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MADS-box transcription factors play crucial roles in regulating development processes and biosynthesis of secondary metabolites in eukaryotes. However, the role of MADS-box transcription factors vary among fungal species, and their function remains unclear in the medicinally and economically important fungus Ganoderma lucidum . In this study, we characterized a MADS-box gene, GlMADS1 , in G. lucidum . Analyses using quantitative real-time polymerase chain reaction (qRT-PCR) showed that GlMADS1 expression levels were up-regulated from the mycelia to the primordia stage. In order to further evaluate the effect of MADS-box transcription factors on secondary metabolism, we utilized RNA interference (RNAi) to silence GlMADS1 in G. lucidum . Ganoderic acid (GA) and flavonoid contents were enhanced in GlMADS1 -silenced strains, suggesting that GlMADS1 negatively regulates GA and flavonoid accumulation.

AGLl9 is an important regulator for flowering in plants and critical in controlling the morphogenesis of flower organs. The fulllength cDNAs of AGL19 in conventional Lonicera macranthoides ( Lm-AGL19 ) and the mutant ' Xianglei ' cultivar ( Lm-XL-AGL19 ) were obtained using rapid amplification of cDNA ends and the expression vectors for Lm-XL-AGL19 were constructed to investigate the roles of AGL19 in the 'Xianglei' cultivar. Lm-AGL19 (GenBank: MK419948) and Lm-XL-AGL19 (GenBank: MK419948) were isolated from the conventional variety and ' Xianglei ' cultivar of L. macranthoides, respectively. Lm-AGL19 is 1274 bp in length, whereas Lm-XL-AGL19 is 1264-bp long, and both include a 654 bp open reading frame, encoding 217 amino acids, which has a highly conserved MADS_MEF2_like domain and a moderately conserved K-box domain, belonging to the type II MADS-box family of genes. Quantitative real-time polymerase chain reaction indicated that the expression levels of these genes at different flowering stages were significantly different, and that the genes were also expressed in stems and leaves. Lm-XL-AGL19 is underexpressed at flowering period 5 that the key time node for corolla expansion and nonexpansion, while LM-AGL19 is overexpressed during this flowering period. AGL19 was speculated to be a functional gene causing different phenotypes in the two L. macranthoides varieties. The successfully constructed plant expression vector provides an experimental reference for further research on the function of this gene and the basis for the excellent phenotype of L. macranthoides ' Xianglei '.

Fruit ripening is a complex and genetically programmed process modulated by transcription factors, hormones, and other regulators. However, the mechanism underlying the regulatory loop involving the membrane-protein targets of RIPENING-INHIBITOR (RIN) remains poorly understood. To unravel the function of tomato ( S olanum l ycopersicum ) FERONIA Like ( SlFERL ), a putative MADS-box transcription factor target gene, we investigated and addressed the significance of SlFERL in fruit ripening by combining reverse genetics, biochemical, and cytological analyses. Here, we report that RIN and Tomato AGAMOUS-LIKE1 (TAGL1) directly bind to the promoter region of SlFERL and further activate its expression transcriptionally, suggesting a potential role of SlFERL in fruit ripening. Overexpression of SlFERL significantly accelerated the ripening process of tomato fruit, whereas RNA interference knockdown of SlFERL resulted in delayed fruit ripening. Moreover, a surface plasmon resonance assay coupled with tandem mass spectrometry and a protein interaction assay revealed that SlFERL interacts with the key enzyme S -adenosyl-Met synthetase 1 (SlSAMS1) in the ethylene biosynthesis pathway, leading to increased S -adenosyl-Met accumulation and elevated ethylene production. Thus, SlFERL serves as a positive regulator of ethylene production and fruit ripening. This study provides clues to the molecular regulatory networks underlying fruit ripening., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

The E class MADS-box genes SEPALLATA (SEP)-like play critical roles in angiosperm reproductive growth, especially in floral organ differentiation. To analyze the sequence characteristics and spatio-temporal expression patterns of E-function MADS-box SEP-like genes during kale (Brassica oleracea L. var. acephala) flower development, BroaSEP1/2/3 (GenBank No. KC967957, KC967958, KC967960) homologues, three kale SEP MADS-box gene, were isolated from the kale variety 'Fourteen Line' using Rapid amplification of cDNA ends (RACE). Sequence and phylogenetic analysis indicated that these three SEP genes had a high degree of identity with SEP1, SEP2, SEP3 from Brassica oleracea var. oleracea, Brassica rapa, Raphanus sativus and Brassica napus, respectively. Alignment of the predicted amino acid sequences from these genes, along with previously published subfamily members, demonstrated that these genes comprise four regions of the typical MIKC-type MADS-box proteins: the MADS domain, intervening (I) domain and keratin-like (K) domain, and the C-terminal domain SEPⅠ and SEP Ⅱ motif. The longest open reading frame deduced from the cDNA sequences of BroaSEP1, BroaSEP2, and BroaSEP3 appeared to be 801 bp, 759 bp, 753 bp in length, respectively, which encoded proteins of 266, 252, and 250 amino acids respectively. Expression analyses using semi-quantitative RT-PCR and quantitative real-time PCR indicate that BroaSEP1/2/3 are specifically expressed in floral buds of kale during flower development process. The expression levels of the three genes are very different at different developmental stages, also in wild type, mutant flower with increased petals, and mutant flower with decreased petals. These different patterns of gene expression maybe cause the flowers to increase or decrease the petal number.

Sugarcane is the most important sugar and biofuel crop. MADS-box genes encode transcription factors that are involved in developmental control and signal transduction in plants. Systematic analyses of MADS-box genes have been reported in many plant species, but its identification and characterization were not possible until a reference genome of autotetraploid wild type sugarcane specie, Saccharum spontaneum is available recently. We identified 182 MADS-box sequences in the S. spontaneum genome, which were annotated into 63 genes, including 6 (9.5%) genes with four alleles, 21 (33.3%) with three, 29 (46%) with two, 7 (11.1%) with one allele. Paralogs (tandem duplication and disperse duplicated) were also identified and characterized. These MADS-box genes were divided into two groups; Type-I (21 Mα, 4 Mβ, 4 Mγ) and Type-II (32 MIKCc, 2 MIKC*) through phylogenetic analysis with orthologs in Arabidopsis and sorghum. Structural diversity and distribution of motifs were studied in detail. Chromosomal localizations revealed that S. spontaneum MADS-box genes were randomly distributed across eight homologous chromosome groups. The expression profiles of these MADS-box genes were analyzed in leaves, roots, stem sections and after hormones treatment. Important alleles based on promoter analysis and expression variations were dissected. qRT-PCR analysis was performed to verify the expression pattern of pivotal S. spontaneum MADS-box genes and suggested that flower timing genes (SOC1 and SVP) may regulate vegetative development.

In trees, stem secondary growth depends on vascular cambium proliferation activity and subsequent cell differentiation, in which an auxin concentration gradient across the cambium area plays a crucial role in regulating the process. However, the underlying molecular mechanism for the establishment of auxin concentration is not fully understood. In this study, we identified two function-unknown MADS-box genes, VCM1 and VCM 2, which are expressed specifically in the vascular cambium and modulate the subcellular homeostasis of auxin. Simultaneous knockdown of both VCM1 and VCM2 enhanced vascular cambium proliferation activity and subsequent xylem differentiation. Overexpression of VCM1 suppressed vascular cambium activity and wood formation by regulating PIN5 expression, which tuned the soluble auxin concentration in the vascular cambium area. This study reveals the role of VCM1 and VCM2 in regulating the proliferation activity of the vascular cambium and secondary growth by modulating the subcellular auxin homeostasis in Populus ., (© 2020 The Author(s).)

The large sunflower family, Asteraceae, is characterized by compressed, flower-like inflorescences that may bear phenotypically distinct flower types. The CYCLOIDEA (CYC)/TEOSINTE BRANCHED1-like transcription factors (TFs) belonging to the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) protein family are known to regulate bilateral symmetry in single flowers. In Asteraceae, they function at the inflorescence level, and were recruited to define differential flower type identities. Here, we identified upstream regulators of GhCYC3 , a gene that specifies ray flower identity at the flower head margin in the model plant Gerbera hybrida We discovered a previously unidentified expression domain and functional role for the paralogous CINCINNATA-like TCP proteins. They function upstream of GhCYC3 and affect the developmental delay of marginal ray primordia during their early ontogeny. At the level of single flowers, the Asteraceae CYC genes show a unique function in regulating the elongation of showy ventral ligules that play a major role in pollinator attraction. We discovered that during ligule development, the E class MADS-box TF GRCD5 activates GhCYC3 expression. We propose that the C class MADS-box TF GAGA1 contributes to stamen development upstream of GhCYC3 Our data demonstrate how interactions among and between the conserved floral regulators, TCP and MADS-box TFs, contribute to the evolution of the elaborate inflorescence architecture of Asteraceae., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Winter varieties of plants can flower only after exposure to prolonged cold. This phenomenon is known as vernalization and has been widely studied in the model plant Arabidopsis thaliana as well as in monocots. Through the repression of floral activator genes, vernalization prevents flowering in winter. In Arabidopsis, FLOWERING LOCUS C or FLC is the key repressor during vernalization, while in monocots vernalization is regulated through VRN1, VRN2 and VRN3 (or FLOWERING LOCUS T). Interestingly, VRN genes are not homologous to FLC but FLC homologs are found to have a significant role in vernalization response in cereals. The presence of FLC homologs in monocots opens new dimensions to understand, compare and retrace the evolution of vernalization pathways between monocots and dicots. In this review, we discuss the molecular mechanism of vernalization-induced flowering along with epigenetic regulations in Arabidopsis and temperate cereals. A better understanding of cold-induced flowering will be helpful in crop breeding strategies to modify the vernalization requirement of economically important temperate cereals., (© 2020 Scandinavian Plant Physiology Society.)

Key Message: EgMADS21 regulates PUFA accumulation in oil palm. Oil palm (Elaeis guineensis Jacq.) is the most productive world oil crop, accounting for 36% of world plant oil production. However, the molecular mechanism of the transcriptional regulation of fatty acid accumulation and lipid synthesis in the mesocarp of oil palm by up- or downregulating the expression of genes involved in related pathways remains largely unknown. Here, an oil palm MADS-box gene, EgMADS21, was screened in a yeast one-hybrid assay using the EgDGAT2 promoter sequence as bait. EgMADS21 is preferentially expressed in early mesocarp developmental stages in oil palm fruit and presents a negative correlation with EgDGAT2 expression. The direct binding of EgMADS21 to the EgDGAT2 promoter was confirmed by electrophoretic mobility shift assay. Subsequently, transient expression of EgMADS21 in oil palm protoplasts revealed that EgMADS21 not only binds to the EgDGAT2 promoter but also negatively regulates the expression of EgDGAT2. Furthermore, EgMADS21 was stably overexpressed in transgenic oil palm embryoids by Agrobacterium-mediated transformation. In three independent transgenic lines, EgDGAT2 expression was significantly suppressed by the expression of EgMADS21. The content of linoleic acid (C18:2) in the three transgenic embryoids was significantly decreased, while that of oleic acid (C18:1) was significantly increased. Combined with the substrate preference of EgDGAT2 identified in previous research, the results demonstrate the molecular mechanism by which EgMADS21 regulates EgDGAT2 expression and ultimately affects fatty acid accumulation in the mesocarp of oil palm.

Interactions between MADS box transcription factors are critical in the regulation of floral development, and shifting MADS box protein-protein interactions are predicted to have influenced floral evolution. However, precisely how evolutionary variation in protein-protein interactions affects MADS box protein function remains unknown. To assess the impact of changing MADS box protein-protein interactions on transcription factor function, we turned to the grasses, where interactions between B-class MADS box proteins vary. We tested the functional consequences of this evolutionary variability using maize ( Zea mays ) as an experimental system. We found that differential B-class dimerization was associated with subtle, quantitative differences in stamen shape. In contrast, differential dimerization resulted in large-scale changes to downstream gene expression. Differential dimerization also affected B-class complex composition and abundance, independent of transcript levels. This indicates that differential B-class dimerization affects protein degradation, revealing an important consequence for evolutionary variability in MADS box interactions. Our results highlight complexity in the evolution of developmental gene networks: changing protein-protein interactions could affect not only the composition of transcription factor complexes but also their degradation and persistence in developing flowers. Our results also show how coding change in a pleiotropic master regulator could have small, quantitative effects on development., (© 2020 American Society of Plant Biologists. All rights reserved.)

Orchids (members of the Orchidaceae family) possess unique flower morphology and adaptive reproduction strategies. Although the mechanisms underlying their perianth development have been intensively studied, the molecular basis of reproductive organ development in orchids remains largely unknown. Here, we report the identification and functional characterization of two AGAMOUS ( AG )-like MADS-box genes, Dendrobium 'Orchid' AG1 ( DOAG1 ) and DOAG2 , which are putative C- and D-class genes, respectively, from the orchid Dendrobium 'Chao Praya Smile'. Both DOAG1 and DOAG2 are highly expressed in the reproductive organ, known as the column, compared to perianth organs, while DOAG2 expression gradually increases in pace with pollination-induced ovule development and is localized in ovule primordia. Ectopic expression of DOAG1 , but not DOAG2 , rescues floral defects in the Arabidopsis ( Arabidopsis thaliana ) ag-4 mutant, including reiteration of stamenoid perianth organs in inner whorls and complete loss of carpels. Downregulation of DOAG1 and DOAG2 in orchids by artificial microRNA interference using l-Met sulfoximine selection-based gene transformation systems shows that both genes are essential for specifying reproductive organ identity, yet they, exert different roles in mediating floral meristem determinacy and ovule development, respectively, in Dendrobium spp. orchids. Notably, knockdown of DOAG1 and DOAG2 also affects perianth organ development in orchids. Our findings suggest that DOAG1 and DOAG2 not only act as evolutionarily conserved C- and D-class genes, respectively, in determining reproductive organ identity, but also play hitherto unknown roles in mediating perianth organ development in orchids., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

B-box transcription factors (BBXs) are important regulators of flowering, photomorphogenesis, shade-avoidance, abiotic and biotic stresses and plant hormonal pathways. In Arabidopsis, 32 BBX proteins have been identified and classified into five groups based on their structural domains. Little is known about the fifth group members (BBX26-BBX32) and the detailed molecular mechanisms relevant to their functions. Here we identified B-box transcription factor 28 (BBX28) that interacts with Constans (CO), a transcriptional activator of Flowering Locus T (FT). Overexpressing BBX28 leads to late flowering with dramatically decreased FT transcription, and bbx28 deficient mutant displays a weak early flowering phenotype under long days (LD), indicating that BBX28 plays a negative and redundant role in flowering under LD. Additionally, the interaction between BBX28 and CO decreases the recruitment of CO to FT locus without affecting the transcriptional activation activity of CO. Moreover, the N-terminal cysteines, especially those within the B-box domain, are indispensable for the heterodimerization between BBX28 and CO and activation of CO on FT transcription. Genetic evidences show that the later flowering caused by BBX28 overexpression is compromised by CO ectopic expression. Collectively, these results supported that BBX28 functions with CO and FT to negatively regulate Arabidopsis flowering, in which the N-terminal conserved cysteines of BBX28 might play a central role.

Fruit development involves chloroplast development, carotenoid accumulation and fruit coloration. Although genetic regulation of fruit development has been extensively investigated, epigenetic regulation of fruit coloration remains largely unexplored. Here, we report a naturally occurring epigenetic regulation of TAGL1, and its impact on chloroplast development and fruit coloration. We used a genome-wide association study in combination with map-based cloning to identify the GREEN STRIPE (GS) locus, a methylated isoform of TAGL1 regulating diversified chloroplast development and carotenoid accumulation. Nonuniform pigmentation of fruit produced by GS was highly associated with methylation of the TAGL1 promoter, which is linked to a SNP at SL2.50ch07_63842838. High degrees of methylation of the TAGL1 promoter downregulated its expression, leading to green stripes. By contrast, low degrees of methylation led to light green stripes in gs. RNA-seq and ChIP collectively showed that the expression of genes involved with Chl synthesis and chloroplast development were significantly upregulated in green stripes relative to light green stripes. Quantitative PCR and dual luciferase assay confirmed that TAGL1 downregulates expression of SlMPEC, SlPsbQ, and SlCAB, and upregulates expression of PSY1 - genes which are associated with chloroplast development and carotenoid accumulation. Altogether, our findings regarding the GS locus demonstrate that naturally occurring methylation of TAGL1 has diverse effects on plastid development in fruit., (© 2020 The Authors. New Phytologist © 2020 New Phytologist Trust.)

Flower malformation represented by phyllody is a common symptom of phytoplasma infection induced by a novel family of phytoplasma effectors called phyllogens. Despite the accumulation of functional and structural phyllogen information, the molecular mechanisms of phyllody have not yet been integrated with their evolutionary aspects due to the limited data on their homologs across diverse phytoplasma lineages. Here, we developed a novel universal PCR-based approach to identify 25 phytoplasma phyllogens related to nine "Candidatus Phytoplasma" species, including four species whose phyllogens have not yet been identified. Phylogenetic analyses showed that the phyllogen family consists of four groups (phyl-A, -B, -C, and -D) and that the evolutionary relationships of phyllogens were significantly distinct from those of phytoplasmas, suggesting that phyllogens were transferred horizontally among phytoplasma strains and species. Although phyllogens belonging to the phyl-A, -C, and -D groups induced phyllody, the phyl-B group lacked the ability to induce phyllody. Comparative functional analyses of phyllogens revealed that a single amino acid polymorphism in phyl-B group phyllogens prevented interactions between phyllogens and A- and E-class MADS domain transcription factors (MTFs), resulting in the inability to degrade several MTFs and induce phyllody. Our finding of natural variation in the function of phytoplasma effectors provides new insights into molecular mechanisms underlying the aetiology of phytoplasma diseases., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

The end of the reproductive phase in monocarpic plants is determined by a coordinated arrest of all active meristems, a process known as global proliferative arrest (GPA). GPA is linked to the correlative control exerted by developing seeds and, possibly, the establishment of strong source-sink relationships. It has been proposed that the meristems that undergo arrest at the end of the reproductive phase behave at the transcriptomic level as dormant meristems, with low mitotic activity and high expression of abscisic acid response genes. Meristem arrest is also controlled genetically. In Arabidopsis ( Arabidopsis thaliana ), the MADS-box transcription factor FRUITFULL induces GPA by directly repressing genes of the APETALA2 ( AP2 ) clade. The AP2 genes maintain shoot apical meristem (SAM) activity in part by keeping WUSCHEL expression active, but the mechanisms downstream of this pathway remain elusive. To identify target genes, we performed a transcriptomic analysis, inducing AP2 activity in meristems close to arrest. Our results suggest that AP2 controls meristem arrest by repressing genes related to axillary bud dormancy in the SAM and negative regulators of cytokinin signaling. In addition, our analysis indicates that genes involved in the response to environmental signals also respond to AP2, suggesting that it could modulate the end of flowering by controlling responses to both endogenous and exogenous signals. Our results support the previous observation that at the end of the reproductive phase the arrested SAM behaves as a dormant meristem, and they strongly support AP2 as a master regulator of this process., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Vernalisation is the process in which long-term cold exposure makes plants competent to flower. In vernalisation of Arabidopsis thaliana, a floral repressor, AtFLC, undergoes epigenetic silencing. Although the silencing of AtFLC is maintained under warm conditions after a sufficient duration of cold, FLC orthologues are reactivated under the same conditions in perennial plants, such as A. halleri. In contrast to the abundant knowledge on cold requirements in AtFLC silencing, it has remained unknown how cold duration affects the reactivation of perennial FLC. Here, we analysed the dynamics of A. halleri FLC (AhgFLC) mRNA, H3K4me3, and H3K27me3 over 8 weeks and 14 weeks cold followed by warm conditions. We showed that the minimum levels of AhgFLC mRNA and H3K4me3 were similar between 8 and 14 weeks vernalisation; however, the maximum level of H3K27me3 was higher after 14 weeks than after 8 weeks vernalisation. Combined with mathematical modelling, we showed that H3K27me3 prevents a rapid increase in AhgFLC expression in response to warm temperatures after vernalisation, which controls AhgFT expression and the initiation of flowering. Thus, the duration of cold defines the rate of AhgFLC reactivation via the buffering function of H3K27me3 against temperature increase.

The auxin-induced embryogenic reprogramming of plant somatic cells is associated with extensive modulation of the gene expression in which epigenetic modifications, including DNA methylation, seem to play a crucial role. However, the function of DNA methylation, including the role of auxin in epigenetic regulation of the SE-controlling genes, remains poorly understood. Hence, in the present study, we analysed the expression and methylation of the TF genes that play a critical regulatory role during SE induction ( LEC1 , LEC2 , BBM , WUS and AGL15 ) in auxin-treated explants of Arabidopsis. The results showed that auxin treatment substantially affected both the expression and methylation patterns of the SE-involved TF genes in a concentration-dependent manner. The auxin treatment differentially modulated the methylation of the promoter (P) and gene body (GB) sequences of the SE-involved genes. Relevantly, the SE-effective auxin treatment (5.0 µM of 2,4-D) was associated with the stable hypermethylation of the P regions of the SE-involved genes and a significantly higher methylation of the P than the GB fragments was a characteristic feature of the embryogenic culture. The presence of auxin-responsive (AuxRE) motifs in the hypermethylated P regions suggests that auxin might substantially contribute to the DNA methylation-mediated control of the SE-involved genes.

In Arabidopsis thaliana, winter is registered during vernalization through the temperature-dependent repression and epigenetic silencing of floral repressor FLOWERING LOCUS C (FLC) . Natural Arabidopsis accessions show considerable variation in vernalization. However, which aspect of the FLC repression mechanism is most important for adaptation to different environments is unclear. By analysing FLC dynamics in natural variants and mutants throughout winter in three field sites, we find that autumnal FLC expression, rather than epigenetic silencing, is the major variable conferred by the distinct Arabidopsis FLC haplotypes. This variation influences flowering responses of Arabidopsis accessions resulting in an interplay between promotion and delay of flowering in different climates to balance survival and, through a post-vernalization effect, reproductive output. These data reveal how expression variation through non-coding cis variation at FLC has enabled Arabidopsis accessions to adapt to different climatic conditions and year-on-year fluctuations., Competing Interests: JH, RA, KB, CS, ET, BY, DC, BC, JI, MH, TS, SH, CD No competing interests declared, (© 2020, Hepworth et al.)

Investigating the evolution of complex phenotypes and the underlying molecular bases of their variation is critical to understand how organisms adapt to their environment. Applying classical quantitative genetics on a segregating population derived from a Can-0xCol-0 cross, we identify the MADS-box transcription factor FLOWERING LOCUS M (FLM) as a player of the phenotypic variation in plant growth and color. We show that allelic variation at FLM modulates plant growth strategy along the leaf economics spectrum, a trade-off between resource acquisition and resource conservation, observable across thousands of plant species. Functional differences at FLM rely on a single intronic substitution, disturbing transcript splicing and leading to the accumulation of non-functional FLM transcripts. Associations between this substitution and phenotypic and climatic data across Arabidopsis natural populations, show how noncoding genetic variation at a single gene might be adaptive through pleiotropic effects.

Abietane diterpenoids are tricyclic diterpenes whose biological functions in angiosperms are largely unknown. Here, we show that dehydroabietinal (DA) fosters transition from the vegetative phase to reproductive development in Arabidopsis thaliana by promoting flowering time. DA's promotion of flowering time was mediated through up-regulation of the autonomous pathway genes FLOWERING LOCUS D (FLD), RELATIVE OF EARLY FLOWERING 6 (REF6), and FVE, which repress expression of FLOWERING LOCUS C (FLC), a negative regulator of the key floral integrator FLOWERING LOCUS T (FT). Our results further indicate that FLD, REF6, and FVE are also required for systemic acquired resistance (SAR), an inducible defense mechanism that is also activated by DA. However, unlike flowering time, FT was not required for DA-induced SAR. Conversely, salicylic acid, which is essential for the manifestation of SAR, was not required for the DA-promoted flowering time. Thus, although the autonomous pathway genes FLD, REF6, and FVE are involved in SAR and flowering time, these biological processes are not interdependent. We suggest that SAR and flowering time signaling pathways bifurcate at a step downstream of FLD, REF6, and FVE, with an FLC-dependent arm controlling flowering time, and an FLC-independent pathway controlling SAR., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Ovules are essential for sexual plant reproduction and seed formation, and are fundamental for agriculture. However, our understanding of the molecular mechanisms governing ovule development is far from complete. In Arabidopsis, ovule identity is determined by homeotic MADS-domain proteins that define the floral C- (AG) and D- (SHP1/SHP2, STK) functions. Pre-mRNA processing of these genes is critical and mediated by HUA-PEP activity, composed of genes encoding RNA-binding proteins. In strong hua-pep mutants, functional transcripts for C- and D-function genes are reduced, resulting in homeotic transformation of ovules. Thus, hua-pep mutants provide an unique sensitized background to study ovule morphogenesis when C- and D-functions are simultaneously compromised. We found that hua-pep ovules are morphologically sepaloid and show ectopic expression of the homeotic class-A gene AP1. Inactivation of AP1 or AP2 (A-function genes) in hua-pep mutants reduced homeotic conversions, rescuing ovule identity while promoting carpelloid traits in transformed ovules. Interestingly, increased AG dosage led to similar results. Our findings strongly suggest that HUA-PEP activity is required for correct C and D floral functions, which in turn prevents ectopic expression of class-A genes in ovules for their proper morphogenesis, evoking the classic A-C antagonism of the ABC model for floral organ development., (© 2020 The Authors. New Phytologist © 2020 New Phytologist Trust.)

Flowering time regulation is one of the most important processes in the whole life of flowering plants and FLOWERING LOCUS C (FLC) is a central repressor of flowering time. However, whether metabolic acetate level affects flowering time is unknown. Here we report that ALDEHYDE DEHYDROGENASE ALDH3F1 plays essential roles in floral transition via FLC-dependent pathway. In the aldh3f1-1 mutant, the flowering time was significant earlier than Col-0 and the FLC expression level was reduced. ALDH3F1 had aldehyde dehydrogenase activity to affect the acetate level in plants, and the amino acids of E214 and C252 are essential for its catalytic activity. Moreover, aldh3f1 mutation reduced acetate level and the total acetylation on histone H3. The H3K9Ac level on FLC locus was decreased in aldh3f1-1, which reduced FLC expression. Expression of ALDH3F1 could rescue the decreased H3K9Ac level on FLC, FLC expression and also the early-flowering phenotype of aldh3f1-1, however ALDH3F1 E214A or ALDH3F1 C252A could not. Our findings demonstrate that ALDH3F1 participates in flowering time regulation through modulating the supply of acetate for acetyl-CoA, which functions as histone acetylation donor to modulate H3K9Ac on FLC locus., (© 2019 Institute of Botany, Chinese Academy of Sciences.)

Actin depolymerizing factor (ADF) is a key modulator for dynamic organization of actin cytoskeleton. Interestingly, it was found that the ADF1 gene silencing delays flowering, but its mechanism remains unclear. In this study, ADF1 was used as a bait to screen its interacting proteins by the yeast two-hybrid (Y2H) system. One of them, the REM16 transcription factor was identified. As one of the AP2/B3-like transcriptional factor family members, the REM16 contains two B3 domains and its transcript levels kept increasing during the floral transition stage. Overexpression of REM16 accelerates flowering while silencing of REM16 delays flowering. Gene expression analysis indicated that the key flowering activation genes such as CONSTANS (CO), FLOWERING LOCUS T (FT), LEAFY (LFY) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC1) were upregulated in the REM16 overexpression lines, while the transcription of the flowering suppression gene FLOWERING LOCUS C (FLC) was decreased. In contrast, the REM16 gene silencing lines contained lower transcript levels of the CO, FT, LFY and SOC1 but higher transcript levels of the FLC compared with the wild-type plants. It was proved that REM16 could directly bind to the promoter regions of SOC1 and FT by in vitro and in vivo assays. Genetic analysis supported that REM16 acts upstream of SOC1 and FT in flowering pathways. All these studies provided strong evidence demonstrating that REM16 promotes flowering by directly activating SOC1 and FT., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

Plants can develop root systems with distinct anatomical features and morphological plasticity to forage nutrients distributed heterogeneously in soils. Lateral root proliferation is a typical nutrient-foraging response to a local supply of nitrate, which has been investigated across many plant species. However, the underlying mechanism in maize roots remains largely unknown. Here, we report on identification of a maize truncated MIKC-type MADS-box transcription factor (ZmTMM1) lacking K- and C-domains, expressed preferentially in the lateral root branching zone and induced by the localized supply of nitrate. ZmTMM1 belongs to the AGL17-like MADS-box transcription factor family that contains orthologs of ANR1, a key regulator for root nitrate foraging in Arabidopsis. Ectopic overexpression of ZmTMM1 recovers the defective growth of lateral roots in the Arabidopsis anr1 agl21 double mutant. The local activation of glucocorticoid receptor fusion proteins for ZmTMM1 and an artificially truncated form of AtANR1 without the K- and C-domains stimulates the lateral root growth of the Arabidopsis anr1 agl21 mutant, providing evidence that ZmTMM1 encodes a functional MADS-box that modulates lateral root development. However, no phenotype was observed in ZmTMM1-RNAi transgenic maize lines, suggesting a possible genetic redundancy of ZmTMM1 with other AGL17-like genes in maize. A comparative genome analysis further suggests that a nitrate-inducible transcriptional regulation is probably conserved in both truncated and non-truncated forms of ZmTMM1-like MADS-box transcription factors found in grass species., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Coconut palm has two distinct types-"tall" and "dwarf"-which differ morphologically. Tall coconut varieties need 8-10 years to start flowering, while dwarf coconut varieties only require 3-5 years. We compared seedling and reproductive stage transcriptomes for both coconut types to determine potential molecular mechanisms underlying control of flowering time in coconut. Several key genes in the photoperiod pathway were differentially expressed between seedling and reproductive leaf samples in both tall and dwarf coconut. These genes included suppressor of overexpression of constans (SOC1), flowering locus T (FT), and Apetala 1 (AP1). Alternative splicing analysis of genes in the photoperiod pathway further revealed that the FT gene produces different transcripts in tall compared to dwarf coconut. The shorter alternative splice variant of FT [which included a 6 bp deletion, alternative 3' splicing sites (A3SS)] was found to be exclusively present in dwarf coconut varieties but absent in most tall coconut varieties. Our results provide a valuable information resource as well as suggesting a probable mechanism for differentiation of flowering time onset in coconut, providing a target for future breeding work in accelerating time to flowering in this crop species.

Stamen development is an important developmental process controlled by multiple internal and external factors. Developmental abnormalities of stamens can disrupt the structure and function of anthers, and then result in male sterility. As well known, APETELA 3 (AP3) has a clear function in regulating stamen development, which may impact in male sterility. However, the mechanisms of stamen development and male sterility controlled by AP3 are still not very clear, particular in Pak-choi (Brassica rapa ssp. chinensis). In this work, BcAP3 encoded a protein containing a MADS-box domain, which was a homolog of AtAP3, was identified in Pak-choi. Sequence alignments and phylogenetic analysis indicated that BcAP3 was highly similar to AtAP3. BcAP3 was shown to be localized to the nucleus and exhibited the potential of transcription factor. The transcript of BcAP3 was only expressed in flowers of Pak-choi, indicating that it may act in flower development. Overexpression of BcAP3 in Arabidopsis resulted in developmental abnormalities of anther wall and low vigor pollen, which were associated with the phenotype of male sterility. Expression levels of NST1 and NST2, involved in secondary wall thickening in anther walls, were significantly higher in the BcAP3-transgenic plants than in control plants, suggesting that BcAP3 may affect anther wall development by regulating NST1 and NST2. Taken together, our study demonstrated that BcAP3 could play an essential role in stamen development and male sterility., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Polycomb-group (PcG) proteins are evolutionarily conserved in higher organisms and play essential roles in many developmental processes by catalyzing the trimethylation of histone H3 lysine 27 (H3K27me3), a key repressive histone mark. In Arabidopsis ( Arabidopsis thaliana ), histone methyltransferase CURLY LEAF (CLF) is one of the major PcG catalytic components, playing critical roles in plant growth and development, especially the floral transition. We have recently profiled the genome-wide occupancy of CLF by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). Interestingly, AGAMOUS-LIKE 17 ( AGL17 ) that encodes a known flowering activator was found to be a CLF direct target. Based on this observation, we set out to examine to what extent this genetic module regulates the flowering. First, we validated the ChIP-seq results by ChIP-qPCR and show that CLF indeed targets AGL17 , and the level of H3K27me3 is decreased when CLF is lost. Further, we show that the expression of AGL17 is significantly up-regulated in the clf-29 mutant compared to wild-type (WT). Finally, our clf agl17 double mutant analysis suggests that AGL17 is a significant downstream target of CLF in floral transition control.

To elucidate the molecular mechanism of juvenility and annual flowering of fruit trees, FLOWERING LOCUS C ( FLC ), an integrator of flowering signals, was investigated in apple as a model. We performed sequence and expression analyses and transgenic experiments related to juvenility with annual flowering to characterize the apple FLC homologs MdFLC . The phylogenetic tree analysis, which included other MADS-box genes, showed that both MdFLC1 and MdFLC3 belong to the same FLC group. MdFLC1c from one of the MdFLC1 splice variants and MdFLC3 contain the four conserved motives of an MIKC-type MADS protein. The mRNA of variants MdFLC1a and MdFLC1b contain intron sequences, and their deduced amino acid sequences lack K- and C-domains. The expression levels of MdFLC1a , MdFLC1b , and MdFLC1c decreased during the flowering induction period in a seasonal expression pattern in the adult trees, whereas the expression level of MdFLC3 did not decrease during that period. This suggests that MdFLC1 is involved in flowering induction in the annual growth cycle of adult trees. In apple seedlings, because phase change can be observed in individuals, seedlings can be used for analysis of expression during phase transition. The expression levels of MdFLC1b , MdFLC1c , and MdFLC3 were high during the juvenile phase and low during the transitional and adult phases. Because the expression pattern of MdFLC3 suggests that it plays a specific role in juvenility, MdFLC3 was subjected to functional analysis by transformation of Arabidopsis . The results revealed the function of MdFLC3 as a floral repressor. In addition, MdFT had CArG box-like sequences, putative targets for the suppression of flowering by MdFLC binding, in the introns and promoter regions. These results indicate that apple homologs of FLC, which might play a role upstream of the flowering signals, could be involved in juvenility as well as in annual flowering. Apples with sufficient genome-related information are useful as a model for studying phenomena unique to woody plants such as juvenility and annual flowering.

Flowering of many plant species depends on interactions between basic leucine zipper (bZIP) transcription factors and systemically transported florigen proteins. Members of the genus Arabidopsis contain two of these bZIPs, FD and FDP, which we show have largely complementary expression patterns in shoot apices before and during flowering. CRISPR-Cas9-induced null mutants for FDP flower slightly earlier than wild-type, whereas fd mutants are late flowering. Identical G-box sequences are enriched at FD and FDP binding sites, but only FD binds to genes involved in flowering and only fd alters their transcription. However, both proteins bind to genes involved in responses to the phytohormone abscisic acid (ABA), which controls developmental and stress responses. Many of these genes are differentially expressed in both fd and fdp mutant seedlings, which also show reduced ABA sensitivity. Thus, florigen-interacting bZIPs have distinct functions in flowering dependent on their expression patterns and, at earlier stages in development, play common roles in phytohormone signaling., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Plant reproductive development from the first appearance of reproductively committed axes through to floral maturation requires massive and rapid remarshalling of gene expression. In dioecious species such as poplar this is further complicated by divergent male and female developmental programs. We used seven time points in male and female balsam poplar (Populus balsamifera L.) buds and catkins representing the full annual flowering cycle, to elucidate the effects of time and sex on gene expression during reproductive development. Time (developmental stage) is dominant in patterning gene expression with the effect of sex nested within this. Here, we find (1) evidence for five successive waves of alterations to the chromatin landscape which may be important in setting the overall reproductive trajectory, regardless of sex. (2) Each individual developmental stage is further characterized by marked sex-differential gene expression. (3) Consistent sexually differentiated gene expression regardless of developmental stage reveal candidates for high-level regulators of sex and include the female-specific poplar ARR17 homologue. There is also consistent male-biased expression of the MADS-box genes PISTILLATA and APETALA3. Our work provides insights into expression trajectories shaping reproductive development, its potential underlying mechanisms, and sex-specific translation of the genome information into reproductive structures in balsam poplar.

The BASIC PENTACYSTEINE (BPC) GAGA (C-box) binding proteins belong to a small plant transcription factor family. We previously reported that class I BPCs bind directly to C-boxes in the SEEDSTICK (STK) promoter and the mutagenesis of these cis-elements affects STK expression in the flower. The MADS-domain factor SHORT VEGETATIVE PHASE (SVP) is another key regulator of STK. Direct binding of SVP to CArG-boxes in the STK promoter are required to repress its expression during the first stages of flower development. Here we show that class II BPCs directly interact with SVP and that MADS-domain binding sites in the STK promoter region are important for the correct spatial and temporal expression of this homeotic gene. Furthermore, we show that class I and class II BPCs act redundantly to repress STK expression in the flower, most likely by recruiting TERMINAL FLOWER 2/LIKE HETEROCHROMATIN PROTEIN 1 (TFL2/LHP1) and mediating the establishment and the maintenance of H3K27me3 repressive marks on DNA. We investigate the role of LHP1 in the regulation of STK expression. In addition to providing a better understanding of the role of BPC transcription factors in the regulation of STK expression, our results suggest the existence of a more general regulatory complex composed of BPCs, MADS-domain factors and Polycomb Repressive Complexes that co-operate to regulate gene expression in reproductive tissues. We believe that our data along with the molecular model described here could provide significant insights for a more comprehensive understanding of gene regulation in plants., (© 2020 The Authors The Plant Journal © 2020 John Wiley & Sons Ltd.)

Main Conclusion: Simultaneous knockdown or knockout of Torenia fournieri PLENA (TfPLE) and FALINELLI (TfFAR) genes with RNAi or genome-editing technologies generated a multi-petal phenotype in torenia. The MADS-box gene AGAMOUS (AG) is well known to play important roles in the development of stamens and carpels in Arabidopsis. Mutations in AG cause the morphological transformation of stamens and carpels into petaloid organs. In contrast, torenia (Torenia fournieri Lind.) has two types of class-C MADS-box genes, PLENA (PLE) and FALINELLI (FAR); however, their functions were previously undetermined. To examine the function of TfPLE and TfFAR in torenia, we used RNAi to knockdown expression of these two genes. TfPLE and TfFAR double-knockdown transgenic torenia plants had morphologically altered stamens and carpels that developed into petaloid organs. TfPLE knockdown transgenic plants also exhibited morphological transformations that included shortened styles, enlarged ovaries, and absent stigmata. Furthermore, simultaneous disruption of TfPLE and TfFAR genes by CRISPR/Cas9-mediated genome editing also resulted in the conversion of stamens and carpels into petaloid organs as was observed in the double-knockdown transgenic plants mediated by RNAi. In addition, the carpels of one TfPLE knockout mutant had the same morphological abnormalities as TfPLE knockdown transgenic plants. TfFAR knockdown genome-edited mutants had no morphological changes in their floral organs. These results clearly show that TfPLE and TfFAR cooperatively play important roles in the development of stamens and carpels. Simultaneous disruption of TfPLE and TfFAR functions caused a multi-petal phenotype, which is expected to be a highly valuable commercial floral trait in horticultural flowers.

RIPENING INHIBITOR (RIN)-deficient fruits generated by CRISPR/Cas9 initiated partial ripening at a similar time to wild-type (WT) fruits but only 10% WT concentrations of carotenoids and ethylene (ET) were synthesized. RIN-deficient fruit never ripened completely, even when supplied with exogenous ET. The low amount of endogenous ET that they did produce was sufficient to enable ripening initiation and this could be suppressed by the ET perception inhibitor 1-MCP. The reduced ET production by RIN-deficient tomatoes was due to an inability to induce autocatalytic system-2 ET synthesis, a characteristic feature of climacteric ripening. Production of volatiles and transcripts of key volatile biosynthetic genes also were greatly reduced in the absence of RIN. By contrast, the initial extent and rates of softening in the absence of RIN were similar to WT fruits, although detailed analysis showed that the expression of some cell wall-modifying enzymes was delayed and others increased in the absence of RIN. These results support a model where RIN and ET, via ERFs, are required for full expression of ripening genes. Ethylene initiates ripening of mature green fruit, upregulates RIN expression and other changes, including system-2 ET production. RIN, ET and other factors are required for completion of the full fruit-ripening programme., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Main Conclusion: Tartary buckwheat rice-type cultivars, which allow easy dehulling, lacked periclinal cell divisions that proceed underneath the epidermis in the proximity of ovary midribs in non-rice-type cultivars. The easy dehulling in these cultivars was associated with a G→A substitution in an AGAMOUS ortholog. Ease of dehulling in Tartary buckwheat (Fagopyrum tataricum) can affect the quality of its products. Tartary buckwheat cultivars that allow easy dehulling are called rice-type cultivars. The rice and non-rice hull types are determined by a single gene, but this gene is unclear. Here, we show that cells underneath the epidermis in the proximity of ovary midribs undergo periclinal cell divisions in non-rice-type cultivars but do not in a rice-type cultivar. The cells that arose from the periclinal cell divisions later underwent lignification, which should increase mechanical strength of hulls. In RNA sequencing, a partial mRNA of an AGAMOUS ortholog in Tartary buckwheat (FtAG) was found to be absent in the rice-type cultivar. Cloning of this gene revealed that this is a 42-bp deletion due to a G→A substitution at a splice acceptor site in the FtAG genomic region. In F2 progeny derived from a cross between non-rice-type and rice-type cultivars, all the rice-type plants exhibited the homozygous A/A allele at this site, whereas all the Tartary-type plants exhibited either the homozygous G/G allele or the heterozygous A/G allele. These results suggest that FtAG is a candidate for the gene that determines ease of dehulling in Tartary buckwheat. The DNA marker that we developed to distinguish the FtAG alleles can be useful in breeding Tartary buckwheat cultivars.

The fungal pathogen Fusarium oxysporum f. sp. cubense (Foc) causes Fusarium wilt that affects banana plants. However, the detailed molecular mechanisms of Foc virulence determinants have not been elucidated. In this study, we identified the MADS-box transcription factor FoRlm1 that is conserved among mitogen-activated protein kinases. Our data revealed that FoRlm1 is essential for aerial hyphal growth and virulence. Transcriptional analysis revealed that FoRlm1 deletion altered the expression of anti-oxidant enzymes, chitin synthases, fusaric acid (FA), and beauvericin biosynthesis genes. Furthermore, FoRlm1 deletion promoted tolerance to Congo red and increased sensitivity to hydrogen peroxide. Transcriptome analysis of ΔFoRlm1 mutant and wild-type strain indicated that the expression of many genes associated with fungal physiology and virulence was up- or down-regulated. Overall, these results suggested that FoRlm1 plays a critical role in the regulation of hyphal growth, anti-oxidation mechanisms, cell wall biosynthesis, transcription of mycotoxin biosynthetic genes encoding FA and beauvericin, and virulence in Foc., (Copyright © 2020 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Flowering is a prerequisite for pear fruit production. Therefore, the development of flower buds and the control of flowering time are important for pear trees. However, the molecular mechanism of pear flowering is unclear. SOC1, a member of MADS-box family, is known as a flowering signal integrator in Arabidopsis. We identified eight SOC1-like genes in Pyrus bretschneideri and analyzed their basic information and expression patterns. Some pear SOC1-like genes were regulated by photoperiod in leaves. Moreover, the expression patterns were diverse during the development of pear flower buds. Two members of the pear SOC1-like genes, PbSOC1d and PbSOC1g, could lead to early flowering phenotype when overexpressed in Arabidopsis. PbSOC1d and PbSOC1g were identified as activators of the floral meristem identity genes AtAP1 and AtLFY and promote flowering time. These results suggest that PbSOC1d and PbSOC1g are promoters of flowering time and may be involved in flower bud development in pear., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2019. Published by Elsevier Inc.)

Upon fertilization, the ovary increases in size and undergoes a complex developmental process to become a fruit. We show that cytokinins (CKs), which are required to determine ovary size before fertilization, have to be degraded to facilitate fruit growth. The expression of CKX7, which encodes a cytosolic CK-degrading enzyme, is directly positively regulated post-fertilization by the MADS-box transcription factor STK. Similar to stk, two ckx7 mutants possess shorter fruits than wild type. Quantification of CKs reveals that stk and ckx7 mutants have high CK levels, which negatively control cell expansion during fruit development, compromising fruit growth. Overexpression of CKX7 partially complements the stk fruit phenotype, confirming a role for CK degradation in fruit development. Finally, we show that STK is required for the expression of FUL, which is essential for valve elongation. Overall, we provide insights into the link between CKs and molecular pathways that control fruit growth., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

As one of the most important events during the life cycle of flowering plants, the floral transition is of crucial importance for plant propagation and requires the precise coordination of multiple endogenous and external signals. There have been at least four flowering pathways (i.e. photoperiod, vernalization, gibberellin, and autonomous) identified in Arabidopsis. We previously reported that two Arabidopsis RNA-binding proteins, KHZ1 and KHZ2, redundantly promote flowering. However, the underlying mechanism was unclear. Here, we found that the double mutant khz1 khz2 flowered late under both long-day and short-day conditions, but responded to vernalization and gibberellin treatments. The late-flowering phenotype was almost completely rescued by mutating FLOWERING LOCUS C (FLC) and fully rescued by overexpressing FLOWERING LOCUS T (FT). Additional experiments demonstrated that the KHZs could form homodimers or interact to form heterodimers, localized to nuclear dots, and repressed the splicing efficiency of FLC pre-mRNA. Together, these data indicate that the KHZs could promote flowering via the autonomous pathway by repressing the splicing efficiency of FLC pre-mRNA., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Background: Floral transition initiates reproductive development of plants and occurs in response to environmental and endogenous signals. In Arabidopsis thaliana, this process is accelerated by several environmental cues, including exposure to long days. The photoperiod-dependent promotion of flowering involves the transcriptional induction of FLOWERING LOCUS T (FT) in the phloem of the leaf. FT encodes a mobile protein that is transported from the leaves to the shoot apical meristem, where it forms part of a regulatory complex that induces flowering. Whether FT also has biological functions in leaves of wild-type plants remains unclear., Results: In order to address this issue, we first studied the leaf transcriptomic changes associated with FT overexpression in the companion cells of the phloem. We found that FT induces the transcription of SWEET10, which encodes a bidirectional sucrose transporter, specifically in the leaf veins. Moreover, SWEET10 is transcriptionally activated by long photoperiods, and this activation depends on FT and one of its earliest target genes SUPPRESSOR OF CONSTANS OVEREXPRESSION 1 (SOC1). The ectopic expression of SWEET10 causes early flowering and leads to higher levels of transcription of flowering-time related genes in the shoot apex., Conclusions: Collectively, our results suggest that the FT-signaling pathway activates the transcription of a sucrose uptake/efflux carrier during floral transition, indicating that it alters the metabolism of flowering plants as well as reprogramming the transcription of floral regulators in the shoot meristem.

The palea and lemma are floral organ structures unique to grasses; these structures form the hull and directly affect grain size and quality. However, the molecular mechanisms controlling the development of the hull are not well understood. In this study, we characterized the rice (Oryza sativa) abnormal flower and grain1 (afg1) mutant, a new allele of OsMADS6. Similar to previously characterized osmads6 alleles, in the afg1 floret, the palea lost its marginal region and acquired the lemma identity. However, in contrast to other osmads6 alleles, the afg1 mutant showed altered grain size and grain quality, with decreased total starch and amylose contents, and increased protein and soluble sugar contents. The analysis of transcriptional activity suggested that AFG1 is a transcriptional activator and may affect grain size by regulating the expression levels of several genes related to cell expansion and proliferation in the afg1 mutant. These results revealed that AFG1 plays an important role in determining palea identity and affecting grain yield and quality in rice.

Key Message: A MADS-domain transcription factorLoSVP, which could delay flowering through vernalization pathway, was isolated from lily. MADS-domain transcription factors play important roles in plant growth and development, especially in the transition from vegetative phase to reproductive phase. However, their functions in bulbous flowering plants are largely unknown. In this work, a SHORT VEGETATIVE PHASE (SVP) encoding genes LoSVP from oriental lily was isolated. Bioinformatic analyses demonstrated that LoSVP encodes a type II MADS-box protein containing a conserved MADS-box, as well as a conserved K-box domain. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) revealed ubiquitous expression of LoSVP in various tissues, including petals, stamens, pistils, leaves and scales. Real-time polymerase chain reaction (PCR) analyses demonstrated that LoSVP was predominantly expressed in the early stage of developing flowers. Constitutive expression of LoSVP in Arabidopsis led to significantly delayed flowering of transgenic plants. These results suggest that LoSVP is involved in plant flowering and could be used as a potential candidate gene for the genetic regulation of flowering time in higher plants.

MADS box transcription factors (TFs) are subdivided into type I and II based on phylogenetic analysis. The type II TFs regulate floral organ identity and flowering time, but type I TFs are relatively less characterized. Here, we report the functional characterization of two type I MADS box TFs in rice ( Oryza sativa ), MADS78 and MADS79 Transcript abundance of both these genes in developing seed peaked at 48 h after fertilization and was suppressed by 96 h after fertilization, corresponding to syncytial and cellularized stages of endosperm development, respectively. Seeds overexpressing MADS78 and MADS 79 exhibited delayed endosperm cellularization, while CRISPR-Cas9-mediated single knockout mutants showed precocious endosperm cellularization. MADS78 and MADS 79 were indispensable for seed development, as a double knockout mutant failed to make viable seeds. Both MADS78 and 79 interacted with MADS89, another type I MADS box, which enhances nuclear localization. The expression analysis of Fie1 , a rice FERTILIZATION-INDEPENDENT SEED-POLYCOMB REPRESSOR COMPLEX2 component, in MADS78 and 79 mutants and vice versa established an antithetical relation, suggesting that Fie1 could be involved in negative regulation of MADS78 and MADS 79 Misregulation of MADS78 and MADS 79 perturbed auxin homeostasis and carbon metabolism, as evident by misregulation of genes involved in auxin transport and signaling as well as starch biosynthesis genes causing structural abnormalities in starch granules at maturity. Collectively, we show that MADS78 and MADS 79 are essential regulators of early seed developmental transition and impact both seed size and quality in rice., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Floral bud dormancy release of fruit tree species is greatly influenced by climate change. The lack of chilling accumulation often results in the occurrence of abnormal flower and low yields of sweet cherries ( Prunus avium L.) in warm regions. To investigate the regulation of dormancy in sweet cherries, six DAM genes with homology to peach DAM , designated PavDAM1-6 , have been identified and characterized. Phylogenetic analysis indicate that these genes are similar to DAMs in peach, apple and pear. The expression patterns of the PavDAMs in the low-chill cultivar 'Royal Lee' were different from that in the high-chill cultivar 'Hongdeng'. 'Royal Lee' exhibits lower transcriptional level of PavDAM1 compared to 'Hongdeng', especially at the stage of chilling accumulation, and transcriptional levels of PavDAM4/5 were high in both cultivars during the endodormancy. Ectopic expression of PavDAM1 and PavDAM5 in Arabidopsis resulted in plants with abnormal flower and seed development, especially the PavDAM5 . Higher transcriptional levels of SOC1 were observed in transgenic PavDAM1/5 lines, and ectopic expression of PavSOC1 had the similar floral phenotype. Further, protein interaction analysis demonstrated that PavDAM1/5 could interact with PavSOC1 in vivo and in vitro, which will help clarify the molecular mechanism of the flower development in sweet cherry or other fruit trees.

Flowering transition is regulated by complex genetic networks in response to endogenous and environmental signals. Pre-mRNA splicing is an essential step for the post-transcriptional regulation of gene expression. Alternative splicing of key flowering genes has been investigated in detail over the past decade. However, few splicing factors have been identified as being involved in flowering transition. Human heterodimeric splicing factor U2 snRNP auxiliary factor (U2AF) consists of two subunits, U2AF35 and U2AF65, and functions in 3' splice site recognition in mRNA splicing. Recent studies reveal that Arabidopsis U2AF65a/b and U2AF35a/b play important roles in the splicing of key flowering genes. We summarize recent advances in research on splicing-regulated flowering transition by focusing on the role of Arabidopsis U2AF in the splicing of key flowering-related genes at ambient temperature and in the abscisic acid signaling pathways., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

MADS-box genes interact with TB1 to regulate plant organ morphogenesis. In rice, OsMADS57 interacts with OsTB1 to control OsD14 transcription. In this study, we aimed to determine the relationships among these genes in barley. We identified a natural mutant of HvTB1 ( HvTB1 x) formed by a C→A transition at position 230, which resulted in a premature stop codon. We cloned the HvMADS57 and HvD14 genes and studied their expression in the tb1 mutant. The results showed that HvMADS57 is a MIKC c -type MADS-box gene, and the expression levels of both HvMADS57 and HvD14 were significantly reduced in the tb1 mutant when compared to those in the wildtype gene. These results indicate that, HvMADS57 regulates plant growth and development by interacting with HvTB1 to suppress the transcription of HvD14 in barley which is similar to the relationships among the orthologs of these genes in rice.

The timing of flowering is vital for plant reproductive success and is therefore tightly regulated by endogenous and exogenous cues. In summer annual Arabidopsis ( Arabidopsis thaliana ) accessions, like Columbia-0, rapid flowering is promoted by repression of the floral repressor FLOWERING LOCUS C ( FLC ). This is through the activity of the autonomous pathway, a group of proteins with diverse functions including RNA 3'-end processing factors, spliceosome components, a transcription elongation factor, and chromatin modifiers. These factors function at the FLC locus linking alternative processing of an antisense long noncoding RNA, called COOLAIR , with delivery of a repressive chromatin environment that affects the transcriptional output. The transcriptional output feeds back to influence the chromatin environment, reinforcing and stabilizing that state. This review summarizes our current knowledge of the autonomous pathway and compares it with similar cotranscriptional mechanisms in other organisms., (© 2020 The authors. All Rights Reserved.)

Wheat (Triticum aestivum) is one of the most important crops worldwide. Given a growing global population coupled with increasingly challenging cultivation conditions, facilitating wheat breeding by fine-tuning important traits is of great importance. MADS-box genes are prime candidates for this, as they are involved in virtually all aspects of plant development. Here, we present a detailed overview of phylogeny and expression of 201 wheat MIKC-type MADS-box genes. Homoeolog retention is significantly above the average genome-wide retention rate for wheat genes, indicating that many MIKC-type homoeologs are functionally important and not redundant. Gene expression is generally in agreement with the expected subfamily-specific expression pattern, indicating broad conservation of function of MIKC-type genes during wheat evolution. We also found extensive expansion of some MIKC-type subfamilies, especially those potentially involved in adaptation to different environmental conditions like flowering time genes. Duplications are especially prominent in distal telomeric regions. A number of MIKC-type genes show novel expression patterns and respond, for example, to biotic stress, pointing towards neofunctionalization. We speculate that conserved, duplicated and neofunctionalized MIKC-type genes may have played an important role in the adaptation of wheat to a diversity of conditions, hence contributing to the importance of wheat as a global staple food., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Seed germination is a crucial transition point in plant life and is tightly regulated by environmental conditions through the coordination of two phytohormones, gibberellin and abscisic acid (ABA). To avoid unfavorable conditions, plants have evolved safeguard mechanisms for seed germination. The present contribution reports a novel function of the Arabidopsis MCM1/AGAMOUS/DEFICIENS/SRF(MADS)-box transcription factor ARABIDOPSIS NITRATE REGULATED 1 (ANR1) in seed germination. ANR1 knockout mutant is insensitive to ABA, salt and osmotic stress during the seed germination and early seedling development stages, whereas ANR1-overexpressing lines are hypersensitive. ANR1 is responsive to ABA and abiotic stresses and upregulates the expression of ABA Intolerant (ABI)3 to suppress seed germination. ANR1 and ABI3 have similar expression pattern during seed germination. Genetically, ABI3 acts downstream of ANR1. Chromatin immunoprecipitation and yeast-one-hybrid assays showed that ANR1 could bind to the ABI3 promoter to regulate its expression. In addition, ANR1 acts synergistically with AGL21 to suppress seed germination in response to ABA as evidenced by anr1 agl21 double mutant. Taken together, the results herein demonstrate that the ANR1 plays an important role in regulating seed germination and early postgermination growth. ANR1 and AGL21 together constitutes a safeguard mechanism for seed germination to avoid unfavorable conditions., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Plants with winter annual life history germinate in summer or autumn and require a period of prolonged winter cold to initiate flowering, known as vernalization. In the Brassicaceae, the requirement for vernalization is conferred by high expression of orthologs of the FLOWERING LOCUS C (FLC) gene, the expression of which is known to be silenced by prolonged exposure to winter-like temperatures [1]. Based on a wealth of vernalization experiments, typically carried out in the range of 5°C-10°C, we would expect field environments during winter to induce flowering in crops with winter annual life history. Here, we show that, in the case of winter oilseed rape, expression of multiple FLC orthologs declines not during winter but predominantly during October when the average air temperature is 10°C-15°C. We further demonstrate that plants proceed through the floral transition in early November and overwinter as inflorescence meristems, which complete floral development in spring. To validate the importance of pre-winter temperatures in flowering time control, we artificially simulated climate warming in field trial plots in October. We found that increasing the temperature by 5°C in October results in raised FLC expression and delays the floral transition by 3 weeks but only has a mild effect on flowering date the following spring. Our work shows that winter annuals overwinter as a floral bud in a manner that resembles perennials and highlights the importance of studying signaling events in the field for understanding how plants transition to flowering under real environmental conditions., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)