Can a late bloomer become an early bird? Tools for flowering time adjustment

The transition from the vegetative to reproductive stage followed by inflorescence is a critical step in plant life; therefore, studies of the genes that influence flowering time have always been of great interest to scientists. Flowering is a process controlled by many genes interacting mutually in a genetic network, and several hypothesis and models of flowering have been suggested so far. Plants in temperate climatic conditions must respond mainly to changes in the day length (photoperiod) and unfavourable winter temperatures. To avoid flowering before winter, some plants exploit a specific mechanism called vernalization. This review summarises current achievements in the study of genes controlling flowering in the dicot model species thale cress (Arabidopsis thaliana), as well as in monocot model species rice (Oryza sativa) and temperate cereals such as barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.). The control of flowering in crops is an attractive target for modern plant breeding efforts aiming to prepare locally well-adapted cultivars. The recent progress in genomics revealed the importance of minor-effect genes (QTLs) and natural allelic variation of genes for fine-tuning flowering and better cultivar adaptation. We briefly describe the up-to-date technologies and approaches that scientists may employ and we also indicate how these modern biotechnological tools and “-omics” can expand our knowledge of flowering in agronomically important crops.     

Copy number variation affecting the Photoperiod-B1 and Vernalization-A1 genes is associated with altered flowering time in wheat (Triticum aestivum)

The timing of flowering during the year is an important adaptive character affecting reproductive success in plants and is critical to crop yield. Flowering time has been extensively manipulated in crops such as wheat (Triticum aestivum L.) during domestication, and this enables them to grow productively in a wide range of environments. Several major genes controlling flowering time have been identified in wheat with mutant alleles having sequence changes such as insertions, deletions or point mutations. We investigated genetic variants in commercial varieties of wheat that regulate flowering by altering photoperiod response (Ppd-B1 alleles) or vernalization requirement (Vrn-A1 alleles) and for which no candidate mutation was found within the gene sequence. Genetic and genomic approaches showed that in both cases alleles conferring altered flowering time had an increased copy number of the gene and altered gene expression. Alleles with an increased copy number of Ppd-B1 confer an early flowering day neutral phenotype and have arisen independently at least twice. Plants with an increased copy number of Vrn-A1 have an increased requirement for vernalization so that longer periods of cold are required to potentiate flowering. The results suggest that copy number variation (CNV) plays a significant role in wheat adaptation.     

Comparative mapping, genomic structure, and expression analysis of eight pseudo-response regulator genes in Brassica rapa

Circadian clocks regulate plant growth and development in response to environmental factors. In this function, clocks influence the adaptation of species to changes in location or climate. Circadian-clock genes have been subject of intense study in models such as Arabidopsis thaliana but the results may not necessarily reflect clock functions in species with polyploid genomes, such as Brassica species, that include multiple copies of clock-related genes. The triplicate genome of Brassica rapa retains high sequence-level co-linearity with Arabidopsis genomes. In B. rapa we had previously identified five orthologs of the five known Arabidopsis pseudo-response regulator (PRR) genes that are key regulators of the circadian clock in this species. Three of these B. rapa genes, BrPRR1, BrPPR5, and BrPPR7, are present in two copies each in the B. rapa genome, for a total of eight B. rapa PRR (BrPRR) orthologs. We have now determined sequences and expression characteristics of the eight BrPRR genes and mapped their positions in the B. rapa genome. Although both members of each paralogous pair exhibited the same expression pattern, some variation in their gene structures was apparent. The BrPRR genes are tightly linked to several flowering genes. The knowledge about genome location, copy number variation and structural diversity of these B. rapa clock genes will improve our understanding of clock-related functions in this important crop. This will facilitate the development of Brassica crops for optimal growth in new environments and under changing conditions.     

Conserved Function of Core Clock Proteins in the Gymnosperm Norway Spruce (Picea abies L. Karst)

From studies of the circadian clock in the plant model species Arabidopsis (Arabidopsis thaliana), a number of important properties and components have emerged. These include the genes CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), GIGANTEA (GI), ZEITLUPE (ZTL) and TIMING OF CAB EXPRESSION 1 (TOC1 also known as PSEUDO-RESPONSE REGULATOR 1 (PRR1)) that via gene expression feedback loops participate in the circadian clock. Here, we present results from ectopic expression of four Norway spruce (Picea abies) putative homologs (PaCCA1, PaGI, PaZTL and PaPRR1) in Arabidopsis, their flowering time, circadian period length, red light response phenotypes and their effect on endogenous clock genes were assessed. For PaCCA1-ox and PaZTL-ox the results were consistent with Arabidopsis lines overexpressing the corresponding Arabidopsis genes. For PaGI consistent results were obtained when expressed in the gi2 mutant, while PaGI and PaPRR1 expressed in wild type did not display the expected phenotypes. These results suggest that protein function of PaCCA1, PaGI and PaZTL are at least partly conserved compared to Arabidopsis homologs, however further studies are needed to reveal the protein function of PaPRR1. Our data suggest that components of the three-loop network typical of the circadian clock in angiosperms were present before the split of gymnosperms and angiosperms.     

Heat shock protein 101 (HSP101) promotes flowering under nonstress conditions

Heat shock proteins (HSPs) are stress-responsive proteins that are conserved across all organisms. Heat shock protein 101 (HSP101) has an important role in thermotolerance owing to its chaperone activity. However, if and how it functions in development under nonstress conditions is not yet known. By using physiological, molecular, and genetic methods, we investigated the role of HSP101 in the control of flowering in Arabidopsis (Arabidopsis thaliana (L.) Heynh.) under nonstress conditions. Knockout and overexpression of HSP101 cause late and early flowering, respectively. Late flowering can be restored by rescue of HSP101. HSP101 regulates the expression of genes involved in the six known flowering pathways; the most negatively regulated genes are FLOWERING LOCUS C (FLC) and SHORT VEGETATIVE PHASE (SVP); downstream integrators of the flowering pathways are positively regulated. The late-flowering phenotype of loss-of-HSP101 mutants is suppressed by both the mutations of FLC and SVP. The responses of flowering time to exogenous signals do not change in HSP101 mutants. HSP101 is also found in nonspecific regions according to subcellular localization. We found that HSP101 promotes flowering under nonstress conditions and that this promotion depends on FLC and SVP. Our data suggest that this promotion could occur through a multiple gene regulation mechanism.

Heat shock protein 101 promotes flowering under nonstress conditions in Arabidopsis (Arabidopsis thaliana (L.) Heynh.).     

Hybridisation‐based target enrichment of phenology genes to dissect the genetic basis of yield and adaptation in barley

Barley (Hordeum vulgare L.) is a major cereal grain widely used for livestock feed, brewing malts and human food. Grain yield is the most important breeding target for genetic improvement and largely depends on optimal timing of flowering. Little is known about the allelic diversity of genes that underlie flowering time in domesticated barley, the genetic changes that have occurred during breeding, and their impact on yield and adaptation. Here, we report a comprehensive genomic assessment of a worldwide collection of 895 barley accessions based on the targeted resequencing of phenology genes. A versatile target‐capture method was used to detect genome‐wide polymorphisms in a panel of 174 flowering time‐related genes, chosen based on prior knowledge from barley, rice and Arabidopsis thaliana. Association studies identified novel polymorphisms that accounted for observed phenotypic variation in phenology and grain yield, and explained improvements in adaptation as a result of historical breeding of Australian barley cultivars. We found that 50% of genetic variants associated with grain yield, and 67% of the plant height variation was also associated with phenology. The precise identification of favourable alleles provides a genomic basis to improve barley yield traits and to enhance adaptation for specific production areas.     

Molecular mechanisms for the photoperiodic regulation of flowering in soybean

Photoperiodic flowering is one of the most important factors affecting regional adaptation and yield in soybean (Glycine max). Plant adaptation to long-day conditions at higher latitudes requires early flowering and a reduction or loss of photoperiod sensitivity; adaptation to short-day conditions at lower latitudes involves delayed flowering, which prolongs vegetative growth for maximum yield potential. Due to the influence of numerous major loci and quantitative trait loci (QTLs), soybean has broad adaptability across latitudes. Forward genetic approaches have uncovered the molecular basis for several of these major maturity genes and QTLs. Moreover, the molecular characterization of orthologs of Arabidopsis thaliana flowering genes has enriched our understanding of the photoperiodic flowering pathway in soybean. Building on early insights into the importance of the photoreceptor phytochrome A, several circadian clock components have been integrated into the genetic network controlling flowering in soybean: E1, a repressor of FLOWERING LOCUS T orthologs, plays a central role in this network. Here, we provide an overview of recent progress in elucidating photoperiodic flowering in soybean, how it contributes to our fundamental understanding of flowering time control, and how this information could be used for molecular design and breeding of high-yielding soybean cultivars.     

What Has Natural Variation Taught Us about Plant Development,Physiology, and Adaptation?

Nearly 100 genes and functional polymorphisms underlying natural variation in plant development and physiology have been identified. In crop plants, these include genes involved in domestication traits, such as those related to plant architecture, fruit and seed structure and morphology, as well as yield and quality traits improved by subsequent crop breeding. In wild plants, comparable traits have been dissected mainly in Arabidopsis thaliana. In this review, we discuss the major contributions of the analysis of natural variation to our understanding of plant development and physiology, focusing in particular on the timing of germination and flowering, plant growth and morphology, primary metabolism, and mineral accumulation. Overall, functional polymorphisms appear in all types of genes and gene regions, and they may have multiple mutational causes. However, understanding this diversity in relation to adaptation and environmental variation is a challenge for which tools are now available.     

Photoperiodism dynamics during the domestication and improvement of soybean

Soybean (Glycine max) is a facultative short-day plant with a sensitive photoperiod perception and reaction system, which allows it to adjust its physiological state and gene regulatory networks to seasonal and diurnal changes in environmental conditions. In the past few decades, soybean cultivation has spread from East Asia to areas throughout the world. Biologists and breeders must now confront the challenge of understanding the molecular mechanism of soybean photoperiodism and improving agronomic traits to enable this important crop to adapt to geographical and environmental changes. In this review, we summarize the genetic regulatory network underlying photoperiodic responses in soybean. Genomic and genetic studies have revealed that the circadian clock, in conjunction with the light perception pathways, regulates photoperiodic flowering. Here, we provide an annotated list of 844 candidate flowering genes in soybean, with their putative biological functions. Many photoperiod-related genes have been intensively selected during domestication and crop improvement. Finally, we describe recent progress in engineering photoperiod-responsive genes for improving agronomic traits to enhance geographic adaptation in soybean, as well as future prospects for research on soybean photoperiodic responses.     

Comparative Genomics of Flowering Time Pathways Using Brachypodium distachyon as a Model for the Temperate Grasses

Brachypodium distachyon (Brachypodium) is a model for the temperate grasses which include important cereals such as barley, wheat and oats. Comparison of the Brachypodium genome (accession Bd21) with those of the model dicot Arabidopsis thaliana and the tropical cereal rice (Oryza sativa) provides an opportunity to compare and contrast genetic pathways controlling important traits. We analysed the homologies of genes controlling the induction of flowering using pathways curated in Arabidopsis Reactome as a starting point. Pathways include those detecting and responding to the environmental cues of day length (photoperiod) and extended periods of low temperature (vernalization). Variation in these responses has been selected during cereal domestication, providing an interesting comparison with the wild genome of Brachypodium. Brachypodium Bd21 has well conserved homologues of circadian clock, photoperiod pathway and autonomous pathway genes defined in Arabidopsis and homologues of vernalization pathway genes defined in cereals with the exception of VRN2 which was absent. Bd21 also lacked a member of the CO family (CO3). In both cases flanking genes were conserved showing that these genes are deleted in at least this accession. Segmental duplication explains the presence of two CO-like genes in temperate cereals, of which one (Hd1) is retained in rice, and explains many differences in gene family structure between grasses and Arabidopsis. The conserved fine structure of duplications shows that they largely evolved to their present structure before the divergence of the rice and Brachypodium. Of four flowering-time genes found in rice but absent in Arabidopsis, two were found in Bd21 (Id1, OsMADS51) and two were absent (Ghd7, Ehd1). Overall, results suggest that an ancient core photoperiod pathway promoting flowering via the induction of FT has been modified by the recruitment of additional lineage specific pathways that promote or repress FT expression.     

The inheritance of flowering time inAmaranthus species

The inheritance of flowering time was studied in a cross between an early flowering weedy species,Amaranthus retroftexus L., and a late flowering related crop species,A. cruentus L. Segregation ratios were scored in four successive backcrosses of the hybrid to the crop species (BC1 to BC4) and in backcross-derived generations (BC2F2, BC2F3) grown under 8-hr and 16-hr photoperiods and 30/20°C day/night temperatures in growth chambers, and under field and greenhouse environments during summer months at Davis. One major gene for earliness (Ea), dominant over late flowering, was identified conclusively under the 8-hr regime and in some genetic backgrounds under field conditions. Based on segregation patterns within individual progenies and the observed transgressive segregation, a model of three genes seemed to explain all of the ratios consistently. Evidence of segregation distortion at theEa locus, resulting in the deficiency of early individuals, and some role of epistatic interactions among the three postulated major genes requires further testing based on more isogenic and specific progeny materials. Flowering time was closely associated with plant form, inflorescence development, and numerous other traits which differentiate the crop and weed species. This association provides interesting insights into evolutionary changes under domestication and dispersal from lower to higher latitudes.     

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.     

Two new clock proteins, LWD1 and LWD2, regulate Arabidopsis photoperiodic flowering

The “light” signal from the environment sets the circadian clock to regulate multiple physiological processes for optimal rhythmic growth and development. One such process is the control of flowering time by photoperiod perception in plants. In Arabidopsis (Arabidopsis thaliana), the flowering time is determined by the correct interconnection of light input and signal output by the circadian clock. The identification of additional clock proteins will help to better dissect the complex nature of the circadian clock in Arabidopsis. Here, we show LIGHT-REGULATED WD1 (LWD1)/LWD2 as new clock proteins involved in photoperiod control. The lwd1lwd2 double mutant has an early-flowering phenotype, contributed by the significant phase shift of CONSTANS (CO), and, therefore, an increased expression of FLOWERING LOCUS T (FT) before dusk. Under entrainment conditions, the expression phase of oscillator (CIRCADIAN CLOCK ASSOCIATED1 [CCA1], LATE ELONGATED HYPOCOTYL [LHY], TIMING OF CAB EXPRESSION1 [TOC1], and EARLY FLOWERING4 [ELF4]) and output (GIGANTEA, FLAVIN-BINDING, KELCH REPEAT, F-BOX1, CYCLING DOF FACTOR1, CO, and FT) genes in the photoperiod pathway shifts approximately 3 h forward in the lwd1lwd2 double mutant. Both the oscillator (CCA1, LHY, TOC1, and ELF4) and output (COLD, CIRCADIAN RHYTHM, AND RNA BINDING2 and CHLOROPHYLL A/B-BINDING PROTEIN2) genes have a short period length in the lwd1lwd2 double mutant. Our data imply that LWD1/LWD2 proteins function in close proximity to or within the circadian clock for photoperiodic flowering control.     

Flowering phenology as a core domestication trait in soybean

正Flowering time variation in soybean is well characterized within domesticated germplasms and is critical for modern production, but its importance during domestication is unclear. Recently, Lu et al.(Nature Genetics, 2020) reported that two homeologous pseudo-response-regulator genes, Tof12 and Tof11, were sequentially selected in early soybean evolution for ancient flowering time adaptation and intensification of crop cultivation.tary     

DIE NEUTRALIS and LATE BLOOMER 1 Contribute to Regulation of the Pea Circadian Clock

The DIE NEUTRALIS (DNE) locus in garden pea (Pisum sativum) was previously shown to inhibit flowering under noninductive short-day conditions and to affect a graft-transmissible flowering signal. In this study, we establish that DNE has a role in diurnal and/or circadian regulation of several clock genes, including the pea GIGANTEA (GI) ortholog LATE BLOOMER 1 (LATE1) and orthologs of the Arabidopsis thaliana genes LATE ELONGATED HYPOCOTYL and TIMING OF CHLOROPHYLL A/B BINDING PROTEIN EXPRESSION 1. We also confirm that LATE1 participates in the clock and provide evidence that DNE is the ortholog of Arabidopsis EARLY FLOWERING4 (ELF4). Circadian rhythms of clock gene expression in wild-type plants under constant light were weaker in pea than in Arabidopsis, and a number of differences were also seen in the effects of both DNE/ELF4 and LATE1/GI on clock gene expression. Grafting studies suggest that DNE controls flowering at least in part through a LATE1-dependent mobile stimulus, and dne mutants show elevated expression of a FLOWERING LOCUS T homolog under short-day conditions. However, the early flowering of the dne mutant is not associated with altered expression of a previously described CONSTANS-like gene. Collectively, our results characterize the clock system and reveal its importance for photoperiod responsiveness in a model legume.     

Overexpression of Wheat <i>TaELF3-1BL</i> Delays Flowering in Arabidopsis

EARLY FLOWERING 3 (ELF3), a light zeitnehmer (time-taker) gene, regulates circadian rhythm and photoperiodic flowering in Arabidopsis, rice, and barley. The three orthologs of ELF3 (TaELF3-1AL, TaELF3-1BL, and TaELF3-1DL) have been identified in wheat too, and one gene, TaELF3-1DL, has been associated with heading date. However, the basic characteristics of these three genes and the roles of the other two genes, TaELF3-1BL and, TaELF3-1AL, remain unknown. Therefore, the present study obtained the coding sequences of the three orthologs (TaELF3-1AL, TaELF3-1BL, and TaELF3-1DL) of ELF3 from bread wheat and characterized them and investigated the role of TaELF3-1BL in Arabidopsis. Protein sequence comparison revealed similarities among the three TaELF3 genes of wheat; however, they were different from the Arabidopsis ELF3. Real-time quantitative PCR revealed TaELF3 expression in all wheat tissues tested, with the highest expression in young spikes; the three genes showed rhythmic expression patterns also. Furthermore, the overexpression of the TaELF3-1BL gene in Arabidopsis delayed flowering, indicating their importance in flowering. Subsequent overexpression of TaELF3-1BL in the Arabidopsis ELF3 nonfunctional mutant (elf3 mutant) eliminated its early flowering phenotype, and slightly delayed flowering. The wild-type Arabidopsis overexpressing TaELF3-1BL demonstrated reduced expression levels of flowering-related genes, such as CONSTANS (AtCO), FLOWERING LOCUS T (AtFT), and GIGANTEA (AtGI). Thus, the study characterized the three TaELF3 genes and associated TaELF3-1BL with flowering in Arabidopsis, suggesting a role in regulating flowering in wheat too. These findings provide a basis for further research on TaELF3 functions in wheat.     

Mapping-by-Sequencing Identifies HvPHYTOCHROME C as a Candidate Gene for the early maturity 5 Locus Modulating the Circadian Clock and Photoperiodic Flowering in Barley

Phytochromes play an important role in light signaling and photoperiodic control of flowering time in plants. Here we propose that the red/far-red light photoreceptor HvPHYTOCHROME C (HvPHYC), carrying a mutation in a conserved region of the GAF domain, is a candidate underlying the early maturity 5 locus in barley (Hordeum vulgare L.). We fine mapped the gene using a mapping-by-sequencing approach applied on the whole-exome capture data from bulked early flowering segregants derived from a backcross of the Bowman(eam5) introgression line. We demonstrate that eam5 disrupts circadian expression of clock genes. Moreover, it interacts with the major photoperiod response gene Ppd-H1 to accelerate flowering under noninductive short days. Our results suggest that HvPHYC participates in transmission of light signals to the circadian clock and thus modulates light-dependent processes such as photoperiodic regulation of flowering.