Serial Section-Based Three-Dimensional Reconstruction of Anaxagorea (Annonaceae) Carpel Vasculature and Implications for the Morphological Relationship between the Carpel and the Ovule

Elucidating the origin of flowers has been a challenge in botany for a long time. One of the central questions surrounding the origin of flowers is how to interpret the carpel, especially the relationship between the phyllome part (carpel wall) and the ovule. Recently, consensus favors the carpel originating from the fusion of an ovule-bearing part and the phyllome part that subtends it. Considering the carpel is a complex organ, the accurate presentation of the anatomical structure of the carpel is necessary for resolving this question. Anaxagorea is the most basal genus in a primitive angiosperm family, Annonaceae. The conspicuous stipe at the base of each carpel makes it an ideal material for exploring the histological relationships among the receptacle, the carpel, and the ovule. In the present study, floral organogenesis and vasculature were delineated in Anaxagorea luzonensis and Anaxagorea javanica, and a three-dimensional model of the carpel vasculature was reconstructed based on serial sections. The results show that in Anaxagorea, the vasculature in the carpel branches in the form of shoots. The radiosymmetrical vasculature pattern is repeatedly presented in the receptacle, the carpel, and the funiculus of the ovule. This provides anatomical evidence of the composite origin of the carpel.

Arabidopsis LSH8 Positively Regulates ABA Signaling by Changing the Expression Pattern of ABA-Responsive Proteins

Phytohormone ABA regulates the expression of numerous genes to significantly affect seed dormancy, seed germination and early seedling responses to biotic and abiotic stresses. However, the function of many ABA-responsive genes remains largely unknown. In order to improve the ABA-related signaling network, we conducted a large-scale ABA phenotype screening. LSH, an important transcription factor family, extensively participates in seedling development and floral organogenesis in plants, but whether its family genes are involved in the ABA signaling pathway has not been reported. Here we describe a new function of the transcription factor LSH8 in an ABA signaling pathway. In this study, we found that LSH8 was localized in the nucleus, and the expression level of LSH8 was significantly induced by exogenous ABA at the transcription level and protein level. Meanwhile, seed germination and root length measurements revealed that lsh8 mutant lines were ABA insensitive, whereas LSH8 overexpression lines showed an ABA-hypersensitive phenotype. With further TMT labeling quantitative proteomic analysis, we found that under ABA treatment, ABA-responsive proteins (ARPs) in the lsh8 mutant presented different changing patterns with those in wild-type Col4. Additionally, the number of ARPs contained in the lsh8 mutant was 397, six times the number in wild-type Col4. In addition, qPCR analysis found that under ABA treatment, LSH8 positively mediated the expression of downstream ABA-related genes of ABI3, ABI5, RD29B and RAB18. These results indicate that in Arabidopsis, LSH8 is a novel ABA regulator that could specifically change the expression pattern of APRs to positively mediate ABA responses.

Genome-Wide Identification and Analysis of the MADS-Box Gene Family in American Beautyberry (Callicarpa americana)

The MADS-box gene family encodes a number of transcription factors that play key roles in various plant growth and development processes from response to environmental cues to cell differentiation and organ identity, especially the floral organogenesis, as in the prominent ABCDE model of flower development. Recently, the genome of American beautyberry (Callicarpa americana) has been sequenced. It is a shrub native to the southern region of United States with edible purple-colored berries; it is a member of the Lamiaceae family, a family of medical and agricultural importance. Seventy-eight MADS-box genes were identified from 17 chromosomes of the C. americana assembled genome. Peptide sequences blast and analysis of phylogenetic relationships with MADS-box genes of Sesame indicum, Solanum lycopersicum, Arabidopsis thaliana, and Amborella trichopoda were performed. Genes were separated into 32 type I and 46 type II MADS-box genes. C. americana MADS-box genes were clustered into four groups: MIKCC, MIKC*, Mα-type, and Mγ-type, while the Mβ-type group was absent. Analysis of the gene structure revealed that from 1 to 15 exons exist in C. americana MADS-box genes. The number of exons in type II MADS-box genes (5–15) greatly exceeded the number in type I genes (1–9). The motif distribution analysis of the two types of MADS-box genes showed that type II MADS-box genes contained more motifs than type I genes. These results suggested that C. americana MADS-box genes type II had more complex structures and might have more diverse functions. The role of MIKC-type MADS-box genes in flower and fruit development was highlighted when the expression profile was analyzed in different organs transcriptomes. This study is the first genome-wide analysis of the C. americana MADS-box gene family, and the results will further support any functional and evolutionary studies of C. americana MADS-box genes and serve as a reference for related studies of other plants in the medically important Lamiaceae family.

Identification and Characterization of MIKCc-Type MADS-Box Genes in the Flower Organs of Adonis amurensis

Adonis amurensis is a perennial herbaceous flower that blooms in early spring in northeast China, where the night temperature can drop to −15 °C. To understand flowering time regulation and floral organogenesis of A. amurensis, the MIKCc-type MADS (Mcm1/Agamous/ Deficiens/Srf)-box genes were identified and characterized from the transcriptomes of the flower organs. In this study, 43 non-redundant MADS-box genes (38 MIKCc, 3 MIKC*, and 2 Mα) were identified. Phylogenetic and conserved motif analysis divided the 38 MIKCc-type genes into three major classes: ABCDE model (including AP1/FUL, AP3/PI, AG, STK, and SEPs/AGL6), suppressor of overexpression of constans1 (SOC1), and short vegetative phase (SVP). qPCR analysis showed that the ABCDE model genes were highly expressed mainly in flowers and differentially expressed in the different tissues of flower organs, suggesting that they may be involved in the flower organ identity of A. amurensis. Subcellular localization revealed that 17 full-length MADSs were mainly localized in the nucleus: in Arabidopsis, the heterologous expression of three full-length SOC1-type genes caused early flowering and altered the expression of endogenous flowering time genes. Our analyses provide an overall insight into MIKCc genes in A. amurensis and their potential roles in floral organogenesis and flowering time regulation.

Phytohormone and integrated mRNA and miRNA transcriptome analyses and differentiation of male between hermaphroditic floral buds of andromonoecious Diospyros kaki Thunb

Background Persimmon (Diospyros kaki Thunb.) has various labile sex types, and studying its sex differentiation can improve breeding efficiency. However, studies on sexual regulation patterns in persimmon have focused mainly on monoecy and dioecy, whereas little research has been published on andromonoecy. In order to reveal the sex differentiation regulation mechanism of andromonoecious persimmon, we performed histological and cytological observations, evaluated OGI and MeGI expression and conducted phytohormones assays and mRNA and small RNA transcriptome analyses of the male and hermaphroditic floral buds of the andromonoecious persimmon ‘Longyanyeshi 1’. Results Stages 2 and 4 were identified as the critical morphological periods for sex differentiation of ‘Longyanyeshi 1’ by histological and cytological observation. At both stages, OGI was differentially expressed in male and hermaphroditic buds, but MeGI was not. This was different from their expressions in dioecious and monoecious persimmons. Meantime, the results of phytohormones assays showed that high IAA, ABA, GA3, and JA levels at stage 2 may have promoted male floral bud differentiation. However, high JA levels at stage 4 and high ZT levels at stages 2 and 4 may have promoted hermaphroditic floral bud differentiation. In these phytohormone biosynthesis and signaling pathways, 52 and 54 differential expression genes (including Aux/IAA, ARFs, DELLA, AHP, A-ARR, B-ARR, CYP735A, CRE1, PP2C, JAZ, MYC2, COI1, CTR1, SIMKK, ACO, and MPK6) were identified, respectively. During the development of male floral buds, five metacaspases genes may have been involved in pistil abortion. In addition, MYB, FAR1, bHLH, WRKY, and MADS transcription factors might play important roles in persimmon floral bud sex differentiation. Noteworthy, miR169v_1, miR169e_3, miR319_1, and miR319 were predicted to contribute to phytohormone biosynthesis and signaling pathways and floral organogenesis and may also regulate floral bud sex differentiation. Conclusion The present study revealed the differences in morphology and phytohormones content between male and hermaphroditic floral buds of ‘Longyanyeshi 1’ during the process of sex differentiation, and identified a subset of candidate genes and miRNAs putatively associated with its sex differentiation. These findings can provide a foundation for molecular regulatory mechanism researching on andromonoecious persimmon.

Genome-wide identification of MIKC-type genes related to stamen and gynoecium development in Liriodendron

The organogenesis and development of reproductive organs, i.e., stamen and gynoecium, are important floral characteristics that are closely related to pollinators and reproductive fitness. As a genus from Magnoliaceae, Liriodendron has only two relict species: L. chinense and L. tulipifera. Despite the similar flower shapes of these species, their natural seed-setting rates differ significantly, implying interspecies difference in floral organogenesis and development. MADS-box genes, which participate in floral organogenesis and development, remain unexplored in Liriodendron. Here, to explore the interspecies difference in floral organogenesis and development and identify MADS-box genes in Liriodendron, we examined the stamen and gynoecium primordia of the two Liriodendron species by scanning electron microscopy combined with paraffin sectioning, and then collected two types of primordia for RNA-seq. A total of 12 libraries were constructed and 42,268 genes were identified, including 35,269 reference genes and 6,999 new genes. Monoterpenoid biosynthesis was enriched in L. tulipifera. Genome-wide analysis of 32 MADS-box genes was conducted, including phylogenetic trees, exon/intron structures, and conserved motif distributions. Twenty-six genes were anchored on 17 scaffolds, and six new genes had no location information. The expression profiles of MIKC-type genes via RT-qPCR acrossing six stamen and gynoecium developmental stages indicates that the PI-like, AG/STK-like, SEP-like, and SVP-like genes may contribute to the species-specific differentiation of the organogenesis and development of reproductive organs in Liriodendron. Our findings laid the groundwork for the future exploration of the mechanism underlying on the interspecific differences in reproductive organ development and fitness in Liriodendron.

Transcriptional control of local auxin distribution by the CsDFB1-CsPHB module regulates floral organogenesis in cucumber

Plant cystatins are cysteine proteinase inhibitors that play key roles in defense responses. In this work, we describe an unexpected role for the cystatin-like protein DEFORMED FLORAL BUD1 (CsDFB1) as a transcriptional regulator of local auxin distribution in cucumber (Cucumis sativus L.). CsDFB1 was strongly expressed in the floral meristems, floral primordia, and vasculature. RNA interference (RNAi)-mediated silencing of CsDFB1 led to a significantly increased number of floral organs and vascular bundles, together with a pronounced accumulation of auxin. Conversely, accompanied by a decrease of auxin, overexpression of CsDFB1 resulted in a dramatic reduction in floral organ number and an obvious defect in vascular patterning, as well as organ fusion. CsDFB1 physically interacted with the cucumber ortholog of PHABULOSA (CsPHB), an HD-ZIP III transcription factor whose transcripts exhibit the same pattern as CsDFB1. Overexpression of CsPHB increased auxin accumulation in shoot tips and induced a floral phenotype similar to that of CsDFB1-RNAi lines. Furthermore, genetic and biochemical analyses revealed that CsDFB1 impairs CsPHB-mediated transcriptional regulation of the auxin biosynthetic gene YUCCA2 and the auxin efflux carrier PIN-FORMED1, and thus plays a pivotal role in auxin distribution. In summary, we propose that the CsDFB1-CsPHB module represents a regulatory pathway for local auxin distribution that governs floral organogenesis and vascular differentiation in cucumber.

AINTEGUMENTA and AINTEGUMENTA-LIKE6 directly regulate floral homeotic and growth genes in young flowers

Arabidopsis flower primordia give rise to floral organ primordia in stereotypical positions within four concentric whorls. Floral organ primordia in each whorl undergo distinct developmental programs to become one of four organ types (sepals, petals, stamens, and carpels). The Arabidopsis transcription factors AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6 (AIL6) play critical and partially overlapping roles during floral organogenesis. They are required for correct positioning of floral organ initiation, contribute to the specification of floral organ identity, and regulate the growth and morphogenesis of developing floral organs. To gain insight into the molecular means by which ANT and AIL6 contribute to floral organogenesis, we identified the genome-wide binding sites of both ANT and AIL6 in stage 3 flower primordia, the developmental stage at which sepal primordia become visible and class B and C floral homeotic genes are first expressed. AIL6 binds to a subset of ANT sites, suggesting that AIL6 regulates some but not all of the same target genes as ANT. ANT and AIL6 binding sites are associated with genes involved in many biological processes related to meristem and flower organ development. Comparison of genes associated with both ANT and AIL6 ChIP-Seq peaks and those differentially expressed after perturbation of ANT or AIL6 activity identified likely direct targets of ANT and AIL6 regulation. These include the floral homeotic genes APETALA3 (AP3) and AGAMOUS (AG) and four growth regulatory genes: BIG BROTHER (BB), ROTUNDIFOLIA3 (ROT3), ANGUSTIFOLIA3/GRF INTERACTING FACTOR (AN3/GIF1), and XYLOGLUCAN ENDOTRANSGLUCOLSYLASE/HYDROLASE9 (XTH9).One Sentence SummaryThe transcription factors ANT and AIL6 directly regulate genes involved in different aspects of flower development including genes that specify floral organ identity and those that regulate growth.

Genome-wide identification of MADS-box gene family in sacred lotus (Nelumbo nucifera) identifies a SEPALLATA homolog gene involved in floral development

Background Sacred lotus (Nelumbo nucifera) is a vital perennial aquatic ornamental plant. Its flower shape determines the horticultural and ornamental values. However, the mechanisms underlying lotus flower development are still elusive. MADS-box transcription factors are crucial in various features of plant development, especially in floral organogenesis and specification. It is still unknown how the MADS-box transcription factors regulate the floral organogenesis in lotus. Results To obtain a comprehensive insight into the functions of MADS-box genes in sacred lotus flower development, we systematically characterized members of this gene family based on the available genome information. A total of 44 MADS-box genes were identified, of which 16 type I and 28 type II genes were categorized based on the phylogenetic analysis. Furthermore, the structure of MADS-box genes and their expressional patterns were also systematically analyzed. Additionally, subcellular localization analysis showed that they are mainly localized in the nucleus, of which a SEPALLATA3 (SEP3) homolog NnMADS14 was proven to be involved in the floral organogenesis. Conclusion These results provide some fundamental information about the MADS-box gene family and their functions, which might be helpful in not only understanding the mechanisms of floral organogenesis but also breeding of high ornamental value cultivars in lotus.