Floral Organ Identity Genes Research Articles

Single nucleotide polymorphisms in SEPALLATA 2 underlie fruit length variation in cucurbits.

Complete disruption of critical genes is generally accompanied by severe growth and developmental defects, which dramatically hinder its utilization in crop breeding. Identifying subtle changes, such as single-nucleotide polymorphisms (SNPs), in critical genes that specifically modulate a favorable trait is a prerequisite to fulfill breeding potential. Here, we found 2 SNPs in the E-class floral organ identity gene cucumber (Cucumis sativus) SEPALLATA2 (CsSEP2) that specifically regulate fruit length. Haplotype (HAP) 1 (8G2667A) and HAP2 (8G2667T) exist in natural populations, whereas HAP3 (8A2667T) is induced by ethyl methanesulfonate mutagenesis. Phenotypic characterization of 4 near-isogenic lines and a mutant line showed that HAP2 fruits are significantly longer than those of HAP1, and those of HAP3 are 37.8% longer than HAP2 fruit. The increasing fruit length in HAP1-3 was caused by a decreasing inhibitory effect on CRABS CLAW (CsCRC) transcription (a reported positive regulator of fruit length), resulting in enhanced cell expansion. Moreover, a 7638G/A-SNP in melon (Cucumis melo) CmSEP2 modulates fruit length in a natural melon population via the conserved SEP2-CRC module. Our findings provide a strategy for utilizing essential regulators with pleiotropic effects during crop breeding.

BdRCN4, a Brachypodium distachyon TFL1 homologue, is involved in regulation of apical meristem fate.

In higher plants, the shift from vegetative to reproductive development is governed by complex interplay of internal and external signals. TERMINALFLOWER1 (TFL1) plays a crucial role in the regulation of flowering time and inflorescence architecture in Arabidopsis thaliana. This study aimed to explore the function of BdRCN4, a homolog of TFL1 in Brachypodium distachyon, through functional analyses in mutant and transgenic plants. The results revealed that overexpression of BdRCN4 in B. distachyon leads to an extended vegetative phase and reduced production of spikelets. Similar results were found in A. thaliana, where constitutive expression of BdRCN4 promoted a delay in flowering time, followed by the development of hypervegetative shoots, with no flowers or siliques produced. Our results suggest that BdRCN4 acts as a flowering repressor analogous to TFL1, negatively regulating AP1, but no LFY expression. To further validate this hypothesis, a 35S::LFY-GR co-transformation approach on 35::BdRCN4 lines was performed. Remarkably, AP1 expression levels and flower formation were restored to normal in co-transformed plants when treated with dexamethasone. Although further molecular studies will be necessary, the evidence in B. distachyon support the idea that a balance between LFY and BdRCN4/TFL1 seems to be essential for activating AP1 expression and initiating floral organ identity gene expression. This study also demonstrates interesting conservation through the molecular pathways that regulate flowering meristem transition and identity across the evolution of monocot and dicot plants.

A 49-bp deletion of PmAP2L results in a double flower phenotype in Prunus mume.

The double flower is an important trait with substantial ornamental value. While mutations in PETALOSA TOE-type or AG (AGAMOUS) genes play a crucial role in enhancing petal number in ornamental plants, the complete mechanism underlying the formation of double flowers remains to be fully elucidated. Through the application of bulked segregant analysis (BSA), we identified a novel gene, APETALA2-like (PmAP2L), characterized by a 49-bp deletion in double-flowered Prunus mume. β-Glucuronidase (GUS) staining and luciferase reporter assays confirmed that the 49-bp deletion in PmAP2L reduced its binding with Pmu-miRNA172a. Phylogenetic analysis and microsynteny analysis suggested that PmAP2L was not a PETALOSA TOE-type gene, and it might be a new gene controlling the formation of double flower in P. mume. Subsequently, overexpression of PmAP2L-D in tobacco led to a significant rise in the number of stamens and the conversion of stamens to petals. Furthermore, silencing of the homologue of RC5G0530900 in rose significantly reduced the number of petals. Using transient gene expression in P. mume flower buds, we determined the functional differences between PmAP2L-D and PmAP2-S in controlling flower development. Meanwhile, DNA-affinity purification sequencing (DAP-seq), yeast hybrid assays and luciferase reporter assays indicated that PmAP2L negatively regulated the floral organ identity genes by forming a repressor complex with PmTPL and PmHDA6/19. Overall, these findings indicate that the variation in PmAP2L is associated with differences in the regulation of genes responsible for floral organ identity, providing new insights into the double-flower trait and double-flower breeding in plants.

Dynamic cytological and transcriptomic analyses provide novel insights into the mechanisms of sex determination in Castanea henryi.

Castanea henryi is a monoecious woody food tree species whose yield and industrialization potential are limited by its low female-to-male flower ratio. Here, the male flowers on the male inflorescence of C. henryi were converted to female flowers by triple applications of exogenous cytokinin (CK) (N-(2-chloro-4-pyridyl)-N'-phenylurea, CPPU). To study the role of exogenous CK in flower sex determination, cytological and transcriptomic analyses were performed on samples from the five stages after CK treatment. Cytological analysis showed that stage 3 (nine days after the last CK treatment) was the critical stage in the differential development of the pistil primordium and stamen primordium. On this basis, one key module and two modules with significant positive correlations with stage 3 were identified by weighted gene co-expression network analysis (WGCNA), combined with transcriptome data. The CK and GA biosynthesis- and signaling-related genes, three transcription factor (TF) families, and 11 floral organ identity genes were identified in the related modules. In particular, the TFs WRKY47, ERF021, and MYB4, and floral organ identity genes AGL11/15, DEF, and SEP1 with large differences are considered to be critical regulators of sex determination in C. henryi. Based on these results, a genetic regulatory network for exogenous CK in the sex determination of flowers in C. henryi is proposed. This study contributes to the understanding of the role of CK in the sex regulation of flowers and provides new insights into the regulatory network of sex determination in C. henryi.

The regulation of chromatin configuration at AGAMOUS locus by LFR-SYD-containing complex is critical for reproductive organ development in Arabidopsis.

Switch defective/sucrose non-fermentable (SWI/SNF) chromatin remodeling complexes are evolutionarily conserved, multi-subunit machinery that play vital roles in the regulation of gene expression by controlling nucleosome positioning and occupancy. However, little is known about the subunit composition of SPLAYED (SYD)-containing SWI/SNF complexes in plants. Here, we show that the Arabidopsis thaliana Leaf and Flower Related (LFR) is a subunit of SYD-containing SWI/SNF complexes. LFR interacts directly with multiple SWI/SNF subunits, including the catalytic ATPase subunit SYD, in vitro and in vivo. Phenotypic analyses of lfr-2 mutant flowers revealed that LFR is important for proper filament and pistil development, resembling the function of SYD. Transcriptome profiling revealed that LFR and SYD shared a subset of co-regulated genes. We further demonstrate that the LFR and SYD interdependently activate the transcription of AGAMOUS (AG), a C-class floral organ identity gene, by regulating the occupation of nucleosome, chromatin loop, histone modification, and Pol II enrichment on the AG locus. Furthermore, the chromosome conformation capture (3C) assay revealed that the gene loop at AG locus is negatively correlated with the AG expression level, and LFR-SYD was functional to demolish the AG chromatin loop to promote its transcription. Collectively, these results provide insight into the molecular mechanism of the Arabidopsis SYD-SWI/SNF complex in the control of higher chromatin conformation of the floral identity gene essential to plant reproductive organ development.

SUPER WOMAN 2 (SPW2) maintains organ identity in spikelets by inhibiting the expression of floral homeotic genes OsMADS3, OsMADS58, OsMADS13, and DROOPING LEAF

Flower organ identity in rice is mainly determined by the A-, B-, C- and E-class genes, with the majority encoding MADS-box transcription factors. However, few studies have investigated how the expression of these floral organ identity genes is regulated during flower development. In this study, we identified a gene named SUPER WOMAN 2 (SPW2), which is necessary for spikelet/floret development in rice by participating in the regulation of the expression of pistil identity genes such as OsMADS3, OsMADS13, OsMADS58 and DL. In the spw2 mutant, ectopic stigma/ovary-like tissues were observed in the non-pistil organs, including sterile lemma, lemma, palea, lodicule, and stamen, suggesting that the identities of these organs were severely affected by mutations in SPW2. SPW2 was shown to encode a plant-specific EMF1-like protein that is involved in H3K27me3 modification as an important component of the PRC2 complex. Expression analysis showed that the SPW2 mutation led to the ectopic expression of OsMADS3, OsMADS13, OsMADS58, and DL in non-pistil organs of the spikelet. The ChIP-qPCR results showed significant reductions in the levels of H3K27me3 modification on the chromatin of these genes. Thus, we demonstrated that SPW2 can mediate the process of H3K27me3 modification of pistil-related genes to regulate their expression in non-pistil organs of spikelets in rice. The results of this study expand our understanding of the molecular mechanism by which SPW2 regulates floral organ identity genes through epigenetic regulation.

Developmental and genetic basis of the androgynophore in Gynandropsis gynandra.

Flowering plants have evolved a vast array of floral features involved in plant-pollinator interactions. A feature that seemingly increases the chance of pollen transfer is the androgynophore, a stalk-like structure that raises the reproductive organs of the flower. However, little is known about the developmental and genetic basis of this structure despite its presence in multiple, distantly related taxa. Here, we address this gap by investigating Gynandropsis gynandra (Cleomaceae), a species with a prominent androgynophore. We combined morphological and anatomical analyses with a comparative transcriptomic study to provide a detailed description of the androgynophore throughout development, examine global gene expression patterns, and identify candidate genes putatively involved in androgynophore elongation. The radially symmetric androgynophore of G. gynandra rapidly lengthens primarily via cell elongation. Despite its structural uniformity, androgynophore development is characterized by complex gene expression patterns including differential expression of floral organ identity genes and genes associated with organ development and growth in Arabidopsis thaliana. Our morphological characterizations and high-quality transcriptome for G. gynandra suggest that the androgynophore is a novel structure formed via elaboration of both the receptacle and base of reproductive organs because it is structurally like an elongated internode but expresses the genetic repertoire typically associated with the reproductive organs. The drastic increase in cell length and uniform structure elevates the androgynophore as a potentially powerful model for cell elongation.

Pushing the boundaries of organ identity: Homology of the grass lemma

Pushing the boundaries of organ identity: Homology of the grass lemma

Delphinieae flowers originated from the rewiring of interactions between duplicated and diversified floral organ identity and symmetry genes.

Species of the tribe Delphinieae (Ranunculaceae) have long been the focus of morphological, ecological, and evolutionary studies due to their highly specialized, nearly zygomorphic (bilaterally symmetrical) spiral flowers with nested petal and sepal spurs and reduced petals. The mechanisms underlying the development and evolution of Delphinieae flowers, however, remain unclear. Here, by conducting extensive phylogenetic, comparative transcriptomic, expression, and functional studies, we clarified the evolutionary histories, expression patterns, and functions of floral organ identity and symmetry genes in Delphinieae. We found that duplication and/or diversification of APETALA3-3 (AP3-3), AGAMOUS-LIKE6 (AGL6), CYCLOIDEA (CYC), and DIVARICATA (DIV) lineage genes was tightly associated with the origination of Delphinieae flowers. Specifically, an AGL6-lineage member (such as the Delphinium ajacis AGL6-1a) represses sepal spur formation and petal development in the lateral and ventral parts of the flower while determining petal identity redundantly with AGL6-1b. By contrast, two CYC2-like genes, CYC2b and CYC2a, define the dorsal and lateral-ventral identities of the flower, respectively, and form complex regulatory links with AP3-3, AGL6-1a, and DIV1. Therefore, duplication and diversification of floral symmetry genes, as well as co-option of the duplicated copies into the preexisting floral regulatory network, have been key for the origin of Delphinieae flowers.

FaesAP3_1 Regulates the FaesELF3 Gene Involved in Filament-Length Determination of Long-Homostyle Fagopyrum esculentum.

The identification downstream genes of floral organ identity regulators are critical to revealing the molecular mechanisms underlying floral morphogenesis. However, a general regulatory pathway between floral organ identity genes and their downstream targets is still unclear because of the lack of studies in nonmodel species. Here, we screened a direct downstream target gene, FaesELF3, of a stamen identity transcription factor, FaesAP3_1, in long-homostyle (LH) Fagopyrum esculentum moench by using yeast one-hybrid (Y1H) and dual-luciferase reporter (DR) assays. Furthermore, FaesAP3_1-silenced LH plants that produced flowers with part stamens or anthers homeotically converted into a tepaloid structure, and FaesELF3-silenced plants that had flowers with part stamens consisting of a short filament and empty anther (male sterile anther). All these suggested that transcription factor (TF) FaesAP3_1 directly activates FaesELF3 in order to regulate filament elongation and pollen grain development in LH buckwheat. Our data also suggested that other stamen development pathways independent of FaesAP3_1 remain in F. esculentum.

Arabidopsis thaliana SHOOT MERISTEMLESS Substitutes for Medicago truncatula SINGLE LEAFLET1 to Form Complex Leaves and Petals.

LEAFY plant-specific transcription factors, which are key regulators of flower meristem identity and floral patterning, also contribute to meristem activity. Notably, in some legumes, LFY orthologs such as Medicago truncatula SINGLE LEAFLET (SGL1) are essential in maintaining an undifferentiated and proliferating fate required for leaflet formation. This function contrasts with most other species, in which leaf dissection depends on the reactivation of KNOTTED-like class I homeobox genes (KNOXI). KNOXI and SGL1 genes appear to induce leaf complexity through conserved downstream genes such as the meristematic and boundary CUP-SHAPED COTYLEDON genes. Here, we compare in M. truncatula the function of SGL1 with that of the Arabidopsis thaliana KNOXI gene, SHOOT MERISTEMLESS (AtSTM). Our data show that AtSTM can substitute for SGL1 to form complex leaves when ectopically expressed in M. truncatula. The shared function between AtSTM and SGL1 extended to the major contribution of SGL1 during floral development as ectopic AtSTM expression could promote floral organ identity gene expression in sgl1 flowers and restore sepal shape and petal formation. Together, our work reveals a function for AtSTM in floral organ identity and a higher level of interchangeability between meristematic and floral identity functions for the AtSTM and SGL1 transcription factors than previously thought.

Effects of Water-deficit Stress and Gibberellic Acid on Floral Gene Expression and Floral Determinacy in ‘Washington’ Navel Orange

Effects of water-deficit stress and foliar-applied gibberellic acid (GA3) on ‘Washington’ navel orange (Citrus sinensis) floral gene expression and inflorescence number were quantified. Trees subjected to 8 weeks of water-deficit stress [average stem water potential (SWP) −2.86 MPa] followed by 3 weeks of re-irrigation (SWP recovered to > −1.00 MPa) produced more inflorescences in week 11 than trees well-irrigated (SWP > −1.00 MPa) for the full 11 weeks (P < 0.001). After 8 weeks of water-deficit stress, bud expression of flowering locus t (FT), suppressor of overexpression of constans1 (SOC1), leafy (LFY), apetala1 (AP1), apetala2 (AP2), sepallata1 (SEP1), pistillata (PI), and agamous (AG) increased during the re-irrigation period (weeks 9 and 10), but only AP1, AP2, SEP1, PI, and AG expression increased to levels significantly greater than that of well-irrigated trees. Foliar-applied GA3 (50 mg·L−1) in weeks 2 through 8 of the water-deficit stress treatment did not reduce bud FT, SOC1, or LFY expression, but prevented the upregulation AP1, AP2, SEP1, PI, and AG expression that occurred during re-irrigation in water-deficit stressed trees not treated with GA3. Applications of GA3 to water-deficit stressed trees reduced inflorescence number 95% compared with stressed trees without GA3. Thus, GA3 inhibited citrus (Citrus sp.) floral development in response to water-deficit stress through downregulating AP1 and AP2 expression, which likely led to the failed activation of the downstream floral organ identity genes. The results reported herein suggest that bud determinacy and subsequent floral development in response to water-deficit stress in ‘Washington’ navel orange are controlled by AP1 and AP2 transcript levels, which regulate downstream floral organ identity gene activity and the effect of GA3 on citrus flower formation. The water-deficit stress floral-induction pathway provides an alternative to low-temperature induction that increases the potential for successful flowering in citrus trees grown in areas experiencing warmer, drier winters due to global climate change.

Cloning and expression of floral organ identity genes in Paeonia ostii ‘Fengdan’

Cloning and expression of floral organ identity genes in Paeonia ostii ‘Fengdan’

The Roles of Floral Organ Genes in Regulating Rosaceae Fruit Development.

The function of floral organ identity genes, APETALA1/2/3, PISTILLATA, AGAMOUS, and SEPALLATA1/2/3, in flower development is highly conserved across angiosperms. Emerging evidence shows that these genes also play important roles in the development of the fruit that originates from floral organs following pollination and fertilization. However, their roles in fruit development may vary significantly between species depending on the floral organ types contributing to the fruit tissues. Fruits of the Rosaceae family develop from different floral organ types depending on the species, for example, peach fruit flesh develops from carpellary tissues, whereas apple and strawberry fruit flesh develop from extra-carpellary tissues, the hypanthium and receptacle, respectively. In this review, we summarize recent advances in understanding floral organ gene function in Rosaceae fruit development and analyze the similarities and diversities within this family as well as between Rosaceae and the model plant species Arabidopsis and tomato. We conclude by suggesting future research opportunities using genomics resources to rapidly dissect gene function in this family of perennial plants.

Genome-Wide Identification and Expression Analysis of MADS-Box Family Genes in Litchi (Litchi chinensis Sonn.) and Their Involvement in Floral Sex Determination.

Litchi possesses unique flower morphology and adaptive reproduction strategies. Although previous attention has been intensively devoted to the mechanisms underlying its floral induction, the molecular basis of flower sex determination remains largely unknown. MADS-box genes are promising candidates for this due to their significant roles in various aspects of inflorescence and flower organogenesis. Here, we present a detailed overview of phylogeny and expression profiles of 101 MADS-box genes that were identified in litchi. These LcMADSs are unevenly located across the 15 chromosomes and can be divided into type I and type II genes. Fifty type I MADS-box genes are subdivided into Mα, Mβ and Mγ subgroups, while fifty-one type II LcMADSs consist of 37 MIKCC -type and 14 MIKC *-type genes. Promoters of both types of LcMADS genes contain mainly ABA and MeJA response elements. Tissue-specific and development-related expression analysis reveal that LcMADS51 could be positively involved in litchi carpel formation, while six MADS-box genes, including LcMADS42/46/47/75/93/100, play a possible role in stamen development. GA is positively involved in the sex determination of litchi flowers by regulating the expression of LcMADS51 (LcSTK). However, JA down-regulates the expression of floral organ identity genes, suggesting a negative role in litchi flower development.

Expression and Functional Analyses of Nymphaea caerulea MADS-Box Genes Contribute to Clarify the Complex Flower Patterning of Water Lilies.

Nymphaeaceae are early diverging angiosperms with large flowers characterized by showy petals and stamens not clearly whorled but presenting a gradual morphological transition from the outer elements to the inner stamens. Such flower structure makes these plant species relevant for studying flower evolution. MADS-domain transcription factors are crucial components of the molecular network that controls flower development. We therefore isolated and characterized MADS-box genes from the water lily Nymphaea caerulea. RNA-seq experiments on floral buds have been performed to obtain the transcript sequences of floral organ identity MADS-box genes. Maximum Likelihood phylogenetic analyses confirmed their belonging to specific MADS-box gene subfamilies. Their expression was quantified by RT-qPCR in all floral organs at two stages of development. Protein interactions among these transcription factors were investigated by yeast-two-hybrid assays. We found especially interesting the involvement of two different AGAMOUS-like genes (NycAG1 and NycAG2) in the water lily floral components. They were therefore functionally characterized by complementing Arabidopsis ag and shp1 shp2 mutants. The expression analysis of MADS-box genes across flower development in N. caerulea described a complex scenario made of numerous genes in numerous floral components. Their expression profiles in some cases were in line with what was expected from the ABC model of flower development and its extensions, while in other cases presented new and interesting gene expression patterns, as for instance the involvement of NycAGL6 and NycFL. Although sharing a high level of sequence similarity, the two AGAMOUS-like genes NycAG1 and NycAG2 could have undergone subfunctionalization or neofunctionalization, as only one of them could partially restore the euAG function in Arabidopsis ag-3 mutants. The hereby illustrated N. caerulea MADS-box gene expression pattern might mirror the morphological transition from the outer to the inner floral organs, and the presence of transition organs such as the petaloid stamens. This study is intended to broaden knowledge on the role and evolution of floral organ identity genes and the genetic mechanisms causing biodiversity in angiosperm flowers.

Genetic containment in vegetatively propagated forest trees: CRISPR disruption of LEAFY function in Eucalyptus gives sterile indeterminate inflorescences and normal juvenile development.

SummaryEucalyptus is among the most widely planted taxa of forest trees worldwide. However, its spread as an exotic or genetically engineered form can create ecological and social problems. To mitigate gene flow via pollen and seeds, we mutated the Eucalyptus orthologue of LEAFY (LFY) by transforming a Eucalyptus grandis × urophylla wild‐type hybrid and two Flowering Locus T (FT) overexpressing (and flowering) lines with CRISPR Cas9 targeting its LFY orthologue, ELFY. We achieved high rates of elfy biallelic knockouts, often approaching 100% of transgene insertion events. Frameshift mutations and deletions removing conserved amino acids caused strong floral alterations, including indeterminacy in floral development and an absence of male and female gametes. These mutants were otherwise visibly normal and did not differ statistically from transgenic controls in juvenile vegetative growth rate or leaf morphology in greenhouse trials. Genes upstream or near to ELFY in the floral development pathway were overexpressed, whereas floral organ identity genes downstream of ELFY were severely depressed. We conclude that disruption of ELFY function appears to be a useful tool for sexual containment, without causing statistically significant or large adverse effects on juvenile vegetative growth or leaf morphology.

Transcriptome-Wide Analyses Provide Insights into Development of the Hedychium coronarium Flower, Revealing Potential Roles of PTL

The flower of Hedychium coronarium possesses highly specialized floral organs: a synsepalous calyx, petaloid staminodes and a labellum. The formation of these organs is controlled by two gene categories: floral organ identity genes and organ boundary genes, which may function individually or jointly during flower development. Although the floral organogenesis of H. coronarium has been studied at the morphological level, the underlying molecular mechanisms involved in particular organ morphologies that emerge in flower development still remain poorly understood. Here, we used comparative transcriptomics combined with Real-time quantitative PCR to investigate gene expression patterns of ABC-class genes in H. coronarium flowers, as well as the homolog of PETAL LOSS (HcPTL). Our studies found that stamen/petal identity or stamen fertility in H. coronarium was not necessarily correlated with the differential expression of HcAP3 and HcAG. We also found a novel spatio-temporal expression pattern for HcPTL mRNA, suggesting it may have evolved a lineage-specific role in the morphogenesis of the Hedychium flower. Our study provides a new transcriptome reference and a functional hypothesis regarding the role of a boundary gene in organ fusion that should be further addressed through phylogenetic analyzes of this gene, as well as functional studies.

Effects of temperature, soil moisture and light intensity on the temporal pattern of floral gene expression and flowering of avocado buds (Persea americana cv. Hass)

For ‘Hass’ avocado (Persea americana Mill.), low temperature is known to induce flowering, but effects of other environmental factors remain equivocal. In addition, documentation of interactions among environmental factors, floral gene expression and subsequent flower development is limited. Thus, in this research, the effects of environmental factors on the temporal expression patterns of genes related to flowering time, floral meristem identity and floral organ identity were quantified in buds relative to inflorescence number. ‘Hass’ avocado trees were subjected to four different environmental conditions: optimal growth condition (OGC) (warm temperature, well-watered, photosynthetic active radiation > 900 μmoles m–2 s–1), low temperature (LT), low soil moisture (LSM) or low light intensity (LLI) for 8 weeks, followed by OGC for 6 weeks. Bud expression of homologs of Arabidopsis thaliana flowering genes FLOWERING LOCUS T (FT), LEAFY (LFY), APETALA1 (AP1), APETALA2 (AP2), APETALA3 (AP3), PISTILLATA.1 (PI.1), AGAMOUS.1 (AG.1) and AGAMOUS.3 (AG.3) were quantified over time. Only buds of LT-treated trees flowered. At the start of the experiment (week 0), buds of all trees expressed PaLFY, PaAP1 and PaAP2. After 4 weeks of treatment, bud PaAP1 expression was greater than in week 0 and continued to increase through week 8 only in LT-treated trees, whereas by week 8 PaLFY expression increased in LT- and LSM-treated trees. By week 10 (2 weeks after transfer to OGC), bud PaFT expression increased only in LT-treated trees, followed by activation of downstream floral organ identity genes PaAP3, PaPI.1, PaAG.1 and PaAG.3 by week 12. For OGC-, LSM- and LLI-treated trees, bud PaAP1, PaFT, PaAP3, PaPI.1 and PaAG.1 expression remained low, with PaAG.3 transcripts undetected at week 12. These trees did not flower. Among environmental factors tested, only LT increased bud expression of both PaLFY and PaAP1, which likely conferred floral meristem determinacy since transfer of LT-treated trees to OGC did not prevent flowering but activated PaFT, downstream floral organ identity genes, and flowering. The results suggest ‘Hass’ avocado floral development is promoted by low temperature-dependent floral induction and warm temperature-dependent floral organogenesis.

Spontaneous homeotic mutants and genetic control of floral organ identity in a ranunculid.

The regulation of floral organ identity was investigated using a forward genetic approach in five floral homeotic mutants of Thalictrum, a noncore eudicot. We hypothesized that these mutants carry defects in the floral patterning genes. Mutant characterization comprised comparative floral morphology and organ identity gene expression at early and late developmental stages, followed by sequence analysis of coding and intronic regions to identify transcription factor binding sites and protein-protein interaction (PPI) motifs. Mutants exhibited altered expression of floral MADS-box genes, which further informed the function of paralogs arising from gene duplications not found in reference model systems. The ensuing modified BCEmodels for the mutants supported instances of neofunctionalization (e.g., B-class genes expressed ectopically in sepals), partial redundancy (E-class), or subfunctionalization (C-class) of paralogs. A lack of deleterious mutations in the coding regions of candidate floral MADS-box genes suggested that cis-regulatory or trans-acting mutations are at play. Consistent with this hypothesis, double-flower mutants had transposon insertions or showed signs of transposon activity in the regulatory intron of AGAMOUS (AG) orthologs. Single amino acid substitutions were also found, yet they did not fall on any of the identified DNA binding or PPI motifs.In conclusion, we present evidence suggesting that transposon activity and regulatory mutations in floral homeotic genes likely underlie the striking phenotypes of these Thalictrum floral homeotic mutants.