In wheat, the transition of the inflorescence meristem to a terminal spikelet (IM→TS) determines the spikelet number per spike (SNS), an important yield component. In this study, we demonstrate that the plant-specific transcription factor LEAFY (LFY) physically and genetically interacts with WHEAT ORTHOLOG OF APO1 (WAPO1) to regulate SNS and floret development. Loss-of-function mutations in either or both genes result in significant and similar reductions in SNS, as a result of a reduction in the rate of spikelet meristem formation per day. SNS is also modulated by significant genetic interactions between LFY and the SQUAMOSA MADS-box genes VRN1 and FUL2, which promote the IM→TS transition. Single-molecule fluorescence in situ hybridization revealed a downregulation of LFY and upregulation of the SQUAMOSA MADS-box genes in the distal part of the developing spike during the IM→TS transition, supporting their opposite roles in the regulation of SNS in wheat. Concurrently, the overlap of LFY and WAPO1 transcription domains in the developing spikelets contributes to normal floret development. Understanding the genetic network regulating SNS is a necessary first step to engineer this important agronomic trait.

Flowering is a crucial stage for plant reproductive success; therefore, the regulation of plant flowering has been widely researched. Although multiple well-defined endogenous and exogenous flowering regulators have been reported, new ones are constantly being discovered. Here, we confirm that a novel plant growth regulator guvermectin (GV) induces early flowering in Arabidopsis. Interestingly, our genetic experiments newly demonstrated that WRKY41 and its homolog WRKY53 were involved in GV-accelerated flowering as positive flowering regulators. Overexpression of WRKY41 or WRKY53 resulted in an early flowering phenotype compared to the wild type (WT). In contrast, the w41/w53 double mutants showed a delay in GV-accelerated flowering. Gene expression analysis showed that flowering regulatory genes SOC1 and LFY were upregulated in GV-treated WT, 35S:WRKY41, and 35S:WRKY53 plants, but both declined in w41/w53 mutants with or without GV treatment. Meanwhile, biochemical assays confirmed that SOC1 and LFY were both direct targets of WRKY41 and WRKY53. Furthermore, the early flowering phenotype of 35S:WRKY41 lines was abolished in the soc1 or lfy background. Together, our results suggest that GV plays a function in promoting flowering, which was co-mediated by WRKY41 and WRKY53 acting as new flowering regulators by directly activating the transcription of SOC1 and LFY in Arabidopsis.

In flowering plants, the LEAFY (LFY) gene controls floral meristem activity. In early land plants such as mosses and ferns, it, however, has a minimum role in cell division and development of diploid sporophyte. Homology modeling, an accurate and efficient protein structure prediction method, was used to construct a 3D model of the LEAFY protein in nonflowering and flowering plants. The present study examines the following species: Charophyte green algae, Physcomitrella, Ceratopteris, Picea, and Arabidopsis, as they are the popularly used model organisms for developmental studies. LEAFY protein sequences from the model organisms were aligned by multiple sequence alignment. 3D models of the LEAFY protein from all the model organisms was constructed using the PHYRE2 program with 100% confidence, and the constructed models were evaluated using the MolProbity tool. On the basis of the conserved regions, Charophyte green algae shared 38-46% sequence similarity with Physcomitrella sp., 37-46% similarity with Ceratopteris sp., 33-41% similarity with Picea sp., and 32-38% similarity with Arabidopsis sp. The Motif Finder server identified the protein family domain FLO_LFY and LFY_SAM, whose function is floral meristem development. Secondary structure prediction analysis indicated that the LEAFY protein belongs to the alpha (α) protein class, which is stable against mutation and thus limits structural changes in the LEAFY protein. The study findings reveal two distinct clusters of the LFY gene from the common ancestor green algae. One cluster is present in nonflowering plants that include mosses, pteridophytes, and gymnosperms, and the other cluster is present in flowering plants that include orchids, monocots, dicots, and angiosperms.

CONCLUSIONS: The AtSF1-FLM module spatially controls temperature-dependent flowering by negatively regulating the expression of FT and LFY in the leaf and shoot apex, respectively. Alternative splicing mediated by various splicing factors is important for the regulation of plant growth and development. Our recent reports have shown that a temperature-dependent interaction between Arabidopsis thaliana splicing factor 1 (AtSF1) and FLOWERING LOCUS M (FLM) pre-mRNA introns controls the differential production of FLM-β transcripts at different temperatures, eventually resulting in temperature-responsive flowering. However, the molecular and genetic interactions between the AtSF1-FLM module and floral activator genes remain unknown. Here, we aimed to identify the interactions among AtSF1, FLM, FLOWERING LOCUS T (FT), and LEAFY (LFY) by performing molecular and genetic analyses. FT and TWIN SISTER OF FT (TSF) expression in atsf1-2 mutants significantly increased in the morning and middle of the night at 16 and 23 °C, respectively, under long-day conditions. In addition, ft mutation suppressed the early flowering of atsf1-2 and atsf1-2 flm-3 mutants and masked the temperature response of atsf1-2 flm-3 mutants, suggesting that FT is a downstream target gene of the AtSF1-FLM module. LFY expression significantly increased in the diurnal samples of atsf1-2 mutants and in the shoot apex regions of atsf1-2 ft-10 mutants at different temperatures. The chromatin immunoprecipitation (ChIP) assay revealed that FLM directly binds to the genomic regions of LFY but not of APETALA1 (AP1). Moreover, lfy mutation suppressed the early flowering of flm-3 mutants, suggesting that LFY is another target of the AtSF1-FLM module. Our results reveal that the AtSF1-FLM module spatially modulates temperature-dependent flowering by regulating FT and LFY expressions.

The important antimalarial drug artemisinin is biosynthesized and stored in Artemisia annua glandular trichomes and the artemisinin content correlates with trichome density; however, the factors affecting trichome development are largely unknown. Here, we demonstrate that the A. annua R2R3 MYB transcription factor TrichomeLess Regulator 1 (TLR1) negatively regulates trichome development. In A. annua, TLR1 overexpression lines had 44.7%-64.0% lower trichome density and 11.5%-49.4% lower artemisinin contents and TLR1-RNAi lines had 33%-93.3% higher trichome density and 32.2%-84.0% higher artemisinin contents compared with non-transgenic controls. TLR1 also negatively regulates the expression of anthocyanin biosynthetic pathway genes in A. annua. When heterologously expressed in Arabidopsis thaliana, TLR1 interacts with GLABROUS3a, positive regulator of trichome development, and represses trichome development. Yeast two-hybrid and pull-down assays indicated that TLR1 interacts with the WUSCHEL homeobox (WOX) protein AaWOX1, which interacts with the LEAFY-like transcription factor TLR2. TLR2 overexpression in Arabidopsis and A. annua showed that TLR2 reduces trichome development by reducing gibberellin levels. Furthermore, artemisinin contents were 19%-43% lower in TLR2-overexpressing A. annua plants compared to controls. These data indicate that TLR1 and TLR2 negatively regulate trichome density by lowering gibberellin levels and may enable approaches to enhance artemisinin yields.

Litchi (Litchi chinensis Sonn.) is an important subtropical fruit crop with high commercial value due to its high nutritional values and favorable tastes. However, irregular bearing attributed to unstable flowering is a major ongoing problem for litchi producers. Previous studies indicate that low-temperature is a key factor in litchi floral induction. In order to reveal the genetic and molecular mechanisms underlying the reproductive process in litchi, we had analyzed the transcriptome of buds before and after low-temperature induction using RNA-seq technology. A key flower bud differentiation associated gene, a homologue of FLORICAULA/LEAFY, was identified and named LcLFY (GenBank Accession No. KF008435). The cDNA sequence of LcLFY encodes a putative protein of 388 amino acids. To gain insight into the role of LcLFY, the temporal expression level of this gene was measured by real-time RT-PCR. LcLFY was highly expressed in flower buds and its expression correlated with the floral developmental stage. Heterologous expression of LcLFY in transgenic tobacco plants induced precocious flowering. Meantime, we investigated the sub-cellular localization of LcLFY. The LcLFY-Green fluorescent protein (GFP) fusion protein was found in the nucleus. The results suggest that LcLFY plays a pivotal role as a transcription factor in controlling the transition to flowering and in the development of floral organs in litchi.

OBJECTIVE: The morphological variability of the flower in angiosperms, combined with its relatively simple structure, makes it an excellent model to study cell specification and the establishment of morphogenetic patterns. Flowers are the products of floral meristems, which are determinate structures that generate four different types of floral organs before terminating. The precise organization of the flower in whorls, each defined by the identity and number of organs it contains, is controlled by a multi-layered network involving numerous transcriptional regulators. In particular, the AGAMOUS (AG) MADS domain-containing transcription factor plays a major role in controlling floral determinacy in Arabidopsis thaliana in addition to specifying reproductive organ identity. This study aims to characterize the genetic interactions between the ULTRAPETALA1 (ULT1) and LEAFY (LFY) transcriptional regulators during flower morphogenesis, with a focus on AG regulation.
METHODS: Genetic and molecular approaches were used to address the question of redundancy and reciprocal interdependency for the establishment of flower meristem initiation, identity and termination. In particular, the effects of loss of both ULT1 and LFY function were determined by analysing flower developmental phenotypes of double-mutant plants. The dependency of each factor on the other for activating developmental genes was also investigated in gain-of-function experiments.
RESULTS: The ULT1 and LFY pathways, while both activating AG expression in the centre of the flower meristem, functioned independently in floral meristem determinacy. Ectopic transcriptional activation by ULT1 of AG and AP3, another gene encoding a MADS domain-containing flower architect, did not depend on LFY function. Similarly, LFY did not require ULT1 function to ectopically determine floral fate.
CONCLUSIONS: The results indicate that the ULT1 and LFY pathways act separately in regulating identity and determinacy at the floral meristem. In particular, they independently induce AG expression in the centre of the flower to terminate meristem activity. A model is proposed whereby these independent contributions bring about a switch at the AG locus from an inactive to an active transcriptional state at the correct time and place during flower development.

The role of resource availability in determining the incidence of masting has been widely studied, but how floral transition and initiation are regulated by the resource level is unclear. We tested the hypothesis that floral transition is stimulated by high resource availabiltiy in Fagus crenata based on a new technique, the expression analyses of flowering genes. We isolated F. crenata orthologues of FLOWERING LOCUS T, LEAFY and APETALA1, and confirmed their functions using transgenic Arabidopsis thaliana. We monitored the gene expression levels for 5 years and detected a cycle of on and off years, which was correlated with fluctuations of the shoot-nitrogen concentration. Nitrogen fertilisation resulted in the significantly higher expression of flowering genes than the control, where all of the fertilised trees flowered, whereas the control did not. Our findings identified nitrogen as a key regulator of mast flowering, thereby providing new empirical evidence to support the resource budget model.

The FLOWERING LOCUS T (FT) gene plays crucial roles in regulating the transition from the vegetative phase to the reproductive phase. In this study, we isolated an FT homologous gene (denoted as ClFT) from Chrysanthemum lavandulifolium. The sequencing analysis indicated that the promoter of the ClFT gene contains many elements, such as light response, abscisic acid, drought-inducibility response and CIRCADIAN clock elements. The expression patterns of ClFT in different tissues/organs at different developmental stages and its responses to different photoperiods were observed. ClFT is expressed in all tested organs/tissues, with the highest expression level being observed in the leaves of plants with visible floral buds under the short day (SD) condition. Next, we studied the rhythmic expression of ClFT during different photoperiod treatments and found that the level of ClFT increases with additional hours of continuous dark. ClFT accumulates when the continuous dark period is 12 h, regardless of the duration of light period. The ectopic expression of the ClFT gene in wild type Arabidopsis (Col-0) results in early flowering, with high expression levels of endogenous LFY and SOC1 being observed in transgenic Arabidopsis. All results indicated that the ClFT gene plays an evolutionarily conserved role in promoting flowering in inductive short days in C. lavandulifolium and that this gene could serve as a vital target for the genetic manipulation of flowering time in chrysanthemums.

The wide variety of plant architectures is largely based on diverse and flexible modes of axillary shoot development. In Arabidopsis, floral transition (flowering) stimulates axillary bud development. The mechanism that links flowering and axillary bud development is, however, largely unknown. We recently showed that FLOWERING LOCUS T (FT) protein, which acts as florigen, promotes the phase transition of axillary meristems, whereas BRANCHED1 (BRC1) antagonizes the florigen action in axillary buds. Here, we present evidences for another possible role of florigen in axillary bud development. Ectopic overexpression of FT or another florigen gene TWIN SISTER OF FT (TSF) with LEAFY (LFY) induces ectopic buds at cotyledonary axils, confirming the previous proposal that these genes are involved in formation of axillary buds. Taken together with our previous report that florigen promotes axillary shoot elongation, we propose that florigen regulates axillary bud development at multiple stages to coordinate it with flowering in Arabidopsis.