Revealing the developmental dynamics in male strobilus transcriptome of Gnetum luofuense using nanopore sequencing technology.

Gnetum is a pantropical distributed gymnosperm genus. As being dioecious, Gnetum species apply female and male strobili to attract and provide nutrition to insect pollinators. Due to its unique gross morphology, a Gnetum male strobilus receives much attention in previous taxonomic and evolutionary studies. However, underlying molecular mechanisms that control male strobilus development and pollination adaptation have not been well studied. In the present study, nine full-length transcriptomes were sequenced from three developmental stages of the G. luofuense male strobili using Oxford Nanopore Technologies. In addition, weighted gene co-expression network analysis (WGCNA), and RT-qPCR analysis were performed. Our results show that a total of 3138 transcription factors and 466 long non-coding RNAs (lncRNAs) were identified, and differentially expressed lncRNAs and TFs reveal a dynamic pattern during the male strobilus development. Our results show that MADS-box and Aux/IAA TFs were differentially expressed at the three developmental stages, suggesting their important roles in the regulation of male strobilus development of G. luofuense. Results of WGCNA analysis and annotation of differentially expressed transcripts corroborate that the male strobilus development of G. luofuense is closely linked to plant hormone changes, photosynthesis, pollination drop secretion and reproductive organ defense. Our results provide a valuable resource for understanding the molecular mechanisms that drive organ evolution and pollination biology in Gnetum.

Deciphering the evolution of the ovule genetic network through expression analyses in Gnetum gnemon.

BACKGROUND AND AIMS: The ovule is a synapomorphy of all seed plants (gymnosperms and angiosperms); however, there are some striking differences in ovules among the major seed plant lineages, such as the number of integuments or the orientation of the ovule. The genetics involved in ovule development have been well studied in the model species Arabidopsis thaliana, which has two integuments and anatropous orientation. This study is approached from what is known in arabidopsis, focusing on the expression patterns of homologues of four genes known to be key for the proper development of the integuments in arabidopsis: AINTEGUMENTA (ANT), BELL1, (BEL1), KANADIs (KANs) and UNICORN (UCN). METHODS: We used histology to describe the morphoanatomical development from ovules to seeds in Gnetum gnemon. We carried out spatiotemporal expression analyses in G. gnemon, a gymnosperm, which has a unique ovule morphology with an integument covering the nucellus, two additional envelopes where the outermost becomes fleshy as the seed matures, and an orthotropous orientation. KEY RESULTS: Our anatomical and developmental descriptions provide a framework for expression analyses in the ovule of G. gnemon. Our expression results show that although ANT, KAN and UCN homologues are expressed in the inner integument, their spatiotemporal patterns differ from those found in angiosperms. Furthermore, all homologues studied here are expressed in the nucellus, revealing major differences in seed plants. Finally, no expression of the studied homologues was detected in the outer envelopes. CONCLUSIONS: Altogether, these analyses provide significant comparative data that allows us to better understand the functional evolution of these gene lineages, providing a compelling framework for evolutionary and developmental studies of seeds. Our findings suggest that these genes were most likely recruited from the sporangium development network and became restricted to the integuments of angiosperm ovules.

A full-length transcriptome and gene expression analysis reveal genes and molecular elements expressed during seed development in Gnetum luofuense.

BACKGROUND: Gnetum is an economically important tropical and subtropical gymnosperm genus with various dietary, industrial and medicinal uses. Many carbohydrates, proteins and fibers accumulate during the ripening of Gnetum seeds. However, the molecular mechanisms related to this process remain unknown. RESULTS: We therefore assembled a full-length transcriptome from immature and mature G. luofuense seeds using PacBio sequencing reads. We identified a total of 5726 novel genes, 9061 alternative splicing events, 3551 lncRNAs, 2160 transcription factors, and we found that 8512 genes possessed at least one poly(A) site. In addition, gene expression comparisons of six transcriptomes generated by Illumina sequencing showed that 14,323 genes were differentially expressed from an immature stage to a mature stage with 7891 genes upregulated and 6432 genes downregulated. The expression of 14 differentially expressed transcription factors from the MADS-box, Aux/IAA and bHLH families was validated by qRT-PCR, suggesting that they may have important roles in seed ripening of G. luofuense. CONCLUSIONS: These findings provide a valuable molecular resource for understanding seed development of gymnosperms.

Single-Molecule Long-Read Sequencing Reveals the Diversity of Full-Length Transcripts in Leaves of Gnetum (Gnetales).

The limitations of RNA sequencing make it difficult to accurately predict alternative splicing (AS) and alternative polyadenylation (APA) events and long non-coding RNAs (lncRNAs), all of which reveal transcriptomic diversity and the complexity of gene regulation. Gnetum, a genus with ambiguous phylogenetic placement in seed plants, has a distinct stomatal structure and photosynthetic characteristics. In this study, a full-length transcriptome of Gnetum luofuense leaves at different developmental stages was sequenced with the latest PacBio Sequel platform. After correction by short reads generated by Illumina RNA-Seq, 80,496 full-length transcripts were obtained, of which 5269 reads were identified as isoforms of novel genes. Additionally, 1660 lncRNAs and 12,998 AS events were detected. In total, 5647 genes in the G. luofuense leaves had APA featured by at least one poly(A) site. Moreover, 67 and 30 genes from the bHLH gene family, which play an important role in stomatal development and photosynthesis, were identified from the G. luofuense genome and leaf transcripts, respectively. This leaf transcriptome supplements the reference genome of G. luofuense, and the AS events and lncRNAs detected provide valuable resources for future studies of investigating low photosynthetic capacity of Gnetum.

Pollination Drop Proteome and Reproductive Organ Transcriptome Comparison in Gnetum Reveals Entomophilous Adaptation.

Gnetum possesses morphologically bisexual but functionally unisexual reproductive structures that exude sugary pollination drops to attract insects. Previous studies have revealed that the arborescent species (G. gnemon L.) and the lianoid species (G. luofuense C.Y.Cheng) possess different pollination syndromes. This study compared the proteome in the pollination drops of these two species using label-free quantitative techniques. The transcriptomes of fertile reproductive units (FRUs) and sterile reproductive units (SRUs) for each species were furthermore compared using Illumina Hiseq sequencing, and integrated proteomic and transcriptomic analyses were subsequently performed. Our results show that the differentially expressed proteins between FRUs and SRUs were involved in carbohydrate metabolism, the biosynthesis of amino acids and ovule defense. In addition, the differentially expressed genes between the FRUs and SRUs (e.g., MADS-box genes) were engaged in reproductive development and the formation of pollination drops. The integrated protein-transcript analyses revealed that FRUs and their exudates were relatively conservative while the SRUs and their exudates were more diverse, probably functioning as pollinator attractants. The evolution of reproductive organs appears to be synchronized with changes in the pollination drop proteome of Gnetum, suggesting that insect-pollinated adaptations are not restricted to angiosperms but also occur in gymnosperms.

Two Independent Plastid accD Transfers to the Nuclear Genome of Gnetum and Other Insights on Acetyl-CoA Carboxylase Evolution in Gymnosperms.

Acetyl-CoA carboxylase (ACCase) is the key regulator of fatty acid biosynthesis. In most plants, ACCase exists in two locations (cytosol and plastids) and in two forms (homomeric and heteromeric). Heteromeric ACCase comprises four subunits, three of them (ACCA-C) are nuclear encoded (nr) and the fourth (ACCD) is usually plastid encoded. Homomeric ACCase is encoded by a single nr-gene (ACC). We investigated the ACCase gene evolution in gymnosperms by examining the transcriptomes of newly sequenced Gnetum ula, combined with 75 transcriptomes and 110 plastomes of other gymnosperms. AccD-coding sequences are elongated through the insertion of repetitive DNA in four out of five cupressophyte families (except Sciadopityaceae) and were functionally transferred to the nucleus of gnetophytes and Sciadopitys. We discovered that, among the three genera of gnetophytes, only Gnetum has two copies of nr-accD. Furthermore, using protoplast transient expression assays, we experimentally verified that the nr-accD precursor proteins in Gnetum and Sciadopitys can be delivered to the plastids. Of the two nr-accD copies of Gnetum, one dually targets plastids and mitochondria, whereas the other potentially targets plastoglobuli. The distinct transit peptides, gene architectures, and flanking sequences between the two Gnetum accDs suggest that they have independent origins. Our findings are the first account of two distinctly targeted nr-accDs of any green plants and the most comprehensive analyses of ACCase evolution in gymnosperms to date.

Proteome-Level Analysis of Metabolism- and Stress-Related Proteins during Seed Dormancy and Germination in Gnetum parvifolium.

Gnetum parvifolium is a rich source of materials for traditional medicines, food, and oil, but little is known about the mechanism underlying its seed dormancy and germination. In this study, we analyzed the proteome-level changes in its seeds during germination using isobaric tags for relative and absolute quantitation. In total, 1,040 differentially expressed proteins were identified, and cluster analysis revealed the distinct time points during which signal transduction and oxidation-reduction activity changed. Gene Ontology analysis showed that "carbohydrate metabolic process" and "response to oxidative stress" were the main enriched terms. Proteins associated with starch degradation and antioxidant enzymes were important for dormancy-release, while proteins associated with energy metabolism and protein synthesis were up-regulated during germination. Moreover, protein-interaction networks were mainly associated with heat-shock proteins. Furthermore, in accord with changes in the energy metabolism- and antioxidant-related proteins, indole-3-acetic acid, Peroxidase, and soluble sugar content increased, and the starch content decreased in almost all six stages of dormancy and germination analyzed (S1-S6). The activity of superoxide dismutase, abscisic acid, and malondialdehyde content increased in the dormancy stages (S1-S3) and then decreased in the germination stages (S4-S6). Our results provide new insights into G. parvifolium seed dormancy and germination at the proteome and physiological levels, with implications for improving seed propagation.

A genome for gnetophytes and early evolution of seed plants.

Gnetophytes are an enigmatic gymnosperm lineage comprising three genera, Gnetum, Welwitschia and Ephedra, which are morphologically distinct from all other seed plants. Their distinctiveness has triggered much debate as to their origin, evolution and phylogenetic placement among seed plants. To increase our understanding of the evolution of gnetophytes, and their relation to other seed plants, we report here a high-quality draft genome sequence for Gnetum montanum, the first for any gnetophyte. By using a novel genome assembly strategy to deal with high levels of heterozygosity, we assembled >4 Gb of sequence encoding 27,491 protein-coding genes. Comparative analysis of the G. montanum genome with other gymnosperm genomes unveiled some remarkable and distinctive genomic features, such as a diverse assemblage of retrotransposons with evidence for elevated frequencies of elimination rather than accumulation, considerable differences in intron architecture, including both length distribution and proportions of (retro) transposon elements, and distinctive patterns of proliferation of functional protein domains. Furthermore, a few gene families showed Gnetum-specific copy number expansions (for example, cellulose synthase) or contractions (for example, Late Embryogenesis Abundant protein), which could be connected with Gnetum's distinctive morphological innovations associated with their adaptation to warm, mesic environments. Overall, the G. montanum genome enables a better resolution of ancestral genomic features within seed plants, and the identification of genomic characters that distinguish Gnetum from other gymnosperms.

High temperature and UV-C treatments affect stilbenoid accumulation and related gene expression levels in Gnetum parvifolium

Background: Gnetum parvifolium stems and roots have been used for a long time in traditional Chinese medicines. Stilbenes are bioactive compounds present in G. parvifolium plants, and they possess antioxidative and anticancer properties. However, little is known about the responses of G. parvifolium stilbene biosynthetic pathways to stress conditions. Therefore, we investigated stilbene biosynthesis, including the expression of relevant genes, in G. parvifolium exposed to high-temperature and ultraviolet-C treatments. Results: High temperatures did not influence the accumulation of total stilbenes in stems but decreased stilbene concentrations in roots at 3 h, with a subsequent restoration to control levels. In contrast, ultraviolet irradiation induced the accumulation of total stilbenes in stems but not in roots. We also observed that high temperatures inhibited the production of resveratrol and piceatannol in G. parvifolium stems and roots, whereas ultraviolet treatments initially inhibited their accumulation (up to 6 h) but induced their production at later time points. Analyses of specific genes (i.e., PAL, C4H, 4CL, STS, and CYP) revealed that their expression levels generally increased in stress-treated stems and roots, although there was some variability in the expression profiles during treatments. Conclusions: Our results indicated that high temperatures and ultraviolet irradiation differentially affect the biosynthesis of specific stilbenes in G. parvifolium stems and roots. Therefore, cultivating G. parvifolium seedlings under optimal stress conditions may increase the biosynthesis of specific stilbene compounds.

The complete chloroplast genome of Gnetum montanum and sequence analysis.

The complete chloroplast sequence of Gnetum montanum (GenBank accession number: NC_021438.1) was determined and characterized in this study. The size of the chloroplast genome of Gnetum montanum is 115 019 bp. Including a large single copy (LSC) region of 66 697 bp and a small single copy (SSC) region of 9494 bp separated by a pair of identical inverted repeat regions (IRs) of 19 414 bp each. A total of 108 genes that were successfully annotated contains 65 protein-coding genes, 40 tRNA genes and 3 rRNA genes. The GC content of the chloroplast genome of Gnetum montanum is 38.16%. Twelve genes contain one introns.

Molecular interactions of orthologues of floral homeotic proteins from the gymnosperm Gnetum gnemon provide a clue to the evolutionary origin of 'floral quartets'.

Several lines of evidence suggest that the identity of floral organs in angiosperms is specified by multimeric transcription factor complexes composed of MADS-domain proteins. These bind to specific cis-regulatory elements ('CArG-boxes') of their target genes involving DNA-loop formation, thus constituting 'floral quartets'. Gymnosperms, angiosperms' closest relatives, contain orthologues of floral homeotic genes, but when and how the interactions constituting floral quartets were established during evolution has remained unknown. We have comprehensively studied the dimerization and DNA-binding of several classes of MADS-domain proteins from the gymnosperm Gnetum gnemon. Determination of protein-protein and protein-DNA interactions by yeast two-hybrid, in vitro pull-down and electrophoretic mobility shift assays revealed complex patterns of homo- and heterodimerization among orthologues of floral homeotic class B, class C and class E proteins and B(sister) proteins. Using DNase I footprint assays we demonstrate that both orthologues of class B with C proteins, and orthologues of class C proteins alone, but not orthologues of class B proteins alone can loop DNA in floral quartet-like complexes. This is in contrast to class B and class C proteins from angiosperms, which require other factors such as class E floral homeotic proteins to 'glue' them together in multimeric complexes. Our findings suggest that the evolutionary origin of floral quartet formation is based on the interaction of different DNA-bound homodimers, does not depend on class E proteins, and predates the origin of angiosperms.

Lineage-specific group II intron gains and losses of the mitochondrial rps3 gene in gymnosperms.

According to PCR assays and sequencing, we now report the shared presence of two rps3 introns, namely the rps3i74 and the rps3i249, in the mitochondria of all the classes representing the surviving lineages of gymnosperms, and unveil several lineages experiencing intron loss. Interestingly, the rps3 intron gains and losses within the four groups of gymnosperms let us sort out the Pinaceae and the non-Pinaceae into intron (+)- and intron (-)-lineages, respectively. Worthy of mention is also the finding that only Gnetum within the Gnetales harbours both the rps3 introns. This intron distribution pattern is consistent with the hypothesis that the two rps3 introns were likely present in the common ancestor of the seed plants and, then, independently lost in the non-Pinaceae during gymnosperm evolution. The derived secondary structural model of the novel group IIA intron improves our understanding of the significance and origin of the extraordinary length polymorphisms observed among rps3i249 orthologs. Despite the remarkable structural plasticity to adopt and reject introns, the rps3 mRNAs undergo accurate processing by splicing and extensive editing in gymnosperm mitochondria. This study provides additional insights into the evolutionarily high dynamics of mitochondrial introns which may come and go in closely related plant species. The turnover of the mitochondrial rps3 group II introns seen among lineages of seed plants further suggests that these introns might be an additional signature to discriminate between particularly cryptical taxonomic groups for which there is a need of a further evaluation of their evolutionary affiliation.

AINTEGUMENTA homolog expression in Gnetum (gymnosperms) and implications for the evolution of ovulate axes in seed plants.

The expression of GpANTL1, a homolog of AINTEGUMENTA (ANT) found in the gymnosperm Gnetum parvifolium, was analyzed by RT-PCR and in situ hybridization. GpANTL1 was expressed in the leaf primordia, root tips, and young ovules. In the ovulate axis, expression was detected as four distinct rings around the outer, middle, and inner envelope primordia as well as around the nucellar tip. This pattern of expression is similar to that of ANT in Arabidopsis thaliana. A comparison of the expression of GpANTL1 with that of PtANTL1 in the conifer Pinus thunbergii suggests that the integrated expression of PtANTL1 may have been caused by congenital fusion of the integument, ovuliferous scale, and bract.

Chloroplast genome (cpDNA) of Cycas taitungensis and 56 cp protein-coding genes of Gnetum parvifolium: insights into cpDNA evolution and phylogeny of extant seed plants.

Phylogenetic relationships among the 5 groups of extant seed plants are presently unsettled. To reexamine this long-standing debate, we determine the complete chloroplast genome (cpDNA) of Cycas taitungensis and 56 protein-coding genes encoded in the cpDNA of Gnetum parvifolium. The cpDNA of Cycas is a circular molecule of 163,403 bp with 2 typical large inverted repeats (IRs) of 25,074 bp each. We inferred phylogenetic relationships among major seed plant lineages using concatenated 56 protein-coding genes in 37 land plants. Phylogenies, generated by the use of 3 independent methods, provide concordant and robust support for the monophylies of extant seed plants, gymnosperms, and angiosperms. Within the modern gymnosperms are 2 highly supported sister clades: Cycas-Ginkgo and Gnetum-Pinus. This result agrees with both the "gnetifer" and "gnepines" hypotheses. The sister relationships in Cycas-Ginkgo and Gnetum-Pinus clades are further reinforced by cpDNA structural evidence. Branch lengths of Cycas-Ginkgo and Gnetum were consistently the shortest and the longest, respectively, in all separate analyses. However, the Gnetum relative rate test revealed this tendency only for the 3rd codon positions and the transversional sites of the first 2 codon positions. A PsitufA located between psbE and petL genes is here first detected in Anthoceros (a hornwort), cycads, and Ginkgo. We demonstrate that the PsitufA is a footprint descended from the chloroplast tufA of green algae. The duplication of ycf2 genes and their shift into IRs should have taken place at least in the common ancestor of seed plants more than 300 MYA, and the tRNAPro-GGG gene was lost from the angiosperm lineage at least 150 MYA. Additionally, from cpDNA structural comparison, we propose an alternative model for the loss of large IR regions in black pine. More cpDNA data from non-Pinaceae conifers are necessary to justify whether the gnetifer or gnepines hypothesis is valid and to generate solid structural evidence for the monophyly of extant gymnosperms.

Dating dispersal and radiation in the gymnosperm Gnetum (Gnetales)--clock calibration when outgroup relationships are uncertain.

Most implementations of molecular clocks require resolved topologies. However, one of the Bayesian relaxed clock approaches accepts input topologies that include polytomies. We explored the effects of resolved and polytomous input topologies in a rate-heterogeneous sequence data set for Gnetum, a member of the seed plant lineage Gnetales. Gnetum has 10 species in South America, 1 in tropical West Africa, and 20 to 25 in tropical Asia, and explanations for the ages of these disjunctions involve long-distance dispersal and/or the breakup of Gondwana. To resolve relationships within Gnetum, we sequenced most of its species for six loci from the chloroplast (rbcL, matK, and the trnT-trnF region), the nucleus (rITS/5.8S and the LEAFY gene second intron), and the mitochondrion (nad1 gene second intron). Because Gnetum has no fossil record, we relied on fossils from other Gnetales and from the seed plant lineages conifers, Ginkgo, cycads, and angiosperms to constrain a molecular clock and obtain absolute times for within-Gnetum divergence events. Relationships among Gnetales and the other seed plant lineages are still unresolved, and we therefore used differently resolved topologies, including one that contained a basal polytomy among gymnosperms. For a small set of Gnetales exemplars (n = 13) in which rbcL and matK satisfied the clock assumption, we also obtained time estimates from a strict clock, calibrated with one outgroup fossil. The changing hierarchical relationships among seed plants (and accordingly changing placements of distant fossils) resulted in small changes of within-Gnetum estimates because topologically closest constraints overrode more distant constraints. Regardless of the seed plant topology assumed, relaxed clock estimates suggest that the extant clades of Gnetum began diverging from each other during the Upper Oligocene. Strict clock estimates imply a mid-Miocene divergence. These estimates, together with the phylogeny for Gnetum from the six combined data sets, imply that the single African species of Gnetum is not a remnant of a once Gondwanan distribution. Miocene and Pliocene range expansions are inferred for the Asian subclades of Gnetum, which stem from an ancestor that arrived from Africa. These findings fit with seed dispersal by water in several species of Gnetum, morphological similarities among apparently young species, and incomplete concerted evolution in the nuclear ITS region.

The internal transcribed spacer of nuclear ribosomal DNA in the gymnosperm Gnetum.

We analyze the structure of the internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal DNA in the gymnosperm Gnetum, using a phylogenetic framework derived mainly from an intron in the nuclear low-copy LEAFY gene. Gnetum comprises 25-35 species in South America, Africa, and Asia, of which we sampled 16, each with two to six clones. Criteria used to assess ITS functionality were highly divergent nucleotide substitution, GC content, secondary structure, and incongruent phylogenetic placement of presumed paralogs. The length of ITS1 ranged from 225 to 986 bp and that of ITS2 from 259 to 305 bp, the largest ranges so far reported from seed plants. Gnetum ITS1 contains two informative sequence motifs, but different from other gymnosperms, there are only few and short (7-13 bp) tandem repeats. Gnetum ITS2 contains two structural motifs, modified in different clades by shortening of stems and loops. Conspecific sequences grouped together except for two recombinant pseudogenes that had ITS1 of one clade and ITS2 of another. Most of the pseudogenic ITS copies, paralogs, and putative chimeras occurred in a clade that according to a fossil-calibrated chloroplast-DNA clock has an age of a few million years. Based on morphology and chromosome numbers, the most plausible causes of the observed high levels of ITS polymorphism are hybridization, allopolyploidy, and introgression.

Distinct MADS-box gene expression patterns in the reproductive cones of the gymnosperm Gnetum gnemon.

Expression patterns from in situ hybridization of four MADS-box genes (GGM7, GGM9, GGM11, and GGM15) from the gymnosperm species Gnetum gnemon are presented. Together with previously published data about putative orthologs of floral homeotic genes from G. gnemon (GGM2, GGM3, GGM13), we describe seven temporally and spatially distinct expression patterns in male, female or both types of reproductive units which very likely reflect the diversity of MADS-box gene function in gymnosperm cones. There is evidence that some aspects of the observed differential expression have been conserved since the last common ancestor of extant angiosperms and gymnosperms about 300 million years ago.

Horizontal gene transfer from flowering plants to Gnetum.

Although horizontal gene transfer is well documented in microbial genomes, no case has been reported in higher plants. We discovered horizontal transfer of the mitochondrial nad1 intron 2 and adjacent exons b and c from an asterid to Gnetum (Gnetales, gymnosperms). Gnetum has two copies of intron 2, a group II intron, that differ in their exons, nucleotide composition, domain lengths, and structural characteristics. One of the copies, limited to an Asian clade of Gnetum, is almost identical to the homologous locus in angiosperms, and partial sequences of its exons b and c show characteristic substitutions unique to angiosperms. Analyses of 70 seed plant nad1 exons b and c and intron 2 sequences, including representatives of all angiosperm clades, support that this copy originated from a euasterid and was horizontally transferred to Gnetum. Molecular clock dating, using calibrations provided by gnetalean macrofossils, suggests an age of 5 to 2 million years for the Asian clade that received the horizontal transfer.

Ancestry and diversity of BEL1-like homeobox genes revealed by gymnosperm ( Gnetum gnemon) homologs.

BEL1-like homeobox genes encode plant-specific transcription factors, at least some of which are important for ovule development. Here we report MELBEL1-MELBEL4,the first BEL1-like genes from a non-flowering plant, the gymnosperm Gnetum gnemon. Our analyses suggest that there was already at least one BEL1-like gene present in the most recent common ancestor of extant seed plants about 300 million years ago. Multiple sequence alignments revealed that since this time, not only the DNA-binding homeodomain, but also a protein-protein interaction domain upstream of the homeodomain, termed the BEL domain, has been highly conserved. Sequence comparison of the BEL domain with upstream domains that have been conserved in other TALE homeodomain proteins, i.e. MEIS, KNOX, and PBC, revealed only weak sequence similarity. However, since homology has been shown for MEIS, KNOX, and PBC domains and since KNOX and BEL domains directly interact in vivo, it appears likely that the BEL domain was also derived from an ancestral upstream (MEINOX) domain.