Disrupting FKF1 homodimerization increases FT transcript levels in the evening by enhancing CO stabilization.

KEY MESSAGE: FKF1 dimerization is crucial for proper FT levels to fine-tune flowering time. Attenuating FKF1 homodimerization increased CO abundance by enhancing its COP1 binding, thereby accelerating flowering under long days. In Arabidopsis (Arabidopsis thaliana), the blue-light photoreceptor FKF1 (FLAVIN-BINDING, KELCH REPEAT, F-BOX 1) plays a key role in inducing the expression of FLOWERING LOCUS T (FT), encoding the main florigenic signal in plants, in the late afternoon under long-day conditions (LDs) by forming dimers with FT regulators. Although structural studies have unveiled a variant of FKF1 (FKF1 I160R) that disrupts homodimer formation in vitro, the mechanism by which disrupted FKF1 homodimer formation regulates flowering time remains elusive. In this study, we determined that the attenuation of FKF1 homodimer formation enhances FT expression in the evening by promoting the increased stability of CONSTANS (CO), a primary activator of FT, in the afternoon, thereby contributing to early flowering. In contrast to wild-type FKF1, introducing the FKF1 I160R variant into the fkf1 mutant led to increased FT expression under LDs. In addition, the FKF1 I160R variant exhibited diminished dimerization with FKF1, while its interaction with GIGANTEA (GI), a modulator of FKF1 function, was enhanced under LDs. Furthermore, the FKF1 I160R variant increased the level of CO in the afternoon under LDs by enhancing its binding to COP1, an E3 ubiquitin ligase responsible for CO degradation. These findings suggest that the regulation of FKF1 homodimerization and heterodimerization allows plants to finely adjust FT expression levels around dusk by modulating its interactions with GI and COP1.

Flowering time genes branching out.

Plants are sessile by nature, and as such they have evolved to sense changes in seasonality and their surrounding environment, and adapt to these changes. One prime example of this is the regulation of flowering time in angiosperms, which is precisely timed by the coordinative action of two proteins: FLOWERING LOCUS T (FT), and TERMINAL FLOWER 1 (TFL1). Both regulators are members of the PHOSPHATIDILETHANOLAMINE BINDING PROTEIN (PEBP) family of proteins. These regulatory proteins do not interact with DNA themselves, but instead interact with transcriptional regulators, such as FLOWERING LOCUS D (FD). FT and TFL1 were initially identified as key regulators of flowering time acting through binding with FD, however PEBP family members are also involved in shaping plant architecture and development. Next to that, PEBPs can interact with TCP transcriptional regulators, such as TEOSINTE BRANCHED 1 (TB1), a well-known regulator of plant architecture and key domestication related gene in many crops. Here, we review the role of PEBP proteins in flowering time, plant architecture and development. As these are also key yield-related traits, we will highlight fascinating examples from the model plant Arabidopsis as well as important food and feed crops such as, rice, barley, wheat, tomato, and potato.

FT-mediated thermal signalling regulates age-dependent inflorescence development in Arabidopsis thaliana.

Many plants show strong heteroblastic changes in the shape and size of organs as they transition from juvenile to reproductive age. Most attention has been focussed on heteroblastic development on leaves, but we wanted to understand heteroblastic changes in reproductive organ size. We therefore studied the progression of reproductive development in the model plant Arabidopsis thaliana, and found strong reductions in the size of flowers, fruit, seed, and internodes during development. These did not arise from correlative inhibition by older fruits, or from changes in inflorescence meristem size, but seemed to stem from changes in the size of floral organ primordia themselves. We hypothesised that environmental conditions might influence this heteroblastic pattern and found that the ambient temperature during organ initiation strongly influences organ size. We show that this temperature-dependent heteroblasty is dependent on FLOWERING LOCUS T (FT) mediated signal integration, adding to the repertoire of developmental processes regulated by this pathway. Our results demonstrate that rising global temperatures will not just affect fertility, as is widely described, but also the size and seed number of fruits produced. However, we also show that such effects are not hard-wired, and that selective breeding for FT expression during reproductive development could mitigate such effects.

FLOWERING LOCUS T genes control floral induction in lotus.

The transition to flowering is a vital process in the lotus life cycle that significantly impacts its ornamental value and seed production. However, the molecular basis of floral transition in lotus remains largely unknown. Here, eight homologous FLOWERING LOCUS T (FT) genes were initially characterized in lotus, which were designated as NnFT1-NnFT8. All of these genes were found to possess the conserved PEBP domain and exhibited high transcript levels in both lotus leaves and floral organs. The proNnFT:ß-glucuronidase (GUS) assay exhibited GUS staining in the vascular tissues of leaves. Furthermore, subcellular localization revealed that NnFT proteins were present in various cellular organelles, including the nucleus, cytoplasm, and endoplasmic reticulum. Overexpression of two NnFT homologs, NnFT2 and NnFT3, rescued the late flowering phenotype in the Arabidopsis ft-10 mutant, indicating the stimulative roles of NnFTs in floral induction. Moreover, NnFTs demonstrated interactions with a bZIP transcription factor, FLOWERING LOCUS D (NnFD), both in vitro and in vivo. These findings will not only deepen our understanding of the regulatory mechanism underlying lotus floral transition, but also provide valuable genetic resources for creating new lotus varieties with extended blooming periods using molecular strategies in the future.

Natural variation in the promoter of FLOWERING LOCUS T-LIKE 2 in pumpkin (Cucurbita moschata Duch.) is associated with flowering time under short-day conditions.

Late flowering is a serious bottleneck in pumpkin (Cucurbita moschata Duch.) agriculture production. Although key genes governing flowering time have been reported in many species, the regulatory network of flowering in pumpkin remains largely obscure, thereby impeding the resolution of industry-wide challenges associated with delayed fruit ripening in pumpkin cultivation. Here, we report an early flowering pumpkin germplasm accession (LXX-4). Using LXX-4 and a late flowering germplasm accession (HYM-9), we constructed an F2 segregation population. A significant difference in FLOWERING LOCUS T-LIKE 2 (FTL2) expression level was identified to be the causal factor of the flowering time trait discrepancy in LXX-4 and HYM-9. Moreover, we have shown that a 21 bp InDel in the FTL2 promoter was the key reason for the waxing and waning of its transcript level. The 21 bp deletion excluded a repressor-AGL19 and recruited activators-BBX7, WRKY40 and SVP to the FTL2 promoter in LXX-4. Together, our data add a useful element to our knowledge which could be used to simplify breeding efforts for early-maturing pumpkin.

Low temperature-mediated repression and far-red light-mediated induction determine morning FLOWERING LOCUS T expression levels.

In order to flower in the appropriate season, plants monitor light and temperature changes and alter downstream pathways that regulate florigen genes such as Arabidopsis (Arabidopsis thaliana) FLOWERING LOCUS T (FT). In Arabidopsis, FT messenger RNA levels peak in the morning and evening under natural long-day conditions (LDs). However, the regulatory mechanisms governing morning FT induction remain poorly understood. The morning FT peak is absent in typical laboratory LDs characterized by high red:far-red light (R:FR) ratios and constant temperatures. Here, we demonstrate that ZEITLUPE (ZTL) interacts with the FT repressors TARGET OF EATs (TOEs), thereby repressing morning FT expression in natural environments. Under LDs with simulated sunlight (R:FR = 1.0) and daily temperature cycles, which are natural LD-mimicking environmental conditions, FT transcript levels in the ztl mutant were high specifically in the morning, a pattern that was mirrored in the toe1 toe2 double mutant. Low night-to-morning temperatures increased the inhibitory effect of ZTL on morning FT expression by increasing ZTL protein levels early in the morning. Far-red light counteracted ZTL activity by decreasing its abundance (possibly via phytochrome A (phyA)) while increasing GIGANTEA (GI) levels and negatively affecting the formation of the ZTL-GI complex in the morning. Therefore, the phyA-mediated high-irradiance response and GI play pivotal roles in morning FT induction. Our findings suggest that the delicate balance between low temperature-mediated ZTL activity and the far-red light-mediated functions of phyA and GI offers plants flexibility in fine-tuning their flowering time by controlling FT expression in the morning.

Temperature-Regulated Flowering Locus T-Like Gene Coordinates the Spike Initiation in Phalaenopsis Orchid.

Phalaenopsis aphrodite can be induced to initiate spike growth and flowering by exposure to low ambient temperatures. However, the factors and mechanisms responsible for spike initiation in P. aphrodite remain largely unknown. In this study, we show that a repressor Flowing Locus T-like (FTL) gene, FTL, can act as a negative regulator of spike initiation in P. aphrodite. The mRNA transcripts of PaFTL are consistently high during high ambient temperature, thereby preventing premature spike initiation. However, during low ambient temperature, PaFTL expression falls while FT expression increases, allowing for spike initiation. Knock-down of PaFTL expression through virus-inducing gene silencing promoted spike initiation at 30/28°C. Moreover, PaFTL interacts with FLOWERING LOCUS D in a similar manner to FT to regulate downstream flowering initiation genes. Transgenic P. aphrodite plants exhibiting high expression of PaFTL do not undergo spike initiation, even when exposed to low ambient temperatures. These findings shed light on the flowering mechanisms in Phalaenopsis and provide new insights into how perennial plants govern spike initiation in response to temperature cues.

How to awaken a sleeping giant: antagonistic expression of Flowering locus T homologs and elements of the age-related pathway are associated with the flowering transition in Agave tequilana.

KEY MESSAGE: Antagonistic expression of Flowering locus T proteins and the ageing pathway via miRNAs and sugar metabolism regulate the initiation of flowering in A. tequilana. Flowering in commercial plantations of Agave tequilana signals that plants are ready to harvest for tequila production. However, time of flowering is often unpredictable and a detailed understanding of the process would be beneficial in the field, for breeding and for the development of future research. This report describes the functional analysis of A. tequilana FLOWERING LOCUS T (FT) genes by heterologous expression in A. thaliana and in situ hybridization in agave plants. The gene structures of the Agave tequilana FT family are also described and putative regulatory promoter elements were identified. Most Agave species have monocarpic, perennial life cycles that can last over 25 years during which plants do not respond to the normal environmental signals which induce flowering, suggesting that the ageing pathway as described in Arabidopsis may play an important role in determining flowering time in these species. Elements of this pathway were analyzed and in silico data is presented that supports the regulation of SQUAMOSA PROMOTER BINDING LIKE proteins (SPL), APETALA2 (AP2) proteins and members of Plant Glycoside Hydrolase Family 32 (PGHF32) by interactions with miRNAs 156, 172 and 164 during the initiation of flowering in A. tequilana.

Analysis of temperature effects on the protein accumulation of the FT-FD module using newly generated Arabidopsis transgenic plants.

Arabidopsis flowering is dependent on interactions between a component of the florigens FLOWERING LOCUS T (FT) and the basic leucine zipper (bZIP) transcription factor FD. These proteins form a complex that activates the genes required for flowering competence and integrates environmental cues, such as photoperiod and temperature. However, it remains largely unknown how FT and FD are regulated at the protein level. To address this, we created FT transgenic plants that express the N-terminal FLAG-tagged FT fusion protein under the control of its own promoter in ft mutant backgrounds. FT transgenic plants complemented the delayed flowering of the ft mutant and exhibited similar FT expression patterns to wild-type Col-0 plants in response to changes in photoperiod and temperature. Similarly, we generated FD transgenic plants in fd mutant backgrounds that express the N-terminal MYC-tagged FD fusion protein under the FD promoter, rescuing the late flowering phenotypes in the fd mutant. Using these transgenic plants, we investigated how temperature regulates the expression of FT and FD proteins. Temperature-dependent changes in FT and FD protein levels are primarily regulated at the transcript level, but protein-level temperature effects have also been observed to some extent. In addition, our examination of the expression patterns of FT and FD in different tissues revealed that similar to the spatial expression pattern of FT, FD mRNA was expressed in both the leaf and shoot apex, but FD protein was only detected in the apex, suggesting a regulatory mechanism that restricts FD protein expression in the leaf during the vegetative growth phase. These transgenic plants provided a valuable platform for investigating the role of the FT-FD module in flowering time regulation.

Differential gene expression during floral transition in pineapple.

Pineapple (Ananas comosus var. comosus) and ornamental bromeliads are commercially induced to flower by treatment with ethylene or its analogs. The apex is transformed from a vegetative to a floral meristem and shows morphological changes in 8 to 10 days, with flowers developing 8 to 10 weeks later. During eight sampling stages ranging from 6 h to 8 days after treatment, 7961 genes were found to exhibit differential expression (DE) after the application of ethylene. In the first 3 days after treatment, there was little change in ethylene synthesis or in the early stages of the ethylene response. Subsequently, three ethylene response transcription factors (ERTF) were up-regulated and the potential gene targets were predicted to be the positive flowering regulator CONSTANS-like 3 (CO), a WUSCHEL gene, two APETALA1/FRUITFULL (AP1/FUL) genes, an epidermal patterning gene, and a jasmonic acid synthesis gene. We confirm that pineapple has lost the flowering repressor FLOWERING LOCUS C. At the initial stages, the SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) was not significantly involved in this transition. Another WUSCHEL gene and a PHD homeobox transcription factor, though not apparent direct targets of ERTF, were up-regulated within a day of treatment, their predicted targets being the up-regulated CO, auxin response factors, SQUAMOSA, and histone H3 genes with suppression of abscisic acid response genes. The FLOWERING LOCUS T (FT), TERMINAL FLOWER (TFL), AGAMOUS-like APETELAR (AP2), and SEPETALA (SEP) increased rapidly within 2 to 3 days after ethylene treatment. Two FT genes were up-regulated at the apex and not at the leaf bases after treatment, suggesting that transport did not occur. These results indicated that the ethylene response in pineapple and possibly most bromeliads act directly to promote the vegetative to flower transition via APETALA1/FRUITFULL (AP1/FUL) and its interaction with SPL, FT, TFL, SEP, and AP2. A model based on AP2/ERTF DE and predicted DE target genes was developed to give focus to future research. The identified candidate genes are potential targets for genetic manipulation to determine their molecular role in flower transition.

A point mutation in MC06g1112 encoding FLOWERING LOCUS T decreases the first flower node in bitter gourd (Momordica charantia L.).

In Cucurbitaceae crops, the first flower node (FFN) is an important agronomic trait which can impact the onset of maturity, the production of female flowers, and yield. However, the gene responsible for regulating FFN in bitter gourd is unknown. Here, we used a gynoecious line (S156G) with low FFN as the female parent and a monoecious line (K8-201) with high FFN as the male parent to obtain F1 and F2 generations. Genetic analysis indicated that the low FFN trait was incompletely dominant over the high FFN trait. A major quantitative trait locus (QTL)-Mcffn and four minor effect QTLs-Mcffn1.1, Mcffn1.2, Mcffn1.3, and Mcffn1.4 were detected by whole-genome re-sequencing-based QTL mapping in the S156G×K8-201 F2 population (n=234) cultivated in autumn 2019. The Mcffn locus was further supported by molecular marker-based QTL mapping in three S156G×K8-201 F2 populations planted in autumn 2019 (n=234), autumn 2020 (n=192), and spring 2022 (n=205). Then, the Mcffn locus was fine-mapped into a 77.98-kb physical region on pseudochromosome MC06 using a large S156G×K8-201 F2 population (n=2,402). MC06g1112, which is a homolog of FLOWERING LOCUS T (FT), was considered as the most likely Mcffn candidate gene according to both expression and sequence variation analyses between parental lines. A point mutation (C277T) in MC06g1112, which results in a P93S amino acid mutation between parental lines, may be responsible for decreasing FFN in bitter gourd. Our findings provide a helpful resource for the molecular marker-assisted selective breeding of bitter gourd.

Hormonal Regulation and Stimulation Response of Jatropha curcas L. Homolog Overexpression on Tobacco Leaf Growth by Transcriptome Analysis.

The Flowering locus T (FT) gene encodes the florigen protein, which primarily regulates the flowering time in plants. Recent studies have shown that FT genes also significantly affect plant growth and development. The FT gene overexpression in plants promotes flowering and suppresses leaf and stem development. This study aimed to conduct a transcriptome analysis to investigate the multiple effects of Jatropha curcas L. homolog (JcFT) overexpression on leaf growth in tobacco plants. The findings revealed that JcFT overexpression affected various biological processes during leaf development, including plant hormone levels and signal transduction, lipid oxidation metabolism, terpenoid metabolism, and the jasmonic-acid-mediated signaling pathway. These results suggested that the effects of FT overexpression in plants were complex and multifaceted, and the combination of these factors might contribute to a reduction in the leaf size. This study comprehensively analyzed the effects of JcFT on leaf development at the transcriptome level and provided new insights into the function of FT and its homologous genes.

The FLOWERING LOCUS T LIKE 2-1 gene of Chenopodium triggers precocious flowering in Arabidopsis seedlings.

The FLOWERING LOCUS T (FT) gene is the essential integrator of flowering regulatory pathways in angiosperms. The paralogs of the FT gene may perform antagonistic functions, as exemplified by BvFT1, that suppresses flowering in Beta vulgaris, unlike the paralogous activator BvFT2. The roles of FT genes in other amaranths were less investigated. Here, we transformed Arabidopsis thaliana with the FLOWERING LOCUS T like (FTL) genes of Chenopodium ficifolium and found that both CfFTL1 and CfFTL2-1 accelerated flowering, despite having been the homologs of the Beta vulgaris floral promoter and suppressor, respectively. The floral promotive effect of CfFTL2-1 was so strong that it caused lethality when overexpressed under the 35S promoter. CfFTL2-1 placed in an inducible cassette accelerated flowering after induction with methoxyphenozide. The spontaneous induction of CfFTL2-1 led to precocious flowering in some primary transformants even without chemical induction. The CqFT2-1 homolog from Chenopodium quinoa had the same impact on viability and flowering as CfFTL2-1 when transferred to A. thaliana. After the FTL gene duplication in Amaranthaceae, the FTL1 copy maintained the role of floral activator. The second copy FTL2 underwent subsequent duplication and functional diversification, which enabled it to control the onset of flowering in amaranths to adapt to variable environments.

The FLOWERINGLOCUS T like 2­1 gene of Chenopodium ficifolium andChenopodium quinoa acts as a strong activator of flowering in Arabidopsis, triggering flowering at cotyledon stage and causing lethality when overexpressed.

Multiple transcriptome comparisons reveal the essential roles of FLOWERING LOCUS T in floral initiation and SOC1 and SVP in floral activation in blueberry.

The flowering mechanisms, especially chilling requirement-regulated flowering, in deciduous woody crops remain to be elucidated. Flower buds of northern highbush blueberry cultivar Aurora require approximately 1,000 chilling hours to bloom. Overexpression of a blueberry FLOWERING LOCUS T (VcFT) enabled precocious flowering of transgenic "Aurora" mainly in non-terminated apical buds during flower bud formation, meanwhile, most of the mature flower buds could not break until they received enough chilling hours. In this study, we highlighted two groups of differentially expressed genes (DEGs) in flower buds caused by VcFT overexpression (VcFT-OX) and full chilling. We compared the two groups of DEGs with a focus on flowering pathway genes. We found: 1) In non-chilled flower buds, VcFT-OX drove a high VcFT expression and repressed expression of a major MADS-box gene, blueberry SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (VcSOC1) resulting an increased VcFT/VcSOC1 expression ratio; 2) In fully chilled flower buds that are ready to break, the chilling upregulated VcSOC1 expression in non-transgenic "Aurora" and repressed VcFT expression in VcFT-OX "Aurora", and each resulted in a decreased ratio of VcFT to VcSOC1; additionally, expression of a blueberry SHORT VEGETATIVE PHASE (VcSVP) was upregulated in chilled flower buds of both transgenic and non-transgenic' "Aurora". Together with additional analysis of VcFT and VcSOC1 in the transcriptome data of other genotypes and tissues, we provide evidence to support that VcFT expression plays a significant role in promoting floral initiation and that VcSOC1 expression is a key floral activator. We thus propose a new hypothesis on blueberry flowering mechanism, of which the ratios of VcFT-to-VcSOC1 at transcript levels in the flowering pathways determine flower bud formation and bud breaking. Generally, an increased VcFT/VcSOC1 ratio or increased VcSOC1 in leaf promotes precocious flowering and flower bud formation, and a decreased VcFT/VcSOC1 ratio with increased VcSOC1 in fully chilled flower buds contributes to flower bud breaking.

Single-nucleus RNA sequencing and mRNA hybridization indicate key bud events and LcFT1 and LcTFL1-2 mRNA transportability during floral transition in litchi.

In flowering plants, floral induction signals intersect at the shoot apex to modulate meristem determinacy and growth form. Here, we report a single-nucleus RNA sequence analysis of litchi apical buds at different developmental stages. A total of 41 641 nuclei expressing 21 402 genes were analyzed, revealing 35 cell clusters corresponding to 12 broad populations. We identify genes associated with floral transition and propose a model that profiles the key events associated with litchi floral meristem identity by analyzing 567 identified floral meristem cells at single cell resolution. Interestingly, single-nucleus RNA-sequencing data indicated that all putative FT and TFL1 genes were not expressed in bud nuclei, but significant expression was detected in bud samples by RT-PCR. Based on the expression patterns and gene silencing results, we highlight the critical role of LcTFL1-2 in inhibiting flowering and propose that the LcFT1/LcTFL1-2 expression ratio may determine the success of floral transition. In addition, the transport of LcFT1 and LcTFL1-2 mRNA from the leaf to the shoot apical meristem is proposed based on in situ and dot-blot hybridization results. These findings allow a more comprehensive understanding of the molecular events during the litchi floral transition, as well as the identification of new regulators.

Spectral light quality regulates the morphogenesis, architecture, and flowering in pepper (Capsicum annuum L.).

Transparent plastic films with poor light transmittance seriously affect the mass composition of visible light in many greenhouses, which leads to the reduction of photosynthesis in vegetable crops. Understanding the regulatory mechanisms of monochromatic light in the vegetative and reproductive growth of vegetable crops is of great importance for the application of light-emitting diodes (LEDs) in the greenhouse. In this study, three monochromatic light treatments (red-, green- and blue-light) were simulated by using LEDs to explore light quality-dependent regulation from the stage of seedling to flowering in pepper (Capsicum annuum L.). The results showed that light quality-dependent regulation guides the growth and morphogenesis in pepper plants. Red- and blue-light played opposite roles in determining the plant height, stomatal density, axillary bud growth, photosynthetic characteristics, flowering time and hormone metabolism, while green light treatment resulted in taller plants and fewer branches, which was similar to the red-light treatment. The weighted correlation network analysis (WGCNA) based on mRNA-seq results revealed that the two modules named "MEred" and "MEmidnightblue" were positively correlated with red- and blue-light treatment, respectively, exhibiting high correlations with the traits such as plant hormone content, branching and flowering. Moreover, our results suggest that the light response factor ELONGATED HYPOCOTYL 5 (HY5) is essential for blue light-induced plant growth and development by regulating photosynthesis in pepper plants. Hence, this study uncovers crucial molecular mechanisms of how light quality determines the morphogenesis, architecture, and flowering in pepper plants, thus providing a basic concept of manipulating light quality to regulate pepper plant growth and flowering under greenhouse conditions.

Omics Profiles of Non-GM Tubers from Transgrafted Potato with a GM Scion.

"Transgrafting" is a grafting procedure whereby a transgenic plant body is grafted to a non-transgenic plant body. It is a novel plant breeding technology that allows non-transgenic plants to obtain benefits usually conferred to transgenic plants. Many plants regulate flowering by perceiving the day-length cycle via expression of FLOWERING LOCUS T (FT) in the leaves. The resulting FT protein is translocated to the shoot apical meristem via the phloem. In potato plants, FT is involved in the promotion of tuber formation. Here we investigated the effects of a genetically modified (GM) scion on the edible parts of the non-GM rootstock by using potato plants transformed with StSP6A, a novel potato homolog of the FT gene. Scions prepared from GM or control (wild-type) potato plants were grafted to non-GM potato rootstocks; these were designated as TN and NN plants, respectively. After tuber harvest, we observed no significant differences in potato yield between TN and NN plants. Transcriptomic analysis revealed that only one gene-with unknown function-was differentially expressed between TN and NN plants. Subsequent proteomic analysis indicated that several members of protease inhibitor families, known as anti-nutritional factors in potato, were slightly more abundant in TN plants. Metabolomic analysis revealed a slight increase in metabolite abundance in NN plants, but we observed no difference in the accumulation of steroid glycoalkaloids, toxic metabolites found in potato. Finally, we found that TN and NN plants did not differ in nutrient composition. Taken together, these results indicate that FT expression in scions had a limited effect on the metabolism of non-transgenic potato tubers.

A straightforward strategy for reducing variability in flowering time at warm ambient temperatures.

Ambient temperature is one of the major environmental factors affecting flowering. As the temperature rises, most plants, including Arabidopsis, flower more rapidly. In addition, phenotypic variability in flowering time tends to increase at warm ambient temperatures. The increased variability of flowering time at warm temperatures prevents accurate flowering time measurements, particularly when evaluating the flowering time of Arabidopsis plants under short-day conditions in order to restrict the photoperiodic effect. Here, we propose a simple method for reducing the variability of flowering time at warm temperatures. Instead of growing plants at different temperatures from germination, the strategy of first vegetative growth at cool temperatures and then shifting to warm temperatures allows plants to respond more stably and robustly to warm temperatures. Consistent with flowering time measurements, plants grown under the modified growth condition exhibited higher levels of FLOWERING LOCUS T (FT) gene expression than plants grown exclusively at warm temperatures. This approach enables more precise thermo-response studies of flowering time control in Arabidopsis.

Multiplex CRISPR/Cas9-mediated mutagenesis of alfalfa FLOWERING LOCUS Ta1 (MsFTa1) leads to delayed flowering time with improved forage biomass yield and quality.

Alfalfa (Medicago sativa L.) is a perennial flowering plant in the legume family that is widely cultivated as a forage crop for its high yield, forage quality and related agricultural and economic benefits. Alfalfa is a photoperiod sensitive long-day (LD) plant that can accomplish its vegetative and reproductive phases in a short period of time. However, rapid flowering can compromise forage biomass yield and quality. Here, we attempted to delay flowering in alfalfa using multiplex CRISPR/Cas9-mediated mutagenesis of FLOWERING LOCUS Ta1 (MsFTa1), a key floral integrator and activator gene. Four guide RNAs (gRNAs) were designed and clustered in a polycistronic tRNA-gRNA system and introduced into alfalfa by Agrobacterium-mediated transformation. Ninety-six putative mutant lines were identified by gene sequencing and characterized for delayed flowering time and related desirable agronomic traits. Phenotype assessment of flowering time under LD conditions identified 22 independent mutant lines with delayed flowering compared to the control. Six independent Msfta1 lines containing mutations in all four copies of MsFTa1 accumulated significantly higher forage biomass yield, with increases of up to 78% in fresh weight and 76% in dry weight compared to controls. Depending on the harvesting schemes, many of these lines also had reduced lignin, acid detergent fibre (ADF) and neutral detergent fibre (NDF) content and significantly higher crude protein (CP) and mineral contents compared to control plants, especially in the stems. These CRISPR/Cas9-edited Msfta1 mutants could be introduced in alfalfa breeding programmes to generate elite transgene-free alfalfa cultivars with improved forage biomass yield and quality.