Analysis of the floral transcriptome of Tarenaya hassleriana (Cleomaceae), a member of the sister group to the Brassicaceae: towards understanding the base of morphological diversity in Brassicales
© Bhide et al.; licensee BioMed Central Ltd. 2014
Received: 8 August 2013
Accepted: 6 February 2014
Published: 19 February 2014
Arabidopsis thaliana, a member of the Brassicaceae family is the dominant genetic model plant. However, while the flowers within the Brassicaceae members are rather uniform, mainly radially symmetrical, mostly white with fixed organ numbers, species within the Cleomaceae, the sister family to the Brassicaceae show a more variable floral morphology. We were interested in understanding the molecular basis for these morphological differences. To this end, the floral transcriptome of a hybrid Tarenaya hassleriana, a Cleomaceae with monosymmetric, bright purple flowers was sequenced, annotated and analyzed in respect to floral regulators.
We obtained a comprehensive floral transcriptome with high depth and coverage close to saturation analyzed using rarefaction analysis a method well known in biodiversity studies. Gene expression was analyzed by calculating reads per kilobase gene model per million reads (RPKM) and for selected genes in silico expression data was corroborated by qRT-PCR analysis. Candidate transcription factors were identified based on differences in expression pattern between A. thaliana and T. hassleriana, which are likely key regulators of the T. hassleriana specific floral characters such as coloration and male sterility in the hybrid plant used. Analysis of lineage specific genes was carried out with members of the fabids and malvids.
The floral transcriptome of T. hassleriana provides insights into key pathways involved in the regulation of late anthocyanin biosynthesis, male fertility, flowering time and organ growth regulation which are unique traits compared the model organism A. thaliana. Analysis of lineage specific genes carried out with members of the fabids and malvids suggests an extensive gene birth rate in the lineage leading to core Brassicales while only few genes were potentially lost during core Brassicales evolution, which possibly reflects the result of the At-β whole genome duplication. Our analysis should facilitate further analyses into the molecular mechanisms of floral morphogenesis and pigmentation and the mechanisms underlying the rather diverse floral morphologies in the Cleomaceae.
KeywordsTarenaya hassleriana Arabidopsis thaliana Floral transcriptome Cleomaceae Brassicaceae Brassicales 454 sequencing Anthocyanins Flower development
Tarenaya hassleriana, formerly known as Cleome hassleriana and sometimes erroneously referred to as Cleome spinosa is a quick growing herbaceous perennial, native to Brazil and adjoining South American countries. The species belongs to the section Tarenaya and the subgenus Neocleome within the Cleomaceae  which includes roughly 300 species distributed throughout the tropical and subtropical regions of the world [3, 4]. The family Cleomaceae belongs to the order Brassicales and previously Cleomaceae were thought to be more closely related to Capparaeceae but recent phylogenetic studies indicate that Cleomaceae are more closely related to and a sister family to Brassicaceae [3, 5]. Molecular clock analyses suggests that Cleomaceae and Brassicaceae diverged from each other around 24.2 – 49.4 Million Years Ago (MYA) [6, 7].
Analysis of normalized expressed sequence tag (EST) sequences in T. hassleriana and comparative genome analysis in Carica papaya, both members of the Brassicales, and in Arabidopsis thaliana belonging to Brassicaceae revealed that Cleomaceae share the most ancient gamma (γ) whole genome duplication (WGD) with both C. papaya and A. thaliana. The sister families Brassicaceae and Cleomaceae also share the more recent beta (β) WGD which is lacking in C. papaya. However, the third and most recent alpha (α) WGD has occurred independently in Brassicaceae and Cleomaceae. The T. hassleriana α WGD (Th- α) is a genome triplication and occurred approximately 13.7 MYA, while the Arabidopsis thalian a α WGD (At- α) happened around 23.3 MYA . In spite of the recent Th- α triplication event the genome of T. hassleriana is only 1.9 times the size of that of A. thaliana and around half the size of the C. papaya genome. The small genome size of T. hassleriana indicates rapid diploidization, and a faster subsequent gene loss when compared to A. thaliana.
Cleomaceae are being intensively studied as C4 type photosynthesis evolved de novo in this group of plants. While A. thaliana and other Brassicaceae are C3 plants, C4 photosynthesis evolved in Cleomaceae at least three times independently in Gynandropsis gynandra, Cleome oxalidea, and Cleome angustifolia. Cleome paradoxa shows a C3 – C4 intermediate anatomy and physiology thus making Cleomaceae a model system to study C3 – C4 evolution . Comparative leaf transcriptome studies by RNA-Seq have been carried out in G. gynandra (C4) and T. hassleriana (C3) to elucidate and identify novel genes and gene networks responsible for the C4 anatomy .
A typical T. hassleriana flower is zygomorphic unlike the disymmetric A. thaliana flower. Each flower has four sepals, four petals, six stamens, and a single gynoecium composed of two fused carpels (Figure 1b). The flower buds are laid out in a disymmetric bauplan during the early developmental stages which changes near anthesis and the mature flowers become zygomorphic. Conversely, in A. thaliana early developmental bud stages are monosymmetric and the flowers become disymmetric near anthesis . C. papaya flowers on the other hand are actinomorphic at anthesis but early development has not been characterized yet. T. hassleriana inflorescences produce hermaphroditic, female, or male only flowers such that fruits are only periodically formed. The synchronous and alternate appearance of male, female, and hermaphroditic flowers in a raceme favors out-crossing, and prevents selfing except in the case of the hermaphroditic flowers . This feature distinguishes T. hassleriana from most plants which are either dioecious (like C. papaya) with separate male and female plants which rarely produce hermaphroditic flowers, or monoecious like A. thaliana which is an obligate self-pollinated plant with hermaphroditic flowers. T. hassleriana plants are very prolific, they reseed and establish in suitable environments very easily and escape from cultivation, often becoming invasive in subtropical countries like Japan, New Zealand, parts of Australia, and the United States of America . Hence many horticultural varieties, possibly like the one used in this study were developed to be sterile so that they cannot establish in non-native environments.
Also unlike most Brassicales T. hassleriana flowers are very colorful due to the presence of various anthocyanins and show ‘petal fading’ i.e. loss of pigmentation and dry matter associated with anthesis. This phenomenon coupled with favored cross pollination may suggest a specific role in flower - pollinator interactions or simply an age related phenomenon .
A close relationship to A. thaliana facilitates the analysis of T. hassleriana specific traits, such as flower coloration, alternating development of three types of flowers and flexible shifts from vegetative to reproductive growth, which are all not found in A. thaliana. Here, we describe the floral transcriptome sequence along with Transcriptome Sequencing Expression (TSE) of a horticultural T. hassleriana hybrid as a starting point for further analysis of Tarenaya flower development. Expression analysis by qRT-PCR documents the robustness of the TSE and rarefaction analysis shows that the transcriptome sequencing covers even rare transcripts. Candidate genes that may be involved in the T. hassleriana-specific flower developmental processes have been identified and are presented here.
Plant material and growth parameters
A T. hassleriana hybrid plant was obtained from a local garden center. It was grown in 3:1 mixture of a peat and sand based potting soil with perlite supplemented with 2 g/l Osmocote® slow release fertilizer (Scotts Deutschland GmbH, Nordhorn, Germany). The plant was grown in a greenhouse under long day growth conditions (17 hours light and 7 hours dark) with light varying between 80 and 700 μmol.m-2.s-1 photons. Supplemental lighting of 70 μmol.m-2.s-1 was provided throughout the photoperiod. The temperatures in the green house varied between 20°C (day) and 16°C (night).
Tissue collection, nucleic acid extraction, and cDNA synthesis
T. hassleriana floral tissue was collected for RNA and DNA extraction. The collected flower tissue was composed of equal quantity (by mass) of flowers at anthesis and each of the 6 floral bud stages defined by bud length as shown in Figure 1c).
For the floral transcriptome sequencing total RNA was extracted from the T. hassleriana floral tissue using guanidium thiocyanate-phenol-chloroform extraction protocol . The polyA+ mRNA was isolated using the Oligotex mRNA Minikit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction. The purified mRNA was analyzed for quality and quantity using the Eukaryote Total RNA Pico assay of the Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). For the qRT-PCRs total RNA was isolated from floral tissues with the plantRNA Kit-OLS® (Omni Life Science, Bremen, Germany) following the manufacturer’s protocol. Genomic DNA was extracted from the floral tissue using the DNeasy® Plant Mini kit (Qiagen, Hilden, Germany) according to the manual.
Library preparation and 454 pyrosequencing
200 ng of purified polyA+ mRNA was used to synthesize the cDNA for sequencing with Roche Rapid Library kit (Roche) following the manual. A massively parallel pyrosequencing run was performed on a GS FLX using Titanium chemicals (Roche) with a split picotiterplate allowing two replicates to run at the same time.
Assembly, annotation and gene expression
All reads together were de novo assembled using CLC Genomics Workbench 4.9 (clcBio, Aarhus Denmark). Default parameters were chosen for the assembly. The resulting 49321 contigs were annotated against TAIR10 coding sequences (representative gene model 20110103). A reciprocal BLATX mapping was performed  and the best bi-directional hit per contig was kept as annotation. Chimeric contigs were determined with the pipeline provided in .
Gene expression was determined by mapping the reads to TAIR10 coding sequences using BLATx. The single best hit for each read was counted. Expression values were normalized to reads per Kilobase gene model per mappable million (RPKM). All reads were additionally mapped to the T. hassleriana floral transcriptome contigs with CLC Genomics Workbench for subsequent rarefaction analysis. The expression data for the T. hassleriana leaf transcriptome was obtained from Bräutigam et al. .
Lineage specific gene detection
Based on the T. hassleriana floral contigs, mappings to the transcriptomes of A. thaliana, B. rapa, C. papaya, and P. trichocarpa with an e-value cutoff of 10-10 were created in proteinspace. From those all against all mappings the 15 overlapping sets and the residual T. hassleriana specific set were determined using R’s set methods .
Quantitative reverse transcription PCR (qRT-PCR)
For the qRT-PCRs the first strand cDNA was synthesized with the RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas, St.Leon-Rot, Germany) according to the manufacturer’s protocol using an universal oligo(dT) (T18) primer. qRT-PCR experiments were performed according to the MIQE guidelines . Exon spanning primers were then generated using PerlPrimer 1.1.21. . A primer efficiency test was carried out and all the primers were tested with genomic DNA to ensure cDNA specificity. (Primer sequences are provided in Additional file 1: Table S2).
The qRT-PCR assay was performed in 96 well plates using the LightCyler®480 II (Roche, Mannheim, Germany) and analyzed with the LCS480 22.214.171.124 software. Each reaction was composed of 10 μl of 2x DyNAmo™ Flash SYBR® Green qPCR Mastermix (Biozym Scientific GmbH, Oldendorf Germany), 2 μl each of 10 μM forward and reverse primers, 1 μl H2O and 5 μl of diluted cDNA template. Standard dose response (SDR) curves were constructed for all the genes by using serial dilutions (1:50 to 1:50,000) of the cDNA template. Each reaction was performed in biological duplicates and technical triplicates along with water and RNA controls for each primer pair. The T. hassleriana ACTIN7 (ACT7) gene served as an internal control. The following PCR program was used: 7 min at 95°C; 45 cycles of 10 s at 95°C, 15 s at 60°C, 15 s at 72°C, followed by a melting curve of 5 s at 95°C, 1 min at 65°C and 30 s at 97°C. The Absolute Quantification analysis and the quantification cycle (Cq) were calculated according to the Fit Points method using the LCS480 126.96.36.199 software. The amplification efficiency was calculated using the SDR for each gene. The raw data were analyzed according to the relative standard curve method and the fold difference between the expression of ACT7 and the genes of interest was calculated using the comparative Cq method (ΔΔCq) . A one way ANOVA was performed to calculate the statistical significance of the difference between the three expression values.
Comparison of A. thaliana and T. hassleriana floral gene expression and GO annotations
In order to identify genes that may play a role in the T. hassleriana specific floral traits, transcripts specific for the T. hassleriana flower, not expressed in the A. thaliana flower and vice versa were identified. Microarray expression data  for A. thaliana flower stages 1–6, 9, 10–11, 12, 15 (ATGE_29_A2, B2, C2; ATGE_31_A2, B2, C2; ATGE_32_A2, B2, C2; ATGE_33_A2, B2, C2; ATGE_39_A2, B2, C2) were downloaded from
The Arabidopsis Information Resource (TAIR), http://arabidopsis.org/servlets/TairObject?type=hyb_descr_collection&id=1006710873#497, on, Nov 20 2012.
Of the 22,746 microarray probes hybridizing to 23,570 genes, only 21,107 probes hybridizing to unique transcripts were considered for the analysis. A dataset corresponding to the expression of these 21,107 transcripts in the A. thaliana floral transcriptome was compiled. Expression of a gene in at least one floral stage and sample subset was considered as presence of the transcript in the A. thaliana floral transcriptome. The presence or absence of homologous transcripts in the T. hassleriana floral transcriptome was analyzed. A list of putative T. hassleriana orthologs of A. thaliana genes expressed in T. hassleriana floral transcriptome but not in the A. thaliana floral transcriptome was constructed. Also, transcripts present in the A. thaliana flower transcriptome but homologs absent in the T. hassleriana transcriptome were identified. Gene Ontology (GO) annotations were assigned to genes expressed exclusively in the A. thaliana or T. hassleriana transcriptome using the online tool for functional annotation Blast2GO®  by performing a BLASTX with a cutoff value of 1e-100 as this value showed robust matches of GO annotations to TAIR annotations.
GO annotations were assigned to T. hassleriana lineage specific sequences, and other sequences shared by Cleomaceae with the Brassicaceae, Brassicales or lost in the Brassicaceae using Blast2GO®  by performing BLASTX and BLASTN with cutoff values of 1e-10.
Rarefaction analysis is commonly used in ecological research defining species richness as a function of sequencing effort. Such an analysis can be broadened to genomics as long as the data are distributed as described in the original paper defining the underlying equation . Hale et al.  already calculated rarefaction curves for transcriptome analysis of a polyploid lake sturgeon. Here, we applied rarefaction analysis to ascertain whether sequencing depth and coverage was sufficient to draw a comprehensive picture of the transcriptome of Cleome. Thus, three different libraries were created: one data set for each biological replica as well as a merged one. Data sets were constructed by listing each gene (defined by a contig) with its read support. Rarefaction curves were calculated using the program aRarefactWin (https://www.uga.edu/strata/software/). Hereby, genes were randomly resampled and it was recorded which gene of the library was identified with which frequency. This procedure was repeated 1,000 times. Then, the average number of each gene found was plotted for different read numbers drawing a curve whose slope indicated if sequencing effort was deep enough. This was the case when the curve flattened and ran into a plateau.
Massively parallel pyrosequencing of two samples of T. hassleriana (Additional file 1: Table S1) yielded 1,254,286 sequencing reads in total. The sequencing raw data are deposited in the DDBJ (DNA Data Bank Japan, http://trace.ddbj.nig.ac.jp/index_e.html) under the experiments SRR1051360 and SRX393170 https://trace.ddbj.nig.ac.jp/DRASearch/run?acc=SRR1051360 and (https://trace.ddbj.nig.ac.jp/DRASearch/experiment?acc=SRX393170) The histogram of reads by length (Additional file 2: Figure S1a) showed an average read length of ~316 nucleotides. Roughly 45% of the reads could be mapped against A. thaliana TAIR10 coding sequences for counting gene expression.
Assembling the reads de novo resulted in 49,237 contigs with an N50 of 690 bases (Additional file 2: Figure S1b). Of these, 41,320 could be annotated by mapping against Arabidopsis. 1.1% (537) chimeric contigs could be detected in the assembly.
qRT-PCR expression analysis validates transcriptome sequencing expression (TSE)
Generally we found a better match of transcript abundance detected by qRT-PCR in T. hassleriana as compared to reads mapped to the A. thaliana orthologs (TSE1) than to the T. hassleriana contigs (TSE2). A correlation plot for the comparison of expression measured by qRT-PCR and TSE was generated (Additional file 2: Figure S3). When all the 14 gene expressions by the two methods were plotted a positive linear correlation was observed (Additional file 2: Figure S3a) as indicated by a R2 value 0.55. The expression of MVP1 and BGLUC19 gene homologs which belong to big gene families with 41 and 66 homologs in A. thaliana respectively was the most significant outlier in this plot. When the expression data for the MVP1 and BGLUC19 gene homologs were removed and the data plotted again a very strong positive linear correlation between TSE1 and qRT-PCR expression values was obtained with an R2 value 0.91 (Additional file 2: Figure S3b). This indicated that TSE1 approach for measuring gene expression was very robust except for genes belonging to large gene families with highly similar homologs in which case the read mapping may be incorrect. Nonetheless a positive linear expression correlation for all genes corroborates the TSE1 expression data. In particular, similar normalized fold expression between qRT-PCR data and reads mapped to the A. thaliana orthologs were observed in the genes RBCS1A (high expression), ATP3, SCE1A, SFGH (moderate expression), and ARF6, PGLUHYD, OMR1, and SPL7 (low expression) P >0,01 (Additional file 1: Table S4 shows the comparative P values for the ANOVA tests). In case of T. hassleriana homologs of genes GAMMAVPE, MVP1 and TT4 the transcript abundance detected by qRT-PCR was more similar when reads were mapped to the T. hassleriana contigs (TSE2) P > 0,01. In case of the GAPC1 and BGLUC19 homologs the difference between qRT-PCR expression and TSE1 and TSE2 was statistically significant P < 0,01. It was further observed that the number of reads mapped to the T. hassleriana contigs was in all cases, with the exception of TT4, grossly overestimating gene expression.
Expression of genes controlling floral traits in the flower and leaf transcriptome
Genes controlling various floral traits and flower development in A. thaliana, Antirrhinum majus, Fagopyrum esculentum etc. were identified based on literature [26–31]. The expression pattern of their putative T. hassleriana orthologs identified by a bidirectional BLATX search with the A. thaliana CDS sequences was analyzed in the flower and leaf transcriptomes to learn more about the regulation of the special floral traits of T. hassleriana (Figure 4). The selected genes were first grouped into different classes such as homeotic transcription factors, regulators of homeotic genes etc. and ordered within their groups according to transcript abundance. Of the genes analyzed, 49 (41.9%) were specific to the flower transcriptome and not found in the leaf transcriptome. (A. thaliana gene identifier, full gene names are shown in Additional file 1: Table S3).
Amongst the putative class ABCDE homeotic transcription factor orthologs, the highest expression was observed among the class B gene homologs AP3 and PI and the class E gene homologs SEP1 and SEP3. The putative ortholog of the C class gene AG was expressed at a 10 fold lower magnitude compared to the class B and E genes. The expression of the putative orthologs of the D class genes SHP1, SHP2 and STK the expression of which regulates the ovule and fruit development in A. thaliana was found to be considerably lower, when compared to the class ABCE genes. AP3, SEP3, SEP1, and STK transcripts were not present in the leaf transcriptome while PI, AP1, AP2, and SEP4 are expressed at a very low level in leaves. In addition to these, 25 other putative MADS box transcription factors without floral homeotic function that are members of the MIKC, Mα, Mβ, Mγ, Mδ subfamilies were also found to be expressed in the floral transcriptome.
Amongst the genes putatively regulating the class ABCDE homeotic transcription factors, the LUG, LUH and SEU orthologs showed highest expression in the flower transcriptome, while a 10 fold lower expression of these genes was observed in the leaf transcriptome. The expression of the LFY homolog was also observed in the floral transcriptome albeit at very low levels. Interestingly, putative homologs of genes regulating class B gene activities like UFO and SUP and class A gene activity like SAP were not identified in the floral transcriptome library. The orthologs for genes regulating patterning and symmetry also showed expression in the floral transcriptome. The putative orthologs of TCP4, TCP2, and PTF1 showed the highest expression. The expression of these genes was also observed in the leaf transcriptome; in case of the TCP4 ortholog a 100 fold higher expression was observed in the floral transcriptome when compared to the leaf transcriptome, while the TCP2 ortholog expression was almost equal in both the transcriptomes. Comparatively low expression of other putative patterning gene orthologs like TCP14, TCP15, TCP18, CUC2 and CUC3 was also observed specific to the floral transcriptome.
Genes putatively involved in anthocyanin synthesis, regulation, and deposition found in the floral transcriptome of T. hassleriana and the expression of their putative orthologs in A. thaliana tissues and developmental stages
Gene homologs expressed in T. hassleriana floral transcriptome
Expression in A. thaliana
Buds, senescent leaf, seed (globular embryo stage)
Buds, petal, seed (globular and torpedo stage embryo)
Buds, petal, young silique, seeds (globular and torpedo stage embryo)
Senescent leaf, young silique, seed (heart stage embryo)
Senescent leaf, young silique, seed (heart stage embryo)
Buds, petal, seeds (torpedo and walking stick stage embryo)
Buds, petal, carpel, seed (globular and heart stage embryo)
Senescent leaf, seed (curled cotyledon, green cotyledon stage embryo)
Young siliques, seeds (heart, walking stick stage embryo)
All plant organs, high expression in cauline and senescent leaves, young siliques, seeds (heart and torpedo stage embryo)
Cauline leaf, young siliques, seeds (Heart, torpedo, walking stage embryo)
Expression data not available
Shoot apex (vegetative, floral transition, inflorescence), young silique, seeds (globular, torpedo, walking stick stage embryo)
Petals, shoot apex (inflorescence)
All plant organs, high expression in pollen, seeds (curled cotyledon and green cotyledon embryo stage)
Young siliques, seeds (globular and heart stage embryo)
Expression of gene orthologs governing time to flower was also analyzed. Expression of both antagonistic groups of genes that prolong time to flower or enhance the transition into flowering was observed. Among the orthologs inducing flowering AGL20, GI, EBS, and FLK had the highest expression; expression of these genes was also observed in the leaf transcriptome at very comparable levels. Amongst the orthologs of genes delaying flowering SUF4, HUA1, COL9, and FLC had high levels of expression which was also observed at comparable levels in the leaf transcriptome. The orthologs of FRI, HUA2, SMZ, and ATC showed moderate to low floral transcriptome specific expression.
T. hassleriana homologs of meristem activity regulators, such as GAI, ANT and CLV1 which are involved in decreasing meristem proliferation was observed at high levels in the flower and varying levels in the leaf transcriptome while ANT expression was not detected in the leaf transcriptome. Putative homologs of genes BAM1, BAM2, BAM3 and WUS which enhance meristem proliferation were also found to have moderate expression levels in the floral transcriptome. Interestingly, putative homologs for FTA, ERA1, and STM, were found to be expressed in the floral transcriptome as their A. thaliana counterparts show very low expression the flower.
Another important category of gene orthologs analyzed for expression are the genes that co-regulate floral organ development alongside the ABCDE floral homeotic transcription factors. High expression was observed in case of orthologs of YAB2, AFO and PEP in both the floral and leaf transcriptomes whereas the expression of the CAL ortholog was about 100 fold higher in the flower transcriptome. Other floral organ developmental regulators, such as ENP, CRC, INO, NUB, JAG, and SPL were not identified in the T. hassleriana leaf transcriptome, but only in floral transcriptome whereas they are also expressed in A. thaliana leaves at very low levels. No expression was observed for ROXY gene homologs which are responsible for anther and male gametophyte development downstream of SPL.
Other putative homologs of A. thaliana floral regulators were identified amongst them were the highly expressed homologs of genes ASK1, CEV1, SKP1, RBX1, which are part of SCF ubiquitin protein ligase complexes which regulate multiple aspects of flower development together with UFO in A. thaliana. Homologs of genes ER, ERL1 and ERL2 which are protein kinases that influence meristem cell fate and patterning in the inflorescence meristem were also highly expressed. Interestingly the homolog of BPEP was found to be expressed only in the floral transcriptome, while the two distinct BPEP transcripts in A. thaliana are expressed in the floral as well as in vegetative organs respectively. Homologs of genes PIN1 and PID were also expressed which are known to affect size, floral organ number and total number of flowers in A. thaliana.
This in silico expression analysis of genes related to flower development demonstrates that with the chosen RNAseq method we are able to monitor gene expression in logarithmic scales covering more than two magnitudes. In addition, the two library preparations for this sequencing experiment show only rarely any difference in RPKM. Detailed expression analysis of putative T. hassleriana homologs of A. thaliana genes in the T. hassleriana floral transcriptome is provided in Additional file 3 along with the AGI identifiers.
Characterization of genes putatively governing sterility in T. hassleriana
Expression analysis by qRT-PCR indeed revealed that the expression of the ROXY1 homolog was very low (103 fold lower compared to ACT7) and well beyond the scope of detection by RNA seq. ROXY1 expression was down regulated in the sterile hybrid only at bud stage M when compared to the fertile parent the (Figure 5b) whereas it was similar to the parent at the younger and later developmental stages. Along with the down regulation of ROXY1, expression for the DYT1 and MYB35 homologs which most likely act downstream of ROXY1 was also down regulated in stage M buds. In stages other than M, the expression of DYT1 and MYB35 homologs in the sterile T. hassleriana hybrid was several fold higher than the respective expression in fertile parent buds in both the early and late developmental stages. Expression of the SPL homolog in the sterile hybrid buds was 3–4 fold higher than the fertile plant buds in stages S and M whereas in stage latter L the expression was 8 fold. Thus our expression data suggest that the complex network governing stamen development and microsporogenesis is disrupted in the T. hassleriana hybrid which could provide a causal link to its sterility.
Characterization of T. hassleriana floral transcriptome specific genes in comparison to A. thaliana
Selection of homologous gene pairs in which the homologs of T. hassleriana are expressed in the flower and the A. thaliana homolog expression is absent from the flower
T. hassleriana TSE (RPKM)
Expression in A. thaliana
multidrug pheromone mdr abc transporter family
tyrosine transaminase family protein
actin-related protein 2-like
Senescent leaf, cauline leaf, buds, flower, inflorescence shoot apex
homeodomain-leucine zipper protein
Young leaf, mature leaf, cauline leaf, senescent leaf, pedicel, seed (torpedo stage embryo)
leucine-rich repeat extensin-like protein 1
Young leaf, pollen, seed (cotyledon stage embryo)
pectate lyase family protein
Young leaf petiole, mature leaf (distal end), seed (curled cotyledon stage embryo)
protein-s-isoprenylcysteine o-methyltransferase a
Young leaf, cauline leaf, senescent leaf, young silique, seed (heart and torpedo stage embryo)
BTB/POZ domain-containing protein
ap2 erf and b3 domain-containing transcription factor rav2
Cotyledon, young leaf, senescent leaf
jacalin-like lectin domain-containing protein
Seed (curled cotyledon, green cotyledon embryo stage), dry seed
s-adenosyl-l-methionine-dependent methyltransferase-like protein
Seed (walking stick, curled cotyledon, green cotyledon embryo stage)
lrr receptor-like serine threonine-protein kinase rch1-like
Root, seed (torpedo stage embryo), imbibed seed
Root, seed (cotyledon embryo stage), dry seed
u3 small nucleolar rna-interacting protein 2-like
Pollen, seed (walking stick, curled cotyledon, green cotyledon stage embryo)
receptor-like protein kinase 2-like
Imbibed seed, root
Identification and characterization of Cleome lineage specific genes
An astonishing number of 5600 contigs (subset Z) could not be matched with high confidence to any other sequence from P. trichocarpa, C. papaya, A. thaliana and B. rapa. Of these contigs only 82 could be assigned to 353 GO terms, but a vast majority of the contigs could not be annotated attributing to no significant BLAST hits. A sequence length histogram for these contigs (Additional file 2: Figure S2) shows a bias towards shorter sequences when compared to the sequence length histogram of all contigs (Additional file 2: Figure S1b) suggesting that these were too short for proper annotation and/or may represent 5’ and 3’ UTR regions of transcripts. Another reason for the small number of annotated genes is because most of the current annotations are based on A. thaliana, B. rapa and P. trichocarpa and we already subtracted the sequences orthologous to them. The GO annotations for the T. hassleriana specific genes are the following: cellular process (26.98%), metabolic process (29.36%), response to stimulus (4.76%), biological regulation (4.7%), development (1.5%), cell proliferation (1.5%), reproduction (6.34%) and signaling processes (4.76%) (Additional file 1: Table S6).
In this work we present the floral transcriptome sequence of T. hassleriana, which is a member of the Cleomaceae and thus a sister taxon to the Brassicaceae. The transcriptome was analyzed by rarefaction analysis and shown to be of sufficient depth to also identify rare transcripts. As normalization was not carried out, abundance of transcripts could be assessed in silico and compared to qRT-PCR data. The leaf transcriptome of T. hassleriana has been published earlier  allowing for comparison of transcript abundance between the leaf and the floral transcriptome. We also attempted to compare the expression of genes represented in the floral transcriptome with the expression of their respective A. thaliana orthologs based on presence/absence of expression in the microarray dataset  including all flower developmental stages. In addition, we are able to identify 5600 putative transcripts that are specific to the Cleomaceae and 684, which are shared only among the Brassicales C. papaya, T. hassleriana, B. rapa, and A. thaliana.
During assembly, annotation and analysis of the reads obtained by 454 sequencing we observed several challenges. In our study we correlated the in silico floral transcriptome expression in T. hassleriana with the conventional qRT-PCR expression of arbitrarily chosen genes with low, moderate, and high expression levels for validation of the transcriptome sequencing expression data. For the in silico expression data we applied two approaches, one was to map the individual reads to the annotated A. thaliana CDS sequences (TSE1), with the advantage that expression data for putative T. hassleriana orthologs of A. thaliana genes can be generated without a prior genome sequence information of T. hassleriana, thus individual reads are not assembled into contigs. The second approach was the de novo assembly of the reads into contigs that are then annotated and the reads are mapped on to these contigs (TSE2). Comparing both methods with qRT-PCR data, the TSE1 approach clearly matches better than the TSE2 approach. One reason for this finding is the presence of chimeric contigs composed of more than one gene, in such cases reads to multiple genes are mapped onto the same contig flaring up the expression. This problem is avoided in TSE1 when the reads are mapped onto orthologous sequences in A. thaliana. Another reason for the disparity between the qRT-PCR expression and TSE2 is due to assembly of contigs with additional non coding nucleotide sequences. This phenomenon was observed in the case of RBCS1A amongst the genes analyzed. The assembled contig was 838 nucleotides long whereas the coding sequence of this gene is ~540 nucleotides across many plant lineages. The additional 295 nucleotides at the 3’ end could represent the 3’ UTR nonetheless reads would be mapped to such sequences leading to an overestimation of expression. The third reason for the differences in the expression between qRT-PCR and TSE2 may be the length of the assembled contig versus transcript size, as larger transcripts are fragmented prior cDNA library preparation. For TSE1, qRT-PCR expression data were normalized to A. thaliana CDS lengths and in case of TSE2 to T. hassleriana contig length. The necessity of normalization was seen in case of gene SPL7. The SPL7 contig length was 629 nucleotides whereas the coding sequence of SPL7 in A. thaliana is 2406 nucleotides, thus when the expression is normalized for length of the contig it leads to a much higher expression than when normalized to the length of the A. thaliana ortholog. In case where the contig length matched to the coding sequence length and when the contig had very low or almost no unknown sequences incorporated the qRT-PCR expression matched very well to the TSE2 as was observed in case of TT4.
However, since T. hassleriana and A. thaliana have, independent α-WGDs, the retention and loss of gene copies following the duplication will be different. By mapping the T. hassleriana reads onto the A. thaliana orthologs identical sets of orthologous gene copies are assumed for both species leading to over or underestimation of transcript abundance. These illustrated pitfalls for calculating gene expression from RNAseq experiments without the availability of a high-quality reference transcriptome or genome require thorough independent validation of gene expression data.
This work was initiated as a primer to identify genes that may contribute to the morphological differences between the T. hassleriana and the A. thaliana flower. Our focus was mainly on coloration, flowering time, and floral organ size as these are traits that show obvious differences between the two species.
T. hassleriana petals show a deep pink coloration which, when the flower opens, fades into light pink after a few days of exposure to the sun. While in most species only the epidermal petal cell layer is pigmented, T. hassleriana also has pigmented mesophyll cells ; suggesting an expansion of the anthocyanin regulation and biosynthesis pathway from petal to mesophyll cells. The pink pigments found in T. hassleriana flowers are acetylated cyanidin diglucoside (sophorosyl)-5-glucosides and acetylated pelargonidin sophorosyl-5-glucosides . All genes required for the synthesis of pelargonidin-3-glucoside and cyanidin-3-glucoside are present in the flower transcriptome. Genes encoding proteins required for the early steps of anthocyanin up to the flavonoid myricetin are also found expressed in the leaf transcriptome, while the genes participating in later steps such as DFR and LDOX are restricted to the flower. These two enzymes are also not expressed in A. thaliana flowers but during seed development and late stages of leaf senescence .
Transcription factors of the MYB, bHLH, and WD40 families regulate the expression of anthocyanin biosynthesis genes in A. thaliana and Zea mays. While early biosynthesis genes, and their regulators such as AtMYB11, AtMYB12, and AtMYB111 are involved in the production of flavonols, late biosynthesis genes and their regulators are required for the synthesis of anthocyanins from flavonols  and references therein. While the putative T. hassleriana orthologs of AtMYB11 and AtMYB12 are hardly expressed in the flower transcriptome, the putative AtMYB111 ortholog shows very strong and flower specific expression suggesting a more prominent role for this gene in the regulation of early biosynthesis genes than for the putative orthologs of AtMYB11 and AtMYB12. Orthologs of the regulators of late anthocyanin biosynthesis in A. thaliana AtTTG1 (WD40 family member), AtTT8, AtGL3, AtEGL3 (all bHLH family members) and AtPAP2 (MYB family member) are also found expressed in the T. hassleriana flowers. The T. hassleriana orthologs of A. thaliana genes AtTTG1, AtTT8, AtGL3, AtEGL3, and AtPAP2 forming the late anthocyanin biosynthesis regulatory complex show an approximately similar transcript abundance suggesting that they may function in a complex similar to the one in A. thaliana, only with an expression domain expanded to the floral organs.
A. thaliana late regulators are mainly expressed in senescing leaves and during seed development (Table 2), but most likely, their expression domain in T. hassleriana has expanded into the flower leading to the pink coloration of the floral organs. A similar situation is found in petunia, where, among others genes AN1, AN11, AN2 and AN4 form complexes similar to that in A. thaliana to regulate anthocyanin biosynthesis in the flower .
T. hassleriana has, unlike A. thaliana, large oval shaped petals, and indeed orthologs of genes involved in limiting growth of floral organs were found to be hardly expressed in the T. hassleriana floral transcriptome. BIG BROTHER, encoding for a E3 ubiquitin-ligase represses cell proliferation in all A. thaliana proliferating tissues and is expressed strongly and uniformly in all developmental stages of the flower independently of other pathways while being a direct target of the petal organ identity gene AP3[39–41]. In the T. hassleriana floral transcriptome it has a very low expression of 9 RPKM, suggesting that this may be a reasonable candidate to account for the differences in petal size between the two species.
The particular T. hassleriana hybrid used in this study is sterile even though it produces all the floral organ whorls in the right number and position. However, even though the anthers developed, they did not produce any pollen and also did not dehisce rendering the plants male sterile. While orthologs of A. thaliana regulators of anther development were expressed in the the T. hassleriana flower, no expression of ROXY1 and ROXY2 was detected. These two genes redundantly control the anther lobe and pollen mother cell differentiation downstream of SPL in A. thaliana. Moreover, only very low expression (6 RPKM) was observed for the T. hassleriana ortholog of DYT1 which acts directly downstream of the ROXY genes. The phenotype of the T. hassleriana anthers also resembles the roxy1 roxy2 double mutant anther phenotype in A. thaliana, suggesting that our T. hassleriana hybrid may lack functional ROXY genes leading to male sterility. We corroborated this observation by qRT-PCR expression data which not only detected very low ROXY1 expression (the only ROXY ortholog in T. hassleriana genome ) in the mid developmental stage but also showed the de-regulation of expression of the upstream and downstream genes throughout bud development which may provide a cause for the male sterility.
T. hassleriana is perpetually flowering and a sharp transition to flowering as in A. thaliana cannot be observed. Several genes involved in flowering time regulation in A. thaliana are differently regulated in leaves and flowers and we compared the expression of their orthologs in the flower and leaf transcriptomes. FRI is a protein involved in activating transcription via chromatin remodeling of the central floral repressor FLC in A. thaliana and is expressed rather uniformly throughout the plant. However, in T. hassleriana, FRI ortholog expression is not found in leaves. This may suggest a different mechanism for FLC ortholog activation in T. hassleriana leaves, as FLC is expressed there without the presence of FRI.
Interestingly; the expression of two more genes most likely involved in the change from vegetative to reproductive phase in T. hassleriana is different from A. thaliana. The A. thaliana gene SMZ is expressed in young seedlings, during floral transition and seed maturation  unlike its T. hassleriana ortholog which is expressed in flowers and developing buds. Possibly, the T. hassleriana SMZ has function different from its A. thaliana ortholog, which is a rather strong repressor of flowering.
Another candidate gene in the group of flowering time regulators that are differentially regulated in A. thaliana and T. hassleriana is ATC. In A. thaliana, ATC is strongly expressed in the root and a small fraction (1-5%) of its mRNAs moves a long distance to the plant’s apex . Notably, we find a significant amount of reads in flower tissue, too many to attribute them to long distance RNA transport. More likely, the ATC homolog is expressed in T. hassleriana floral tissue and may be transported throughout the plant to enable the vegetative shoots to first reach sufficient size to start flowering.
Taken together we conclude from our expression data that a number of floral regulators show expression distinct from that in A. thaliana suggesting that differences in life history traits such as perpetual flowering and pigmentation may be regulated by similar components of regulatory networks in A. thaliana and T. hassleriana that are highly conserved in coding sequence but expressed in a different way in the two species, suggesting that modifications in expression pattern account for a large part of the diversity in flowers and plant life history traits.
Whole genome duplication
A. thaliana alpha WGD
A. thaliana beta WGD
Expressed sequence tag
Million years ago
Reads per kilobase gene model per mappable million
Standard dose response
T. hassleriana alpha WGD
Transcriptome sequencing expression
TSE mapped to A. thaliana CDS sequences
TSE mapped to T. hassleriana contigs.
We thank the University of Bremen and the Justus-Liebig University for funding of A. Bhide’s position, work in A. Becker’s lab is largely funded by the DFG (German Research Foundation). A.P.M.W. acknowledges funding by DFG Priority Program 1529 (Adaptomics). Library preparation and subsequent 454 sequencing was performed by René Deenen at the Biomedical Research Center (BMFZ) of the Heinrich-Heine-University Düsseldorf.
- Iltis HH, Cochrane TS: Studies in the Cleomaceae V: a new genus and ten new combinations for the flora of North America. J Bot Nomencl. 2007, 17: 447-451.View ArticleGoogle Scholar
- Inda LA, Torrecilla P, Catalán P, Ruiz-Zapata T: Phylogeny of Cleome L. and its close relatives Podandrogyne Ducke and Polanisia Raf. (Cleomoideae, Cleomaceae) based on analysis of nuclear ITS sequences and morphology. Plant Syst Evol. 2008, 274: 111-126. 10.1007/s00606-008-0026-y.View ArticleGoogle Scholar
- Hall JC, Sytsma KJ, Iltis HH: Phylogeny of Capparaceae and Brassicaceae based on chloroplast sequence data. Am J Bot. 2002, 89: 1826-1842. 10.3732/ajb.89.11.1826.PubMedView ArticleGoogle Scholar
- Kers LE: Capparaceae. Flowering Plants Dicotyledons. Edited by: Kubitzki K, Bayer C. 2003, Berlin, Heidelberg: Springer Berlin Heidelberg, 36-56.View ArticleGoogle Scholar
- Rodman J, Soltis P, Soltis D, Sytsma K, Karol K: Parallel evolution of glucosinolate biosynthesis inferred from congruent nuclear and plastid gene phylogenies. Am J Bot. 1998, 85: 997-10.2307/2446366.PubMedView ArticleGoogle Scholar
- Schranz ME: Independent ancient polyploidy events in the sister families Brassicaceae and Cleomaceae. Plant Cell Online. 2006, 18: 1152-1165. 10.1105/tpc.106.041111.View ArticleGoogle Scholar
- Couvreur TLP, Franzke A, Al-Shehbaz IA, Bakker FT, Koch MA, Mummenhoff K: Molecular phylogenetics, temporal diversification, and principles of evolution in the mustard family (Brassicaceae). Mol Biol Evol. 2009, 27: 55-71.View ArticleGoogle Scholar
- Barker MS, Vogel H, Schranz ME: Paleopolyploidy in the Brassicales: analyses of the cleome transcriptome elucidate the history of genome duplications in Arabidopsis and other Brassicales. Genome Biol Evol. 2009, 1: 391-399.PubMed CentralPubMedView ArticleGoogle Scholar
- Koteyeva NK, Voznesenskaya EV, Roalson EH, Edwards GE: Diversity in forms of C4 in the genus Cleome (Cleomaceae). Ann Bot. 2011, 107: 269-283. 10.1093/aob/mcq239.PubMed CentralPubMedView ArticleGoogle Scholar
- Brautigam A, Kajala K, Wullenweber J, Sommer M, Gagneul D, Weber KL, Carr KM, Gowik U, Mass J, Lercher MJ, Westhoff P, Hibberd JM, Weber APM: An mRNA blueprint for C4 photosynthesis derived from comparative transcriptomics of closely related C3 and C4 species. Plant Physiol. 2011, 155: 142-156. 10.1104/pp.110.159442.PubMed CentralPubMedView ArticleGoogle Scholar
- Stout AB: Alternation of Sexes and Intermittent Production of Fruit in the Spider Flower (cleome Spinosa). 1923, New York: New York Botanical Garden, [Contributions from the New York Botanical Garden]Google Scholar
- Patchell MJ, Bolton MC, Mankowski P, Hall JC: Comparative floral development in Cleomaceae reveals two distinct pathways leading to monosymmetry. Int J Plant Sci. 2011, 172: 352-365. 10.1086/658158.View ArticleGoogle Scholar
- Randall RP: A Global Compendium of Weeds. 2012, 2Google Scholar
- Nozzolillo C, Amiguet VT, Bily AC, Harris CS, Saleem A, Andersen , Oyvind M, Jordheim M: Novel aspects of the flowers and floral pigmentation of two Cleome species (Cleomaceae), C. hassleriana and C. serrulata. Biochem Syst Ecol. 2010, 38: 361-369. 10.1016/j.bse.2010.03.005.View ArticleGoogle Scholar
- Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987, 162: 156-159.PubMedView ArticleGoogle Scholar
- Kent WJ: BLAT‒-the BLAST-like alignment tool. Genome Res. 2002, 12: 656-664. 10.1101/gr.229202. Article published online before March 2002.PubMed CentralPubMedView ArticleGoogle Scholar
- Schliesky S, Gowik U, Weber A, Andreas PM, Bräutigam A: RNA-Seq assembly ‒ are we there yet?. Front Plant Sci. 2012, 3:Google Scholar
- R Core Team: R: A Language and Environment for Statistical Computing. 2013, Vienna, AustriaGoogle Scholar
- Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT: The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009, 55: 611-622. 10.1373/clinchem.2008.112797.PubMedView ArticleGoogle Scholar
- Marshall OJ: PerlPrimer: cross-platform, graphical primer design for standard, bisulphite and real-time PCR. Bioinformatics. 2004, 20: 2471-2472. 10.1093/bioinformatics/bth254.PubMedView ArticleGoogle Scholar
- Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative 5PCR6 and the 2-\textgreek5DD6CT method. Methods. 2001, 25: 402-408. 10.1006/meth.2001.1262.PubMedView ArticleGoogle Scholar
- Schmid M, Davison TS, Henz SR, Pape UJ, Demar M, Vingron M, Schölkopf B, Weigel D, Lohmann JU: A gene expression map of Arabidopsis thaliana development. Nat Genet. 2005, 37: 501-506. 10.1038/ng1543.PubMedView ArticleGoogle Scholar
- Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M: Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics. 2005, 21: 3674-3676. 10.1093/bioinformatics/bti610.PubMedView ArticleGoogle Scholar
- Tipper JC: Rarefaction and rarefiction; the use and abuse of a method in paleoecology. Paleobiology. 1979, 5: 423-434.Google Scholar
- Hale MC, McCormick CR, Jackson JR, DeWoody JA: Next-generation pyrosequencing of gonad transcriptomes in the polyploid lake sturgeon (Acipenser fulvescens): the relative merits of normalization and rarefaction in gene discovery. BMC Genomics. 2009, 10: 203-10.1186/1471-2164-10-203.PubMed CentralPubMedView ArticleGoogle Scholar
- Lohmann JU, Weigel D: Building beauty: the genetic control of floral patterning. Dev Cell. 2002, 2: 135-142. 10.1016/S1534-5807(02)00122-3.PubMedView ArticleGoogle Scholar
- Broun P: Transcriptional control of flavonoid biosynthesis: a complex network of conserved regulators involved in multiple aspects of differentiation in Arabidopsis. Curr Opin Plant Biol. 2005, 8: 272-279. 10.1016/j.pbi.2005.03.006.PubMedView ArticleGoogle Scholar
- Weiss J, Delgado-Benarroch L, Egea-Cortines M: Genetic control of floral size and proportions. Int J Dev Biol. 2005, 49: 513-525. 10.1387/ijdb.051998jw.PubMedView ArticleGoogle Scholar
- Zhang X, Feng B, Zhang Q, Zhang D, Altman N, Ma H: Genome-wide expression profiling and identification of gene activities during early flower development in Arabidopsis. Plant Mol Biol. 2005, 58: 401-419. 10.1007/s11103-005-5434-6.PubMedView ArticleGoogle Scholar
- Gou J, Felippes FF, Liu C, Weigel D, Wang J: Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPL transcription factor. Plant Cell Online. 2011, 23: 1512-1522. 10.1105/tpc.111.084525.View ArticleGoogle Scholar
- Logacheva MD, Kasianov AS, Vinogradov DV, Samigullin TH, Gelfand MS, Makeev VJ, Penin AA: De novo sequencing and characterization of floral transcriptome in two species of buckwheat (Fagopyrum). BMC Genomics. 2011, 12: 30-10.1186/1471-2164-12-30.PubMed CentralPubMedView ArticleGoogle Scholar
- Winter D, Vinegar B, Nahal H, Ammar R, Wilson GV, Provart NJ, Baxter I: An “Electronic fluorescent pictograph” browser for exploring and analyzing large-scale biological data sets. PloS One. 2007, 2: e718-10.1371/journal.pone.0000718.PubMed CentralPubMedView ArticleGoogle Scholar
- Ni W: Regulation of flower development in Arabidopsis by SCF complexes. Plant Physiol. 2004, 134: 1574-1585. 10.1104/pp.103.031971.PubMed CentralPubMedView ArticleGoogle Scholar
- Xing S, Zachgo S: ROXY1 and ROXY2, two Arabidopsis glutaredoxin genes, are required for anther development. Plant J. 2008, 53: 790-801. 10.1111/j.1365-313X.2007.03375.x.PubMedView ArticleGoogle Scholar
- Cheng S, van den Bergh E, Zeng P, Zhong X, Xu J, Liu X, Hofberger J, de Bruijn S, Bhide AS, Kuelahoglu C, Bian C, Chen J, Fan G, Kaufmann K, Hall JC, Becker A, Brautigam A, Weber APM, Shi C, Zheng Z, Li W, Lv M, Tao Y, Wang J, Zou H, Quan Z, Hibberd JM, Zhang G, Zhu X, Xu X, et al: The tarenaya hassleriana genome provides insight into reproductive trait and genome evolution of crucifers. Plant Cell. 2013, 25: 2813-2830. 10.1105/tpc.113.113480.PubMed CentralPubMedView ArticleGoogle Scholar
- Beilstein MA, Nagalingum NS, Clements MD, Manchester SR, Mathews S: Dated molecular phylogenies indicate a Miocene origin for Arabidopsis thaliana. Proc Natl Acad Sci. 2010, 107: 18724-18728. 10.1073/pnas.0909766107.PubMed CentralPubMedView ArticleGoogle Scholar
- Petroni K, Tonelli C: Recent advances on the regulation of anthocyanin synthesis in reproductive organs. Plant Sci. 2011, 181: 219-229. 10.1016/j.plantsci.2011.05.009.PubMedView ArticleGoogle Scholar
- Quattrocchio F: PH4 of petunia is an R2R3 MYB protein that activates vacuolar acidification through interactions with basic-helix-loop-helix transcription factors of the anthocyanin pathway. Plant Cell Online. 2006, 18: 1274-1291. 10.1105/tpc.105.034041.View ArticleGoogle Scholar
- Disch S, Anastasiou E, Sharma VK, Laux T, Fletcher JC, Lenhard M: The E3 ubiquitin ligase BIG BROTHER controls Arabidopsis organ size in a dosage-dependent manner. Curr Biol. 2006, 16: 272-279. 10.1016/j.cub.2005.12.026.PubMedView ArticleGoogle Scholar
- Wuest SE, O’Maoileidigh DS, Rae L, Kwasniewska K, Raganelli A, Hanczaryk K, Lohan AJ, Loftus B, Graciet E, Wellmer F: Molecular basis for the specification of floral organs by APETALA3 and PISTILLATA. Proc Natl Acad Sci. 2012, 109: 13452-13457. 10.1073/pnas.1207075109.PubMed CentralPubMedView ArticleGoogle Scholar
- Krizek BA, Anderson JT: Control of flower size. J Exp Bot. 2013, 64: 1427-1437. 10.1093/jxb/ert025.PubMedView ArticleGoogle Scholar
- Choi K, Kim J, Hwang H, Kim S, Park C, Kim SY, Lee I: The FRIGIDA complex activates transcription of FLC, a strong flowering repressor in Arabidopsis, by recruiting chromatin modification factors. Plant Cell Online. 2011, 23: 289-303. 10.1105/tpc.110.075911.View ArticleGoogle Scholar
- Mathieu J, Yant LJ, Mürdter F, Küttner F, Schmid M, Dean C: Repression of flowering by the miR172 target SMZ. PLoS Biol. 2009, 7: e1000148-10.1371/journal.pbio.1000148.PubMed CentralPubMedView ArticleGoogle Scholar
- Huang N, Jane W, Chen J, Yu T: Arabidopsis thaliana CENTRORADIALIS homologue ( ATC ) acts systemically to inhibit floral initiation in Arabidopsis. Plant J. 2012, 72: 175-184. 10.1111/j.1365-313X.2012.05076.x.PubMedView ArticleGoogle Scholar
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