MADS-box Genes Research Articles

Molecular Characterization and Expression Patterns of the HkSVP Gene Reveal Distinct Roles in Inflorescence Structure and Floral Organ Development in Hemerocallis fulva.

SHORT VEGETATIVE PHASE (SVP) genes are members of the well-known MADS-box gene family that play a key role in regulating vital developmental processes in plants. Hemerocallis are perennial herbs that exhibit continuous flowering development and have been extensively used in landscaping. However, there are few reports on the regulatory mechanism of flowering in Hemerocallis. To better understand the molecular basis of floral formation of Hemerocallis, we identified and characterized the SVP-like gene HkSVP from the Hemerocallis cultivar ‘Kanai Sensei’. Quantitative RT-PCR (qRT-PCR) indicated that HkSVP transcript was mainly expressed in the vegetative growth stage and had the highest expression in leaves, low expression in petals, pedicels and fruits, and no expression in pistils. The HkSVP encoded protein was localized in the nucleus of Arabidopsis protoplasts and the nucleus of onion epidermal cells. Yeast two hybrid assay revealed that HKSVP interacted with Hemerocallis AP1 and TFL1. Moreover, overexpression of HkSVP in Arabidopsis resulted in delayed flowering and abnormal phenotypes, including enriched trichomes, increased basal inflorescence branches and inhibition of inflorescence formation. These observations suggest that the HkSVP gene may play an important role in maintaining vegetative growth by participating in the construction of inflorescence structure and the development of flower organs.

Interactions between SQUAMOSA and SHORT VEGETATIVE PHASE MADS-box proteins regulate meristem transitions during wheat spike development.

Inflorescence architecture is an important determinant of crop productivity. The number of spikelets produced by the wheat inflorescence meristem (IM) before its transition to a terminal spikelet (TS) influences the maximum number of grains per spike. Wheat MADS-box genes VERNALIZATION 1 (VRN1) and FRUITFULL 2 (FUL2) (in the SQUAMOSA-clade) are essential to promote the transition from IM to TS and for spikelet development. Here we show that SQUAMOSA genes contribute to spikelet identity by repressing MADS-box genes VEGETATIVE TO REPRODUCTIVE TRANSITION 2 (VRT2), SHORT VEGETATIVE PHASE 1 (SVP1), and SVP3 in the SVP clade. Constitutive expression of VRT2 resulted in leafy glumes and lemmas, reversion of spikelets to spikes, and downregulation of MADS-box genes involved in floret development, whereas the vrt2 mutant reduced vegetative characteristics in spikelets of squamosa mutants. Interestingly, the vrt2 svp1 mutant showed similar phenotypes to squamosa mutants regarding heading time, plant height, and spikelets per spike, but it exhibited unusual axillary inflorescences in the elongating stem. We propose that SQUAMOSA–SVP interactions are important to promote heading, formation of the TS, and stem elongation during the early reproductive phase, and that downregulation of SVP genes is then necessary for normal spikelet and floral development. Manipulating SVP and SQUAMOSA genes can contribute to engineering spike architectures with improved productivity.

The Elite Alleles of OsSPL4 Regulate Grain Size and Increase Grain Yield in Rice

Grain weight and grain number, the two important yield traits, are mainly determined by grain size and panicle architecture in rice. Herein, we report the identification and functional analysis of OsSPL4 in panicle and grain development of rice. Using CRISPR/Cas9 system, two elite alleles of OsSPL4 were obtained, which exhibited an increasing number of grains per panicle and grain size, resulting in increase of rice yield. Cytological analysis showed that OsSPL4 could regulate spikelet development by promoting cell division. The results of RNA-seq and qRT-PCR validations also demonstrated that several MADS-box and cell-cycle genes were up-regulated in the mutation lines. Co-expression network revealed that many yield-related genes were involved in the regulation network of OsSPL4. In addition, OsSPL4 could be cleaved by the osa-miR156 in vivo, and the OsmiR156-OsSPL4 module might regulate the grain size in rice. Further analysis indicated that the large-grain allele of OsSPL4 in indica rice might introgress from aus varieties under artificial selection. Taken together, our findings suggested that OsSPL4 could be as a key regulator of grain size by acting on cell division control and provided a strategy for panicle architecture and grain size modification for yield improvement in rice.

Cloning, Expression, and Tobacco Overexpression Analyses of a PISTILLATA/GLOBOSA-like (OfGLO1) Gene from Osmanthus fragrans.

GLOBOSA (GLO), a B-class MADS-box gene, is involved in floral organ determination but has rarely been studied in Osmanthus fragrans, which is a very popular ornamental tree species in China. Here, the full-length cDNA of a homologous GLO1 gene (named OfGLO1) was cloned from a flower bud of O. fragrans using the RACE technique. The OfGLO1 has a 645 bp open reading frame, encoding 214 amino acids. Similar to other PI/GLO proteins, OfGLO1 has two conserved domains, MADS MEF2-like and K-box, and a 16-amino-acid PI motif in the C terminal region. Our phylogeny analysis classified OfGLO1 as a PI-type member of the B-class MADS-box gene family. The qRT-PCR assay showed that the expression of OfGLO1 in O. fragrans was continuously upregulated from the tight bud stage to the full flowering stage but barely expressed in the pistils, sepals, and non-floral organs, such as root, leaf, and stem. The genetic effect of OfGLO1 was assayed by ectopic expression in tobacco plants. Compared with the wild-type, OfGLO1 transformants showed reduced plant size, earlier flowering, shorter stamens, and lower seed setting rates. Furthermore, some stamens were changed into petal-like structures. These findings indicate that OfGLO1 plays an important role in the regulation of flower development. This study improved our understanding of class B gene function in woody plants.

The Melastoma dodecandrum genome and the evolution of Myrtales

Melastomataceae has abundant morphological diversity with high economic and ornamental merit in Myrtales. The phylogenetic position of Myrtales is still contested. Here, we report the chromosome-level genome assembly of Melastoma dodecandrum in Melastomataceae. The assembled genome size is 299.81 Mb with a contig N50 value of 3.00 Mb. Genome evolution analysis indicated that M. dodecandrum, Eucalyptus grandis, and Punica granatum were clustered into a clade of Myrtales and formed a sister group with the ancestor of fabids and malvids. We found that M. dodecandrum experienced four whole-genome polyploidization events: the ancient event was shared with most eudicots, one event was shared with Myrtales, and the other two events were unique to M. dodecandrum. Moreover, we identified MADS-box genes and found that the AP1-like genes expanded, and AP3-like genes might have undergone subfunctionalization. The SUAR63-like genes and AG-like genes showed different expression patterns in stamens, which may be associated with heteranthery. In addition, we found that LAZY1-like genes were involved in the negative regulation of stem branching development, which may be related to its creeping features. Our study sheds new light on the evolution of Melastomataceae and Myrtales, which provides a comprehensive genetic resource for future research.

Overexpression of the MADS-box gene SIMBP21 alters leaf morphology and affects reproductive development in tomato

Fruit yield is the most important horticultural trait of tomato. SlMBP21, a SEPALLATA subclass MADS-box gene has been reported to have functions in regulating pedicel abscission zone identity and development and controlling sepal size in tomato. However, we generated transgenic tomato plants which overexpress SlMBP21 and found the transformants displayed curly leaves, abnormally shaped flowers with twisted and opened stamens, reduced yield parameters, and small and light seeds. Our studies on the gain-of-function phenotype and gene expression level showed that its novel aspects played important roles in determining leaf morphology, flower and inflorescence architecture, and seed size, as well as the fruit yield. Overexpression of SlMBP21 in tomato resulted in curly leaves with fewer leaflets due to the regulation of the critical leaf polarity genes that cause an imbalance between the midvein adaxial–abaxial cell growth. Defects in the architecture of flowers and inflorescences resulted in reduced fruit set. Furthermore, we demonstrated that SlMBP21 plays its role through inhibiting the expression of the genes involved in the determination of seed development in tomato and SlMBP21 protein can interact with other MADS-box protein (SlAGL11, TAGL1 and SlMBP3) to control seed size. Thus, these results suggest that overexpression of SlMBP21 causes multiple types of damage to plant growth and development, especially fruit yield, in tomato.

Genome-Wide Analysis and the Expression Pattern of the MADS-Box Gene Family in Bletilla striata.

Bletilla striata (Thunb. ex A. Murray) Rchb. f., a species of the perennial herb Orchidaceae, has potent anti-inflammatory and antiviral biological activities. MADS-box transcription factors play critical roles in the various developmental processes of plants. Although this gene family has been extensively investigated in many species, it has not been analyzed for B. striata. In total, 45 MADS-box genes were identified from B. striata in this study, which were classified into five subfamilies (Mδ, MIKC, Mα, Mβ, and Mγ). Meanwhile, the highly correlated protein domains, motif compositions, and exon–intron structures of BsMADSs were investigated according to local B. striata databases. Chromosome distribution and synteny analyses revealed that segmental duplication and homologous exchange were the main BsMADSs expansion mechanisms. Further, RT-qPCR analysis revealed that BsMADSs had different expression patterns in response to various stress treatments. Our results provide a potential theoretical basis for further investigation of the functions of MADS genes during the growth of B. striata.

Genome-Wide Identification and Analysis of the MADS-Box Gene Family in Theobroma cacao

Genome-Wide Identification and Analysis of the MADS-Box Gene Family in Theobroma cacao

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.

Comparative genome-wide analysis of WRKY, MADS-box and MYB transcription factor families in Arabidopsis and rice

Transcription factors (TFs) form the major class of regulatory genes and play key roles in multiple plant stress responses. In most eukaryotic plants, transcription factor (TF) families (WRKY, MADS-box and MYB) activate unique cellular-level abiotic and biotic stress-responsive strategies, which are considered as key determinants for defense and developmental processes. Arabidopsis and rice are two important representative model systems for dicot and monocot plants, respectively. A comprehensive comparative study on 101 OsWRKY, 34 OsMADS box and 122 OsMYB genes (rice genome) and, 71 AtWRKY, 66 AtMADS box and 144 AtMYB genes (Arabidopsis genome) showed various relationships among TFs across species. The phylogenetic analysis clustered WRKY, MADS-box and MYB TF family members into 10, 7 and 14 clades, respectively. All clades in WRKY and MYB TF families and almost half of the total number of clades in the MADS-box TF family are shared between both species. Chromosomal and gene structure analysis showed that the Arabidopsis-rice orthologous TF gene pairs were unevenly localized within their chromosomes whilst the distribution of exon–intron gene structure and motif conservation indicated plausible functional similarity in both species. The abiotic and biotic stress-responsive cis-regulatory element type and distribution patterns in the promoter regions of Arabidopsis and rice WRKY, MADS-box and MYB orthologous gene pairs provide better knowledge on their role as conserved regulators in both species. Co-expression network analysis showed the correlation between WRKY, MADs-box and MYB genes in each independent rice and Arabidopsis network indicating their role in stress responsiveness and developmental processes.

The MADS-box gene PpPI is a key regulator of the double-flower trait in peach.

Double flower is an invaluable trait in ornamental peach, but the mechanism underlying its development remains largely unknown. Here, we report the roles of ABCE model genes in double flower development in peach. A total of nine ABCE regulatory genes, including eight MADS-box genes and one AP2/EREBP gene, were identified in the peach genome. Subcellular localization assay showed that all the ABCE proteins were localized in the nucleus. Four genes, PpAP1, PpAP3, PpSEP3, and PpPI, showed a difference in expression levels between single and double flowers. Ectopic overexpression of PpPI increased petal number in Arabidopsis, while transgenic lines overexpressing PpAP3 or PpSEP3 were morphologically similar to wild-type. Ectopic overexpression of PpAP1 resulted in a significant decrease in the number of basal leaves and caused early flowering. These results suggest that PpPI is likely crucial for double flower development in peach. In addition, double flowers have petaloid sepals and stamens, and single flower could occasionally change to be double flower by converting stamens to petals in peach, suggesting that the double-flower trait is likely to have evolved from an ancestral single-flower structure. Our results provide new insights into mechanisms underlying the double-flower trait in peach.

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.

Genome-Wide Identification MIKC-Type MADS-Box Gene Family and Their Roles during Development of Floral Buds in Wheel Wingnut (Cyclocarya paliurus).

MADS-box transcription factors (TFs) have fundamental roles in regulating floral organ formation and flowering time in flowering plants. In order to understand the function of MIKC-type MADS-box family genes in Cyclocarya paliurus (Batal.) Iljinskaja, we first implemented a genome-wide analysis of MIKC-type MADS-box genes in C. paliurus. Here, the phylogenetic relationships, chromosome location, conserved motif, gene structure, promoter region, and gene expression profile were analyzed. The results showed that 45 MIKC-type MADS-box were divided into 14 subfamilies: BS (3), AGL12 (1), AP3-PI (3), MIKC* (3), AGL15 (3), SVP (5), AGL17 (2), AG (3), TM8 (1), AGL6 (2), SEP (5), AP1-FUL (6), SOC1 (7), and FLC (1). The 43 MIKC-type MADS-box genes were distributed unevenly in 14 chromosomes, but two members were mapped on unanchored scaffolds. Gene structures were varied in the same gene family or subfamily, but conserved motifs shared similar distributions and sequences. The element analysis in promoters’ regions revealed that MIKC-type MADS-box family genes were associated with light, phytohormone, and temperature responsiveness, which may play important roles in floral development and differentiation. The expression profile showed that most MIKC-type MADS-box genes were differentially expressed in six tissues (specifically expressed in floral buds), and the expression patterns were also visibly varied in the same subfamily. CpaF1st24796 and CpaF1st23405, belonging to AP3-PI and SEP subfamilies, exhibited the high expression levels in PA-M and PG-F, respectively, indicating their functions in presenting heterodichogamy. We further verified the MIKC-type MADS-box gene expression levels on the basis of transcriptome and qRT-PCR analysis. This study would provide a theoretical basis for classification, cloning, and regulation of flowering mechanism of MIKC-type MADS-box genes in C. paliurus.

A high-quality genome assembly of Jasminum sambac provides insight into floral trait formation and Oleaceae genome evolution.

As one of the most economically significant Oleaceae family members, Jasminum sambac is renowned for its distinct sweet, heady fragrance. Using Illumina reads, Nanopore long reads, and HiC-sequencing, we efficiently assembled and annotated the J. sambac genome. The high-quality genome assembly consisted of a total of 507Mb sequence (contig N50=17.6Mb) with 13pseudomolecules. A total of 21,143 protein-coding genes and 303Mb repeat sequences were predicted. An ancient whole-genome triplication event at the base of Oleaceae (~66million years ago [Ma], Late Cretaceous) was identified and this may have contributed to the diversification of the Oleaceae ancestor and its divergence from the Lamiales. Stress-related (e.g., WRKY) and flowering-related (e.g., MADS-box) genes were located in the triplicated regions, suggesting that the polyploidy event might have contributed adaptive potential. Genes related to terpenoid biosynthesis, for example, FTA and TPS,were observed to be duplicated to a great extent in the J. sambac genome, perhaps explaining the strong fragrance of the flowers. Copy number changes in distinct phylogenetic clades of the MADS-box family were observed in J. sambac genome, for example, AGL6- and Mα- were lost and SOC- expanded, features that might underlie the long flowering period of J. sambac. The structural genes implicated in anthocyanin biosynthesis were depleted and this may explain the absence of vivid colours in jasmine. Collectively, assembling the J. sambac genome provides new insights into the genome evolution of the Oleaceae family and provides mechanistic insights into floral properties.

Development of the petaloid bracts of a paleoherb species, Saururus chinensis.

Saururus chinensis is a core member of Saururaceae, an ancient, perianthless (lacking petals or sepals) family of the magnoliids in the Mesangiospermae, which is important for understanding the origin and evolution of early flowers due to its unusual floral composition and petaloid bracts. To compare their transcriptomes, RNA-seq abundance analysis identified 43,463 genes that were found to be differentially expressed in S. chinensis bracts. Of these, 5,797 showed significant differential expression, of which 1,770 were up-regulated and 4,027 down-regulated in green compared to white bracts. The expression profiles were also compared using cDNA microarrays, which identified 166 additional differentially expressed genes. Subsequently, qRT-PCR was used to verify and extend the cDNA microarray results, showing that the A and B class MADS-box genes were up-regulated in the white bracts. Phylogenetic analysis was performed on putative S. chinensis A and B-class of MADS-box genes to infer evolutionary relationships within the A and B-class of MADS-box gene family. In addition, nature selection and protein interactions of B class MADS-box proteins were inferred that B-class genes free from evolutionary pressures. The results indicate that petaloid bracts display anatomical and gene expression features normally associated with petals, as found in petaloid bracts of other species, and support an evolutionarily conserved developmental program for petaloid bracts.

Genome-wide analysis of the MADS-box gene family in Rhododendron hainanense Merr. and expression analysis under heat and waterlogging stresses

The MADS-box transcription factor gene family plays an important role in the growth and development of plants as well as in biotic and abiotic stress. We conducted a genome-wide identification of the members of the MADS-box family of Rhododendron hainanense to determine the molecular evolutionary characteristics of the MADS-box gene family and the expression profile of this gene family under different abiotic stress conditions. A total of 55 MADS-box gene family members with complete domains were identified, which were unevenly distributed on 13 chromosomes. The construction of a phylogenetic tree showed that the 55 MADS-box proteins were divided into five categories and distributed on 8 chromosomes unevenly. Conserved motif analysis shows that motif 1 and motif 2 are highly conserved in most RhMADS proteins. Proteins belonging to the same category show similar gene structures. Analysis of gene replication events suggested that there were one pair of tandem repeats and eight pairs of fragment repeat events. In addition, analysis of cis-acting elements and protein interaction networks indicated that some genes are involved in abiotic stress processes in plants. The transcriptome data were used to analyze the expression of the members of the MADS-box family under different temperatures and waterlogging period treatments, which were then verified by qRT-PCR. The expressions of RhMADS24, RhMADS25, RhMADS39 and RhMADS44 were significantly regulated under temperature and waterlogging stresses. The identified MADS-box candidate genes of R. hainanense play an important role in resisting abiotic stress and lay the foundation for future applications in breeding and gardening.

Transcript Profiling of MIKCc MADS-Box Genes Reveals Conserved and Novel Roles in Barley Inflorescence Development.

MADS-box genes have a wide range of functions in plant reproductive development and grain production. The ABCDE model of floral organ development shows that MADS-box genes are central players in these events in dicotyledonous plants but the applicability of this model remains largely unknown in many grass crops. Here, we show that transcript analysis of all MIKCc MADS-box genes through barley (Hordeum vulgare L.) inflorescence development reveals co-expression groups that can be linked to developmental events. Thirty-four MIKCc MADS-box genes were identified in the barley genome and single-nucleotide polymorphism (SNP) scanning of 22,626 barley varieties revealed that the natural variation in the coding regions of these genes is low and the sequences have been extremely conserved during barley domestication. More detailed transcript analysis showed that MADS-box genes are generally expressed at key inflorescence developmental phases and across various floral organs in barley, as predicted by the ABCDE model. However, expression patterns of some MADS genes, for example HvMADS58 (AGAMOUS subfamily) and HvMADS34 (SEPALLATA subfamily), clearly deviate from predicted patterns. This places them outside the scope of the classical ABCDE model of floral development and demonstrates that the central tenet of antagonism between A- and C-class gene expression in the ABC model of other plants does not occur in barley. Co-expression across three correlation sets showed that specifically grouped members of the barley MIKCc MADS-box genes are likely to be involved in developmental events driving inflorescence meristem initiation, floral meristem identity and floral organ determination. Based on these observations, we propose a potential floral ABCDE working model in barley, where the classic model is generally upheld, but that also provides new insights into the role of MIKCc MADS-box genes in the developing barley inflorescence.

Chromosome-scale assembly of the Dendrobium chrysotoxum genome enhances the understanding of orchid evolution

As one of the largest families of angiosperms, the Orchidaceae family is diverse. Dendrobium represents the second largest genus of the Orchidaceae. However, an assembled high-quality genome of species in this genus is lacking. Here, we report a chromosome-scale reference genome of Dendrobium chrysotoxum, an important ornamental and medicinal orchid species. The assembled genome size of D. chrysotoxum was 1.37 Gb, with a contig N50 value of 1.54 Mb. Of the sequences, 95.75% were anchored to 19 pseudochromosomes. There were 30,044 genes predicted in the D. chrysotoxum genome. Two whole-genome polyploidization events occurred in D. chrysotoxum. In terms of the second event, whole-genome duplication (WGD) was also found to have occurred in other Orchidaceae members, which diverged mainly via gene loss immediately after the WGD event occurred; the first duplication was found to have occurred in most monocots (tau event). We identified sugar transporter (SWEET) gene family expansion, which might be related to the abundant medicinal compounds and fleshy stems of D. chrysotoxum. MADS-box genes were identified in D. chrysotoxum, as well as members of TPS and Hsp90 gene families, which are associated with resistance, which may contribute to the adaptive evolution of orchids. We also investigated the interplay among carotenoid, ABA, and ethylene biosynthesis in D. chrysotoxum to elucidate the regulatory mechanisms of the short flowering period of orchids with yellow flowers. The reference D. chrysotoxum genome will provide important insights for further research on medicinal active ingredients and breeding and enhances the understanding of orchid evolution.

High-quality genome assembly of 'Cuiguan' pear (Pyrus pyrifolia) as a reference genome for identifying regulatory genes and epigenetic modifications responsible for bud dormancy

Dormancy-associated MADS-box (DAM) genes serve as crucial regulators of the endodormancy cycle in rosaceous plants. Although pear DAM genes have been identified previously, the lack of a high-quality reference genome and techniques to study gene function have prevented accurate genome-wide analysis and functional verification of such genes. Additionally, the contribution of other genes to the regulation of endodormancy release remains poorly understood. In this study, a high-quality genome assembly for 'Cuiguan' pear (Pyrus pyrifolia), which is a leading cultivar with a low chilling requirement cultivated in China, was constructed using PacBio and Hi-C technologies. Using this genome sequence, we revealed that pear DAM genes were tandemly clustered on Chr8 and Chr15 and were differentially expressed in the buds between 'Cuiguan' and the high-chilling-requirement cultivar 'Suli' during the dormancy cycle. Using a virus-induced gene silencing system, we determined the repressive effects of DAM genes on bud break. Several novel genes potentially involved in the regulation of endodormancy release were identified by RNA sequencing and H3K4me3 chromatin immunoprecipitation sequencing analyses of 'Suli' buds during artificial chilling using the new reference genome. Our findings enrich the knowledge of the regulatory mechanism underlying endodormancy release and chilling requirements and provide a foundation for the practical regulation of dormancy release in fruit trees as an adaptation to climate change.

Genome-wide identification of jujube witches’ broom phytoplasma effectors revealed the role of SJP3 in inducing phyllody

Jujube witches’ broom (JWB), associated with ‘Candidatus Phytoplasma ziziphi’, is a devastating disease in Chinese jujube (Ziziphus jujuba Mill.). One of the symptoms in plants infected with phytoplasmas is the change of floral organs into leaf-like structures, known as phyllody. Effector proteins secreted by phytoplasmas are key to inducing this and other witches’ broom symptoms. However, the phytoplasma effector proteins that induce phyllody in jujube remain unknown. We identified 43 candidate secreted JWB phytoplasma proteins (SJPs) in the JWB phytoplasma genome. Eight of the predicted candidate effector genes exhibited high transcript levels in infected flower buds. Among the eight candidate effectors, SJP3 experimentally showed the potential to induce phyllody. SJP3 expression in infected jujube was at a high level in young leaves and flowers. Ectopic expression of SJP3 in Arabidopsis induced leaf-like flowers and extra secondary flowers and significantly changed the transcript levels of PISTILLATA (PI), SHORT VEGETATIVE PHASE (SVP), AGAMOUS-LIKE 24 (AGL24) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1). The expression levels of these MADS-box homologous genes were also changed in infected jujube flower buds and all four whorls of the floral organs. The data showed that the effector SJP3 disrupts expression of several MADS-box genes associated with floral organ identity and flowering time and is likely key to inducing phyllody in phytoplasma-infected jujube. This study provides new insight into understanding the pathogenesis of JWB disease induced by phytoplasma.