Flower Development Genes Research Articles

Efficient multiplex mutagenesis by RNA-guided Cas9 and its use in the characterization of regulatory elements in the AGAMOUS gene.

BackgroundThe efficiency of multiplex editing in plants by the RNA-guided Cas9 system is limited by efficient introduction of its components into the genome and by their activity. The possibility of introducing large fragment deletions by RNA-guided Cas9 tool provides the potential to study the function of any DNA region of interest in its ‘endogenous’ environment.ResultsHere, an RNA-guided Cas9 system was optimized to enable efficient multiplex editing in Arabidopsis thaliana. We demonstrate the flexibility of our system for knockout of multiple genes, and to generate heritable large-fragment deletions in the genome. As a proof of concept, the function of part of the second intron of the flower development gene AGAMOUS in Arabidopsis was studied by generating a Cas9-free mutant plant line in which part of this intron was removed from the genome. Further analysis revealed that deletion of this intron fragment results 40 % decrease of AGAMOUS gene expression without changing the splicing of the gene which indicates that this regulatory region functions as an activator of AGAMOUS gene expression.ConclusionsOur modified RNA-guided Cas9 system offers a versatile tool for the functional dissection of coding and non-coding DNA sequences in plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-016-0125-7) contains supplementary material, which is available to authorized users.

Arabidopsis Flower and Embryo Developmental Genes are Repressed in Seedlings by Different Combinations of Polycomb Group Proteins in Association with Distinct Sets of Cis-regulatory Elements.

Polycomb repressive complexes (PRCs) play crucial roles in transcriptional repression and developmental regulation in both plants and animals. In plants, depletion of different members of PRCs causes both overlapping and unique phenotypic defects. However, the underlying molecular mechanism determining the target specificity and functional diversity is not sufficiently characterized. Here, we quantitatively compared changes of tri-methylation at H3K27 in Arabidopsis mutants deprived of various key PRC components. We show that CURLY LEAF (CLF), a major catalytic subunit of PRC2, coordinates with different members of PRC1 in suppression of distinct plant developmental programs. We found that expression of flower development genes is repressed in seedlings preferentially via non-redundant role of CLF, which specifically associated with LIKE HETEROCHROMATIN PROTEIN1 (LHP1). In contrast, expression of embryo development genes is repressed by PRC1-catalytic core subunits AtBMI1 and AtRING1 in common with PRC2-catalytic enzymes CLF or SWINGER (SWN). This context-dependent role of CLF corresponds well with the change in H3K27me3 profiles, and is remarkably associated with differential co-occupancy of binding motifs of transcription factors (TFs), including MADS box and ABA-related factors. We propose that different combinations of PRC members distinctively regulate different developmental programs, and their target specificity is modulated by specific TFs.

Comparative expression profiling of three early inflorescence stages of oil palm indicates that vegetative to reproductive phase transition of meristem is regulated by sugar balance.

Breeding and seed production activities in oil palm have been hampered because of the inability of the male parent Pisifera to produce male inflorescence as source of pollen under normal conditions. Researchers are using complete defoliation to induce male inflorescences, but the biological and molecular processes responsible for this morphological change are yet to be revealed. To understand the underlying network of genes that initiate and control this phenotypically documented activity, we initiated a study aimed at identifying differentially expressed genes (DEGs) in three stages of an oil palm inflorescence under complete defoliation stress using RNA-seq. Sequencing on an Illumina platform produced 82631476 reads consisting of 8345779076 bases. A total of 60700 genes were obtained after transcript filtering and normalisation and 54% of them were downregulated. Differences in gene expression levels were significant between tissues under stress. The farther the distance between tissues, the more DEGs recorded. Comparison between stage 2 and stage 1 induced 3893 DEGs whereas 10136 DEGs were induced between stage 3 and stage 1. Stress response genes and flower development genes were among the highly expressed genes. This study suggests a link between complete defoliation and meristem differentiation from vegetative to reproductive phase in oil palm.

The role of WOX genes in flower development.

WOX (Wuschel-like homeobOX) genes form a family of plant-specific HOMEODOMAIN transcription factors, the members of which play important developmental roles in a diverse range of processes. WOX genes were first identified as determining cell fate during embryo development, as well as playing important roles in maintaining stem cell niches in the plant. In recent years, new roles have been identified in plant architecture and organ development, particularly at the flower level. In this review, the role of WOX genes in flower development and flower architecture is highlighted, as evidenced from data obtained in the last few years. The roles played by WOX genes in different species and different flower organs are compared, and differential functional recruitment of WOX genes during flower evolution is considered. This review compares available data concerning the role of WOX genes in flower and organ architecture among different species of angiosperms, including representatives of monocots and eudicots (rosids and asterids). These comparative data highlight the usefulness of the WOX gene family for evo-devo studies of floral development.

Isolation and characterization of the papaya MADS-box E-class genes, CpMADS1 and CpMADS3, and a TM6 lineage gene CpMADS2.

Papaya (Carica papaya L.) plants are polygamous, with female, male, and hermaphroditic flowers. To understand the roles of MADS-box genes in flower development and sex determination, we cloned cDNAs of E-class genes CpMADS1 and CpMADS3 and a TM6 lineage of the B-class gene CpMADS2 from young flower buds of papaya. Reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR analyses revealed that CpMADS1 and CpMADS3 were preferentially expressed in the carpel and also in petals and stamens. CpMADS2 was expressed in both petals and stamens early during floral development. Comparison of 10 papaya genotypes of 5 different sex phenotypes - hermaphrodite, male, female, progeny-all-hermaphrodite, and progeny-all-male - by Southern blot analysis of genomic DNAs with probes of the 3 genes revealed similar restriction patterns and copy number, suggesting a low relationship of the 3 CpMADS genes with sex expression of papaya plants at the genomic level.

SlWUS1; An X-linked Gene Having No Homologous Y-Linked Copy inSilene latifolia

The dioecious plant Silene latifolia has heteromorphic sex chromosomes, and comparison of the positions of sex-linked genes indicates that at least three large inversions have occurred during the evolution of the Y chromosome. In this article, we describe the isolation of a new sex-linked gene from S. latifolia, which provides new information on the evolution of this plant’s young sex chromosomes. By using reverse-transcription polymerase chain reaction degenerate primers based on the Arabidopsis thaliana sequence of WUSCHEL, a flower-development gene, we found two copies in S. latifolia, which we named SlWUS1 and SlWUS2. Southern blot and genetic segregation analysis showed that SlWUS1 is located on the X chromosome and SlWUS2 is autosomal. No Y-linked copy of SlWUS1 was found by either Southern blot analysis under low-stringency conditions or polymerase chain reaction with degenerate primers, so we conclude that SlWUS1 probably has no Y-linked homolog. It is unknown whether the Y chromosome lost the SlWUS1 copy by degeneration of this individual gene or whether deletion of a larger genome region was involved. Several tests lead us to conclude that dosage compensation has not evolved for this sex-linked gene. We mapped the ortholog in the nondioecious relative S. vulgaris (SvWUS1), to compare the location in a species that has no history of having sex chromosomes. SvWUS1 maps to the same linkage group as other fully X-linked genes, indicating that it was not added to the X, but was lost from the Y. Its location differs in the maps from the two species, raising the possibility that the X chromosome, as well as the Y, may have been rearranged.

Flower-specific KNOX phenotype in the orchid Dactylorhiza fuchsii

The KNOTTED1-like homeobox (KNOX) genes are best known for maintaining a pluripotent stem-cell population in the shoot apical meristem that underlies indeterminate vegetative growth, allowing plants to adapt their development to suit the prevailing environmental conditions. More recently, the function of the KNOX gene family has been expanded to include additional roles in lateral organ development such as complex leaf morphogenesis, which has come to dominate the KNOX literature. Despite several reports implicating KNOX genes in the development of carpels and floral elaborations such as petal spurs, few authors have investigated the role of KNOX genes in flower development. Evidence is presented here of a flower-specific KNOX function in the development of the elaborate flowers of the orchid Dactylorhiza fuchsii, which have a three-lobed labellum petal with a prominent spur. Using degenerate PCR, four Class I KNOX genes (DfKN1–4) have been isolated, one from each of the four major Class I KNOX subclades and by reverse transcription PCR (RT-PCR), it is demonstrated that DfKNOX transcripts are detectable in developing floral organs such as the spur-bearing labellum and inferior ovary. Although constitutive expression of the DfKN2 transcript in tobacco produces a wide range of floral abnormalities, including serrated petal margins, extra petal tissue, and fused organs, none of the vegetative phenotypes typical of constitutive KNOX expression were produced. These data are highly suggestive of a role for KNOX expression in floral development that may be especially important in taxa with elaborate flowers.

Rice MADS6 Interacts with the Floral Homeotic Genes SUPERWOMAN1, MADS3, MADS58, MADS13, and DROOPING LEAF in Specifying Floral Organ Identities and Meristem Fate

AGAMOUS-LIKE6 (AGL6) genes play essential roles in flower development, but whether and how they work with floral organ identity genes remain less understood. Here, we describe interactions of the rice (Oryza sativa) AGL6 gene MADS6 with other rice floral homeotic genes in flower development. Genetic analyses revealed that MADS6 specifies the identity of the three inner whorls and floral meristem determinacy redundantly with SUPERWOMAN1/MADS16 (B-gene) or MADS3 (C-gene). MADS6 was shown to define carpel/ovule development and floral determinacy by interacting with MADS13 (D-gene) and control the palea and floral meristem identities together with the YABBY gene DROOPING LEAF. Expression analyses revealed that the transcript levels of six B-, C-, and E-class genes were reduced in mads6-1 at the early flower developmental stage, suggesting that MADS6 is a key regulator of early flower development. Moreover, MADS6 can directly bind to a putative regulatory motif on MADS58 (C-gene), and mads6-1 mads58 displayed phenotypes similar to that of mads6-1. These results suggest that MADS6 is a key player in specifying flower development via interacting with other floral homeotic genes in rice, thus providing new insights into the mechanism by which flower development is controlled.

Generation of serrated and wavy petals by inhibition of the activity of TCP transcription factors in Arabidopsis thaliana

The final shape of shoot lateral organs, namely, leaves and flowers, is determined by coordinated growth after the initiation of primordia from shoot meristems in seed plants. This coordination is achieved by the complex action of many transcription factors, which include the TEOSINTE BRANCHED1, CYCLOIDEA, and PCF (TCP) family. We have recently reported that CINCINNATA-like (CIN-like) TCP genes act dose-dependently to regulate the flat and smooth morphology of leaves in Arabidopsis thaliana. In contrast, the roles of CIN-like TCP genes in flower development are poorly understood. In this report, using multiple tcp mutants and transgenic plants in which the activity of CIN-like TCP transcription factors is dominantly inhibited, we found that these TCPs regulate the smooth and flat morphology of petals. Based on these findings, we discuss a possible strategy to generate a fringed morphology in floricultural plants.

Characterization of Expressed Sequence Tags from Flower Buds of Alpine Lilium formosanum using a Subtractive cDNA Library

Formosan lily (Lilium formosanum), a species endemic in Taiwan, is characterized by showy and fragrant flowers. To understand the gene expression at its reproductive phase, we constructed a suppression subtractive cDNA library of immature flower buds, from which 1,324 expressed sequence tags (ESTs) were randomly selected and sequenced. These EST sequences were clustered into 974 nonredundant sequences. Based on BLAST searching, functions of 376 sequences (39%) were determined, and 80 sequences showed high similarities to genes encoding hypothetical proteins without known functions. Another 518 sequences did not show significant homology to any known sequences and were therefore classified as novel sequences. Further analyses of the 376 ESTs sequences revealed high abundance of stress-related and flower-development genes. The highly expressing stress-related transcripts include 39 with high similarities to lipid transfer proteins, five to ascorbate peroxidases, and five to heat shock proteins 70. Using real-time quantitative RT-PCR analysis, we further revealed the expression of these three genes in the immature flower buds and in the pistils or stamens of the blooming flower of Formosan lily collected from alpine regions. These results suggest that the flower of L. formosanum possesses a significantly elevated level of stress genes in response to alpine environment and the ESTs analyzed here represent a valuable resource for studying a resistance mechanism of the reproductive organs of Formosan lily.

Differential gene expression analysis of a new Ogura CMS line and its maintainer in non-heading Chinese cabbage by cDNA-AFLP

CMS is induced by the coordinated expression of certain mitochondrial and nuclear genes in flower development. Mitochondrial genes regulate manifestation of CMS, whereas nuclear genes regulate fertility phenotype and thus affect negatively. In this article, the buds of newly bred Ogura CMS of non-heading Chinese cabbage and its maintainer line as plant materials, genes differentially expressed transcripts were analyzed by cDNA-AFLP. Seventeen differently expressed genes were found in new Ogura CMS and nine genes in maintainer line. These genes were involved in energy metabolism, signal transduction, flower development, stress-related metabolism, transcription, etc. Expression patterns of three genes encoding BrCAM6, BrANK, BrTUB3 were verified by qRT-PCR in different organs and various stage flower buds of CMS and its maintainer line. The results revealed that two genes related to signal transduction, BrCAM6 and BrANK, were highly expressed in stamens and microspores of CMS than in maintainer line. As believed, these two genes involved in signal transduction of male sterile in CMS line. In comparison, BrTUB3 gene was accumulated in stamens and was expressed in significantly lower level in CMS line than in maintainer line. It expressed significantly lower in CMS than maintainer line after tetrad stage. This expression profile suggests that BrTUB3 played an important role in the development of the pollen, and may be closely related to male sterility.

Cell‐type specific analysis of translating RNAs in developing flowers reveals new levels of control

Determining both the expression levels of mRNA and the regulation of its translation is important in understanding specialized cell functions. In this study, we describe both the expression profiles of cells within spatiotemporal domains of the Arabidopsis thaliana flower and the post-transcriptional regulation of these mRNAs, at nucleotide resolution. We express a tagged ribosomal protein under the promoters of three master regulators of flower development. By precipitating tagged polysomes, we isolated cell type-specific mRNAs that are probably translating, and quantified those mRNAs through deep sequencing. Cell type comparisons identified known cell-specific transcripts and uncovered many new ones, from which we inferred cell type-specific hormone responses, promoter motifs and coexpressed cognate binding factor candidates, and splicing isoforms. By comparing translating mRNAs with steady-state overall transcripts, we found evidence for widespread post-transcriptional regulation at both the intron splicing and translational stages. Sequence analyses identified structural features associated with each step. Finally, we identified a new class of noncoding RNAs associated with polysomes. Findings from our profiling lead to new hypotheses in the understanding of flower development.

A Role for Auxin in Flower Development

AbstractAuxin has long been implicated in many aspects of plant growth and development including flower development. However, the exact roles of auxin in flower development have not been well defined until the recent identification of auxin biosynthesis mutants. Auxin is necessary for the initiation of floral primordia, and the disruption of auxin biosynthesis, polar auxin transport or auxin signaling leads to the failure of flower formation. Auxin also plays an essential role in specifying the number and identity of floral organs. Further analysis of the relationship between the auxin pathways and the known flower development genes will provide critical information regarding mechanisms of organogenesis and pattern formation in plants.

Tomato Flower Abnormalities Induced by Stolbur Phytoplasma Infection Are Associated with Changes of Expression of Floral Development Genes

Tomato (Lycopersicon esculentum cv. Micro-Tom) plants infected by the stolbur phytoplasma (isolate PO) display floral abnormalities, including sepal hypertrophy, virescence, phyllody, and aborted reproductive organs, which are reminiscent of those observed in Arabidopsis thaliana mutants affected in flower development genes. Semiquantitative reverse transcription-polymerase chain reaction and in situ RNA hybridization were used to compare expressions of meristem and flower development genes in healthy and stolbur phytoplasma-infected tomatoes. In infected plants, FALSIFLORA (FA), controlling the identity of the inflorescence meristem, was up-regulated, whereas LeWUSCHEL (LeWUS) and LeCLAVATA1 (LeCLV1), regulating the meristem development, and LeDEFICIENS (LeDEF), responsible for the organ (petals and stamens) identity within the flower, were down-regulated regardless of the development stage of the flower bud. In contrast, expression of TAG1, which regulates stamen and carpel identities and negatively controls LeWUS, was up-regulated at the early stages and down-regulated at the late stages. In situ RNA hybridization analyses revealed that TAG1 transcripts were restricted to the same floral meristem territories in healthy and infected tomatoes, indicating that tissue-specific expression of TAG1 was not affected by the stolbur phytoplasma infection. Taken together, these data indicate that flower malformations of stolbur phytoplasma-infected tomatoes are associated with early changes in the expression of key flower development genes. The possible mechanisms by which the multiplication of stolbur phytoplasma in tomato sieve tubes deregulates floral development are discussed.

Expression Patterns of Purple Acid Phosphatase Genes in Arabidopsis Organs and Functional Analysis of AtPAP23 Predominantly Transcribed in Flower

Purple acid phosphatases (PAPs) are metallo-phosphoesterases. Their expression and function have not been systematically investigated in higher plants. In this work, we compared the transcript levels of 28 Arabidopsis PAP (AtPAP) genes in five Arabidopsis organs. The 28 members, although differed in their expression patterns in vegetative organs, were all transcribed in flower. Furthermore, the transcription of seven members (AtPAPs 6, 11, 14, 19, 23, 24 and 25) occurred predominantly in the flower. To begin dissecting the role of AtPAP genes in flower development, further expression and functional analyses were conducted using AtPAP23. Histochemical staining of transgenic plants expressing AtPAP23 promoter-beta-glucuronidase (GUS) gene construct revealed that AtPAP23 transcription was strong in flower apical meristems, but became restricted to petals and anther filaments in fully developed flower. A GST (glutathione S-transferase) fusion protein of AtPAP23 (GST:AtPAP23) was expressed in bacterial cells, and was found to contain significant amounts of Fe and Mn (whereas the control GST protein contained none). In biochemical tests, GST:AtPAP23 showed typical acid phosphatase activities. The fusion protein was also highly active on phosphoserine, but not phosphotyrosine. Despite its highly specific expression pattern and the demonstrated biochemical function of its protein product, the RNAi (RNA interference), T-DNA knock-out and overexpression lines of AtPAP23 were indistinguishable from wild type plants in the development of flower (or other organs). Interestingly, the Fe and Mn contents were found significantly increased in AtPAP23 overexpression lines, which may offer a new direction for further functional studies of AtPAPs in Arabidopsis.

PETAL LOSS gene regulates initiation and orientation of second whorl organs in the Arabidopsis flower.

PETAL LOSS is a new class of flower development gene whose mutant phenotype is confined mostly to the second whorl. Two properties are disrupted, organ initiation and organ orientation. Initiation is frequently blocked, especially in later-formed flowers, or variably delayed. The few petals that arise occupy a wider zone of the flower primordium than normal. Also, a minority of petals are trumpet-shaped, thread-like or stamenoid. Studies of ptl combined with homeotic mutants have revealed that the mutant effect is specific to the second whorl, not to organs with a petal identity. We propose that the PTL gene normally promotes the induction of organ primordia in specific regions of the second floral whorl. In ptl mutants, these regions are enlarged and organ induction is variably reduced, often falling below a threshold. A dominant genetic modifier of the ptl mutant phenotype was found in the Landsberg erecta strain that significantly boosts the mean number of petals per flower, perhaps by reinforcing induction so that the threshold is now more often reached. The second major disruption in ptl mutants relates to the orientation adopted by second whorl organs from early in their development. In single mutants the full range of orientations is seen, but when B function (controlling organ identity) is also removed, most second whorl organs now face outwards rather than inwards. Orientation is unaffected in B function single mutants. Thus petals apparently perceive their orientation within the flower primordium by a mechanism requiring PTL function supported redundantly by that of B class genes.

Evolution of the cycloidea gene family in Antirrhinum and Misopates.

Studies at the nucleotide level on the nuclear flower development gene cycloidea (cyc) in seven Antirrhinum, two Misopates, one Linaria, one Cymbalaria, and one Digitalis species revealed that cyc is a member of a gene family composed of at least five apparently functional genes. The estimated ages of the duplication events that created this gene family are from 7.5 Myr to more than 75 Myr. We also report the first estimates of DNA sequence diversity for species of Antirrhinum and Misopates. Low between-species variability suggests that this group of species may have diverged recently.

Genetic Diversity in the Common Morning Glory

Abstract Populations of the common morning glory in the southeastern US are characterized by a striking diversity of flower color polymorphisms. This diversity is probably a consequence of horticultural escapes from cultivation in the 18th and 19th centuries. More than 15 years of research in our laboratory has shown that some color phenotypes are selected by virtue of their differential attraction to insect pollinators. We have studied genetic diversity at isozyme and ribosomal DNA loci and we find reduced diversity in the southeastern US compared to Mexican populations. In an effort to link ecological genetics to molecular evolution, we have cloned and characterized the chalcone synthase (CHS) gene family in morning glory and we have studied the expression of CHS genes in flower development. We have also initiated an investigation of spatial patterns of diversity at CHS genes by sampling and sequencing genes from US and Mexican populations. These investigations reveal (1) that the four CHS genes (CHS A, B, C, and PS) characterized to date evolve rapidly in morning glory and that the gene family in Ipomoea is of relatively recent origin (approximately 21 million years); (2) the duplicate genes in Ipomoea group into two categories (CHS A, C versus CHS B, PS) that may indicate a functional divergence between chalcone synthase and stilbene synthase activities; (3) levels of molecular diversity for CHS A genes sampled from Mexico are much higher than observed in US collections suggesting a major population bottleneck associated with the introduction of morning glory into the southeastern US; and (4) the ratio of amino acid substitution to synonymous substitution between Ipomoea species is remarkably high (about 5.4 synonymous to amino acid substitutions) compared to CHS genes in other plant species. Taken together these data portray a rapidly evolving gene family where functional divergence may arise repeatedly in evolution, despite the central role of chalcone synthase in flavonoid metabolism.

Flower development and evolution: new answers and new questions.

Certain genes have a way of rewarding continued study, as can be seen from the long histories of discoveries that have resulted from work on mammalian hemoglobin genes, on the Escherichia coli lacZ gene, and on many others. A plant gene that may fit this mold is the homeotic flower-development gene AGAMOUS (AG). In the past few years, study of the genetics and molecular genetics of this Arabidopsis thaliana gene and of its orthologues in other plant species has led to a stream of discoveries that have revealed some of the mechanisms and some of the complexity of flower development. Two recent papers (1, 2), one in this issue, describe a new level of regulation of AG and raise new questions about the function and evolutionary history of this gene.

The Role of Homeotic Genes in Flower Development and Evolution

The Role of Homeotic Genes in Flower Development and Evolution