowering of Arabidopsis thaliana compared with short days (SD) by activating the photoperiodic pathway. Here we show that growth under very‐SD (3 h) or darkness (on sucrose) also accelerates flowering on a biological scale, indicating that SD actively repress flowering compared with very‐SD. CONSTANS (CO) repressed flowering under SD, and the early flowering of co under SD required FLOWERING LOCUS T (FT). FT was expressed at a basal level in the leaves under SD, but these levels were not enhanced in co. This indicates that the action of CO in A. thaliana is not the mirror image of the action of its homologue in rice. In the apex, CO enhanced the expression of TERMINAL FLOWER 1 (TFL1) around the time when FT expression is important to promote flowering. Under SD, the tfl1 mutation was epistatic to co and in turn ft was epistatic to tfl1. These observations are consistent with the long‐standing but not demonstrated model where CO can inhibit FT induction of flowering by affecting TFL1 expression.     

Validation of the <Emphasis Type="Italic">VRN-H2</Emphasis>/<Emphasis Type="Italic">VRN-H1</Emphasis> epistatic model in barley reveals that intron length variation in <Emphasis Type="Italic">VRN-H1</Emphasis> may account for a continuum of vernalization sensitivity

The epistatic interaction of alleles at the VRN-H1 and VRN-H2 loci determines vernalization sensitivity in barley. To validate the current molecular model for the two-locus epistasis, we crossed homozygous vernalization-insensitive plants harboring a predicted “winter type” allele at either VRN-H1 (Dicktoo) or VRN-H2 (Oregon Wolfe Barley Dominant), or at both VRN-H (Calicuchima-sib) loci and measured the flowering time of unvernalized F₂ progeny under long-day photoperiod. We assessed whether the spring growth habit of Calicuchima-sib is an exception to the two-locus epistatic model or contains novel “spring” alleles at VRN-H1 (HvBM5A) and/or VRN-H2 (ZCCT-H) by determining allele sequence variants at these loci and their effects relative to growth habit. We found that (a) progeny with predicted “winter type” alleles at both VRN-H1 and VRN-H2 alleles exhibited an extremely delayed flowering (i.e. vernalization-sensitive) phenotype in two out of the three F₂ populations, (b) sequence flanking the vernalization critical region of HvBM5A intron 1 likely influences degree of vernalization sensitivity, (c) a winter habit is retained when ZCCT-Ha has been deleted, and (d) the ZCCT-H genes have higher levels of allelic polymorphism than other winterhardiness regulatory genes. Our results validate the model explaining the epistatic interaction of VRN-H2 and VRN-H1 under long-day conditions, demonstrate recovery of vernalization-sensitive progeny from crosses of vernalization-insensitive genotypes, show that intron length variation in VRN-H1 may account for a continuum of vernalization sensitivity, and provide molecular markers that are accurate predictors of “winter vs spring type” alleles at the VRN-H loci.     

Genes conferring late flowering inArabidopsis thaliana

Three naturally occurring late flowering, vernalization responsive ecotypes ofArabidopsis thaliana, Pitztal, Innsbruck and Kiruna-2, were each crossed with the early flowering ecotypes of Landsbergerecta, Columbia and Niederzenz. Analysis of the subsequent generations suggested that late flowering in Kiruna-2 is recessive and mainly determined by a single, late flowering gene. This late flowering gene is not, however, the same as that in any of the late flowering mutants generated in the Landsbergerecta background. Both Pitztal and Innsbruck appear to contain the same dominant gene which confers late flowering to these ecotypes. The early flowering parents Niederzenz and Landsberg both contain genes which modify the phenotype of this dominant late flowering locus, causing F₁ plants to flower either earlier (Landsberg) or later (Niederzenz) than the late parent. Mapping of the dominant late flowering locus from Pitztal demonstrated that late flowering co-segregated with an RFLP marker from one end of chromosome 4. This is a similar position to that ofFLA, the gene responsible for late flowering of theArabidopsis ecotypes Sf-2 and Le-O.     

QTL analysis of flowering time inArabidopsis thaliana

Quantitative trait loci (QTL) analyses based on restriction fragment length polymorphism maps have been used to resolve the genetic control of flowering time in a cross between twoArabidopsis thaliana ecotypes H51 and Landsbergerecta, differing widely in flowering time. Five quantitative trait loci affecting flowering time were identified in this cross (RLN1-5), four of which are located in regions containing mutations or loci previously identified as conferring a late-flowering phenotype. One of these loci is coincident with theFRI locus identified as the major determinant for late flowering and vernalization responsiveness in theArabidopsis ecotype Stockholm.RLN5, which maps to the lower half of chromosome five (between markers mi69 and m233), only affected flowering time significantly under short day conditions following a vernalization period. The late-flowering phenotype of H51 compared to Landsbergerecta was due to alleles conferring late flowering at only two of the five loci. At the three other loci, H51 possessed alleles conferring early flowering in comparison to those of Landsbergerecta. Combinations of alleles conferring early and late flowering from both parents accounted for the transgressive segregation of flowering time observed within the F₂ population. Three QTL,RLN1,RLN2 andRLN3 displayed significant genotype-by-environment interactions for flowering time. A significant interaction between alleles atRLN3 andRLN4 was detected.     

Low-temperature tolerance and genetic potential in wheat (Triticum aestivum L.): response to photoperiod, vernalization, and plant development

It is frequently observed that winter habit types are more low-temperature (LT) tolerant than spring habit types. This raises the question of whether this is due to pleiotropic effects of the vernalization loci or to the linkage of LT-tolerance genes to these vernalization loci. Reciprocal near-isogenic lines (NILs) for alleles at the Vrn-A1 locus, Vrn-A1 and vrn-A1, determining spring and winter habit respectively, in two diverse genetic backgrounds of wheat (Triticum aestivum L.) were used to separate the effects of vernalization, photoperiod, and development on identical, or near identical, genetic backgrounds. The vrn-A1 allele in the winter lines allowed full expression of genotype dependent LT tolerance potential. The winter allele (vrn-A1) in a very cold tolerant genetic background resulted in 11°C, or a 2.4-fold, greater LT tolerance compared to the spring allele. Similarly, the delay in development caused by short-day (SD) versus long-day (LD) photoperiod in the identical spring habit NIL resulted in an 8.5°C or 2.1-fold, increase in LT tolerance. The duration of time in early developmental stages was shown to underlie full expression of genetic LT-tolerance potential. Therefore, pleiotropic effects of the vernalization loci can explain the association of LT tolerance and winter habit irrespective of either the proposed closely linked Fr-A1 or the more distant Fr-A2 LT-tolerance QTLs. Plant development progressively reduced LT-acclimation ability, particularly after the main shoot meristem had advanced to the double ridge reproductive growth stage. The Vrn-1 genes, or other members of the flowering induction pathway, are discussed as possible candidates for involvement in LT-tolerance repression.     

Mapping loci controlling vernalization requirement in Brassica rapa

Brassica cultivars are classified as biennial or annual based on their requirement for a period of cold treatment (vernalization) to induce flowering. Genes controlling the vernalization requirement were identified in a Brassica rapa F₂ population derived from a cross between an annual and a biennial oilseed cultivar by using an RFLP linkage map and quantitative trait locus (QTL) analysis of flowering time in F₃ lines. Two genomic regions were strongly associated with variation for flowering time of unvernalized plants and alleles from the biennial parent in these regions delayed flowering. These QTLs had no significant effect on flowering time after plants were vernalized for 6 weeks, suggesting that they control flowering time through the requirement for vernalization. The two B. rapa linkage groups containing these QTLs had RFLP loci in common with two B. napus linkage groups that were shown previously to contain QTLs for flowering time. An RFLP locus detected by the cold-induced gene COR6.6 cloned from Arabidopsis thaliana mapped very near to one of the B. rapa QTLs for flowering time.     

The relationship between vernalization-and photoperiodically-regulated genes and the development of frost tolerance in wheat and barley

The review summarizes the level of current knowledge of impacts of vernalization and photoperiod on the induction and maintenance of frost tolerance (FrT) in wheat and barley. The phenomenon of vernalization is briefly described and the major vernalization (VRN) loci are characterised. Vernalization requirement and the three major growth habits of Triticeae (facultative, winter and spring) are defined on the basis of the two-locus VRN-2/VRN-1 epistatic model. Major photoperiodically regulated genes, which influence the transition to flowering, are characterised and their interactions with VRN genes are briefly discussed. The phenomenon of induction of FrT during the process of cold acclimation (CA) is described and the major cold-induced Cor/Lea genes are listed. Important regulatory mechanisms, i.e., CBF pathway, controlling the expression of Cor/Lea genes under cold, are discussed. The major loci affecting the development of FrT in Triticeae, the Fr loci, are characterised. In conclusion, current progress in this research field is summarized and new questions arising in the area are formulated.     

Interactions of temperature and photoperiod in the control of flowering of latitudinal and altitudinal populations of wild strawberry (Fragaria vesca)

Floral induction and development requirements of a range of latitudinal and altitudinal Norwegian populations of the wild strawberry Fragaria vesca L. have been studied in controlled environments. Rooted runner plants were exposed to a range of photoperiods and temperatures for 5 weeks for floral induction and then transferred to long day (LD) at 20°C for flower development. A pronounced interaction of temperature and photoperiod was shown in the control of flowering. At 9°C, flowers were initiated in both short day (SD) and LD conditions, at 15 and 18°C in SD only, whereas no initiation took place at 21°C regardless of daylength conditions. The critical photoperiod for SD floral induction was about 16 h and 14 h at 15 and 18°C, respectively, the induction being incomplete at 18°C. The optimal condition for floral induction was SD at 15°C. A minimum of 4 weeks of exposure to such optimal conditions was required. Although the populations varied significantly in their flowering performance, no clinal relationship was present between latitude of origin and critical photoperiod. Flower development of SD-induced plants was only marginally advanced by LD conditions, while inflorescence elongation and runnering were strongly enhanced by LD at this stage. The main shift in these responses took place at photoperiods between 16 and 17 h. Unlike all other populations studied, a high-latitude population from 70°N ('Alta') had an obligatory vernalization requirement. Although flowering and fruiting in its native Subarctic environment and after overwintering in the field in south Norway, this population did not flower in the laboratory in the absence of vernalization, even with 10 or 15 weeks of exposure to SD at 9°C. Flowering performance in the field likewise indicated a vernalization requirement of this high-latitude population.     

The alleles at the E1 locus impact the expression pattern of two soybean FT-like genes shown to induce flowering in Arabidopsis

A small gene family of phosphatidyl ethanolamine-binding proteins (PEBP) has been shown to function as key regulators in flowering; in Arabidopsis thaliana the FT protein promotes flowering whilst the closely related TFL1 protein represses flowering. Control of flowering time in soybean [Glycine max (L.) Merrill] is important for geographic adaptation and maximizing yield. Soybean breeders have identified a series of loci, the E-genes, that control photoperiod-mediated flowering time, yet how these loci control flowering is poorly understood. The objectives of this study were to evaluate the expression of GmFT-like genes in the E1 near-isogenic line (NIL) background. Of the 20 closely related PEBP proteins in the soybean genome, ten are similar to the Arabidopsis FT protein. Expression analysis of these ten GmFT-like genes confirmed that only two are detectable in the conditions tested. Further analysis of these two genes in the E1 NILs grown under short-day (SD) and long-day (LD) conditions showed a diurnal expression and tissue specificity expression commensurate with soybean flowering time under SD and LD conditions, suggesting that these were good candidates for flowering induction in soybean. Arabidopsis ft mutant lines flowered early when transformed with the two soybean genes, suggesting that the soybean genes can complement the Arabidopsis FT function. Flowering time in E1 NILs is consistent with the differential expression of the two GmFT-like genes under SD and LD conditions, suggesting that the E1 locus, at least in part, impacts time to flowering through the regulation of soybean FT expression.     

Mutagenesis of GmFT2a and GmFT5a mediated by CRISPR/Cas9 contributes for expanding the regional adaptability of soybean

Flowering time is a key agronomic trait that directly influences the successful adaptation of soybean (Glycine max) to diverse latitudes and farming systems. GmFT2a and GmFT5a have been extensively identified as flowering activators and integrators in soybean. Here, we identified two quantitative trait loci (QTLs) regions harbouring GmFT2a and GmFT5a, respectively, associated with different genetic effects on flowering under different photoperiods. We analysed the flowering time of transgenic plants overexpressing GmFT2a or GmFT5a, ft2a mutants, ft5a mutants and ft2aft5a double mutants under long‐day (LD) and short‐day (SD) conditions. We confirmed that GmFT2a and GmFT5a are not redundant, they collectively regulate flowering time, and the effect of GmFT2a is more prominent than that of GmFT5a under SD conditions whereas GmFT5a has more significant effects than GmFT2a under LD conditions. GmFT5a, not GmFT2a, was essential for soybean to adapt to high latitude regions. The ft2aft5a double mutants showed late flowering by about 31.3 days under SD conditions and produced significantly increased numbers of pods and seeds per plant compared to the wild type. We speculate that these mutants may have enormous yield potential for the tropics. In addition, we examined the sequences of these two loci in 202 soybean accessions and investigated the flowering phenotypes, geographical distributions and maturity groups within major haplotypes. These results will contribute to soybean breeding and regional adaptability.     

Non-reversion of Impatiens in the absence of meristem commitment

Purple-flowered plants of Impatiens balsamina maintained floral development on transfer from inductive short days (SD) to long days (LD), a treatment in which red-flowered plants of Impatiens are known to revert to leaf production. An investigation into the non-reverting nature of purple-flowered plants was carried out to establish whether these plants achieved meristem commitment or whether their non-reverting state was controlled by the leaves. When the leaves that had unfolded during the inductive SD treatment were removed at the time of transfer to LD, the purple-flowered plant did revert. This result suggests that, as in red-flowered Impatiens, meristem commitment is absent, but that purple-flowered plants maintain flowering in LD conditions because of a more permanent supply of signal from their leaves than occurs in red-flowered plants. A working hypothesis is proposed to explain how a signal from the leaves can retain a controlling role during flower development.Key words: Floral commitment, Impatiens, floral reversion, floricaula.      

EMF Genes Interact with Late-Flowering Genes to Regulate Arabidopsis Shoot Development

To investigate the genetic mechanisms regulating the transitionfrom vegetative to reproductive phase in Arabidopsis, doublemutants between two embryonic flower (emf) and 12 differentlate-flowering mutants were constructed and analyzed. Doublemutants in all combinations displayed the emf phenotypes withoutforming rosettes during early development; however, clear variationsbetween different double mutants were observed during late development,fwa significantly enhanced the vegetative property of both emfmutants by producing a high number of sessile leaves withoutany further reproductive growth in emf1 fwa double mutants.It also produced numerous leaf-like flower structures similarto those in leafy ap1 double mutant in emf1 fwa double mutants.Nine late-flowering mutants, ft, fca, ld, fd, fpa, fe, fy, fha,and fve, caused different degrees of increase in the numberof sessile leaves, the size of inflorescence, and the numberof flowers only in weak emf1 and emf2 mutant alleles background.Two late-flowering mutants, co and gi, however, had no effecton either emf1 and emf2 mutant alleles in double mutants. Ourresults suggest that FWA function in distinct pathways fromboth EMF genes to regulate flower competence by activating geneswhich specify floral meristem identity. CO and GI negativelyregulate both EMF genes, whereas the other nine late-floweringgenes may interact with EMF genes directly or indirectly toregulate shoot maturation in Arabidopsis. ¹ To whom correspondence should be addressed.     

Control of barley (Hordeum vulgare L.) development and senescence by the interaction between a chromosome six grain protein content locus, day length, and vernalization

Regulatory processes controlling traits such as anthesis timing and whole-plant senescence are of primary importance for reproductive success and for crop quality and yield. It has previously been demonstrated that the presence of alleles associated with high grain protein content (GPC) at a locus on barley chromosome six leads to accelerated leaf senescence, and to strong (>10-fold) up-regulation of several genes which may be involved in senescence control. One of these genes (coding for a glycine-rich RNA-binding protein termed HvGR-RBP1) exhibits a high degree of similarity to Arabidopsis glycine-rich RNA-binding protein 7 (AtGRP7), which has been demonstrated to accelerate flowering under both long-day (LD) and short-day (SD) conditions, but not after vernalization. Development of near-isogenic barley lines, differing in the allelic state of the GPC locus, was compared from the seedling stage to maturity under both SD and LD and after vernalization under LD. Intriguingly, pre-anthesis plant development [measured by leaf emergence timing and pre-anthesis (sequential) leaf senescence] was enhanced in high-GPC germplasm. Differences were more pronounced under SD than under LD, but were eliminated by vernalization, associating observed effects with floral induction pathways. By contrast, differences in post-anthesis flag leaf and whole-plant senescence between low- and high-GPC germplasm persisted under all tested conditions, indicating that the GPC locus, possibly through HvGR-RBP1, impacts on both developmental stages. Detailed molecular characterization of this experimental system may allow the dissection of cross-talk between signalling pathways controlling early plant and floral development on one side, and leaf/whole-plant senescence on the other side.     

Inhibition imposed by developing flowers on further flower-bud initiation in Chamelaucium uncinatum Schau

In Chamelaucium uncinatum, an Australian woody perennial, flower initiation ceases under continuous inductive short-day (SD) conditions after the first flowering flush. The developing flowers were found to be the prime cause of the cessation in flower initiation. Removal of flowering shoots or lowers as soon as the buds appeared resulted in continuous flower formation. Pruning the plants below the young flower buds at the same stage also caused increased flower formation at the tips of the new growth. If pruning was delayed until flower buds were approx. 3 mm in diameter, however, nor further flower initiation took place and the plants, although still under inductive conditions, shifted to vegetative growth. The inhibiting factor is translocated from one branch to another. At least a six-week rest period (a vegetative growth period under long-day conditions) is needed before the plants are able to respond to further SD stimuli.Abbreviations LD long day - SD short day - SE standard error     

Natural variation and CRISPR/Cas9‐mediated mutation in GmPRR37 affect photoperiodic flowering and contribute to regional adaptation of soybean

Flowering time is a critical determinant of the geographic distribution and regional adaptability of soybean (Glycine max) and is strongly regulated by photoperiod and temperature. In this study, quantitative trait locus (QTL) mapping and subsequent candidate gene analysis revealed that GmPRR37, encoding a pseudo‐response regulator protein, is responsible for the major QTL qFT12‐2, which was identified from a population of 308 recombinant inbred lines (RILs) derived from a cross between a very late‐flowering soybean cultivar, ‘Zigongdongdou (ZGDD)’, and an extremely early‐flowering cultivar, ‘Heihe27 (HH27)’, in multiple environments. Comparative analysis of parental sequencing data confirmed that HH27 contains a non‐sense mutation that causes the loss of the CCT domain in the GmPRR37 protein. CRISPR/Cas9‐induced Gmprr37‐ZGDD mutants in soybean exhibited early flowering under natural long‐day (NLD) conditions. Overexpression of GmPRR37 significantly delayed the flowering of transgenic soybean plants compared with wild‐type under long photoperiod conditions. In addition, both the knockout and overexpression of GmPRR37 in soybean showed no significant phenotypic alterations in flowering time under short‐day (SD) conditions. Furthermore, GmPRR37 down‐regulated the expression of the flowering‐promoting FT homologues GmFT2a and GmFT5a, and up‐regulated flowering‐inhibiting FT homologue GmFT1a expression under long‐day (LD) conditions. We analysed haplotypes of GmPRR37 among 180 cultivars collected across China and found natural Gmprr37 mutants flower earlier and enable soybean to be cultivated at higher latitudes. This study demonstrates that GmPRR37 controls soybean photoperiodic flowering and provides opportunities to breed optimized cultivars with adaptation to specific regions and farming systems.     

Flowering and dwarfism induced by DNA demethylation in Pharbitis nil

Flowering and dwarfism induced by 5‐azacytidine and zebularine, which both cause DNA demethylation, were studied in a short‐day (SD) plant Pharbitis nil (synonym Ipomoea nil), var. Violet whose photoinduced flowering state does not last for a long period of time. The DNA demethylating reagents induced flowering under non‐inductive long‐day (LD) conditions. The flower‐inducing effect of 5‐azacytidine did not last for a long period of time, and the plants reverted to vegetative growth. The progeny of the plants that were induced to flower by DNA demethylation did not flower under the non‐inductive photoperiodic conditions. These results suggest that the flowering‐related genes were activated by DNA demethylation and then remethylated again in the progeny. The DNA demethylation also induced dwarfism. The dwarfism did not last for a long period of time, was not heritable and was overcome by gibberellin A₃ but not by t‐zeatin or kinetin. The change in the genome‐wide methylation state was examined by methylation‐sensitive amplified fragment length polymorphism (MS‐AFLP) analysis. The analysis detected many more polymorphic fragments between the DNA samples isolated from the cotyledons treated with SD than from the cotyledons under LD conditions, indicating that the DNA methylation state was altered by photoperiodic conditions. Seven LD‐specific fragments were extracted from the gel of the MS‐AFLP and were sequenced. One of these fragments was highly homologous with the genes encoding ribosomal proteins.     

Genetic interactions among late-flowering mutants of Arabidopsis.

Flowering time in Arabidopsis is controlled by a large number of genes, identified by induced mutations. Forty-two double mutants involving 10 of these loci were obtained and analyzed for their flowering behavior under long-day conditions, with and without vernalization, and under short-day conditions. The genetic interactions between the various mutants proved to be complex, although a major epistatic group (called group A) could be identified corresponding to the mutants, which are relatively insensitive to vernalization and daylength. In contrast, the genetic behavior of the mutants much more responsive to these environmental factors (group B) is more complex. The vernalization responsiveness of the group B mutants did not compensate for the lateness of the group A mutants. This indicated that these genes do not control vernalization sensitivity as such, but provide a factor that becomes limiting in short days. The classification of these mutants in different physiological groups is discussed in relation to the detected genetic interactions, and based on these interactions a more detailed model of their role in flowering initiation is proposed.     

FLD interacts with genes that affect different developmental phase transitions to regulate Arabidopsis shoot development

A new fld mutant allele, fld-2, which significantly delayed flowering, was isolated and characterized in Arabidopsis thaliana. Even under long-day conditions after more than 100 days in the greenhouse, the majority of fld-2 mutant plants had not bolted. In addition, mutant inflorescences produced more than 10 co-florescences that were subtended by a high number of rosette-like leaves before giving rise to flowers. The late-flowering phenotype of the fld-2 mutation could be partially overcome by both vernalization and GA treatment but it was not influenced by 5-azaC treatment. Phenotypic analyses of double mutants indicated that fld-2 is epistatic to early flowering mutants elf1, elf2 and elf3. In addition, fld-2 could enhance vegetative characteristics in embryonic flower 1 (emf1) mutants by causing many small sessile leaves in fld-2 emf1 double mutants. The relief of the terminal flower 1 (tfl1) mutant phenotype in fld-2 tfl1 double mutants, and the enhancement of leafy (lfy) and apetala1 (ap1) mutant phenotypes in fld-2 lfy and fld-2 ap1 double mutants, suggest that FLD is also likely to be involved in the floral transition. Our results strongly suggest that the FLD gene plays a key role in regulating the reproductive competence of the shoot and results in different developmental phase transitions in Arabidopsis.