Arabidopsis histone H3 lysine 9 methyltransferases KYP/SUVH5/6 are involved in leaf development by interacting with AS1-AS2 to repress KNAT1 and KNAT2.

The Arabidopsis H3K9 methyltransferases KRYPTONITE/SUPPRESSOR OF VARIEGATION 3-9 HOMOLOG 4 (KYP/SUVH4), SUVH5 and SUVH6 are redundantly involved in silencing of transposable elements (TEs). Our recent study indicated that KYP/SUVH5/6 can directly interact with the histone deacetylase HDA6 to synergistically regulate TE expression. However, the function of KYP/SUVH5/6 in plant development is still unclear. The transcriptional factors ASYMMETRIC LEAVES1 (AS1) and AS2 form a transcription complex, which is involved in leaf development by repressing the homeobox genes KNOTTED-LIKE FROM ARABIDOPSIS THALIANA 1 (KNAT1) and KNAT2. In this study, we found that KYP and SUVH5/6 directly interact with AS1-AS2 to repress KNAT1 and KNAT2 by altering histone H3 acetylation and H3K9 dimethylation levels. In addition, KYP can directly target the promoters of KNAT1 and KNAT2, and the binding of KYP depends on AS1. Furthermore, the genome-wide occupancy profile of KYP indicated that KYP is enriched in the promoter regions of coding genes, and the binding of KYP is positively correlated with that of AS1 and HDA6. Together, these results indicate that Arabidopsis H3K9 methyltransferases KYP/SUVH5/6 are involved in leaf development by interacting with AS1-AS2 to alter histone H3 acetylation and H3K9 dimethylation from KNAT1 and KNAT2 loci.

Allopatric Lineage Divergence of the East Asian Endemic Herb Conandron ramondioides Inferred from Low-Copy Nuclear and Plastid Markers.

The evolutionary histories of ornamental plants have been receiving only limited attention. We examined the origin and divergence processes of an East Asian endemic ornamental plant, Conandron ramondioides. C. ramondioides is an understory herb occurring in primary forests, which has been grouped into two varieties. We reconstructed the evolutionary and population demography history of C. ramondioides to infer its divergence process. Nuclear and chloroplast DNA sequences were obtained from 21 Conandron populations on both sides of the East China Sea (ECS) to explore its genetic diversity, structure, and population differentiation. Interestingly, the reconstructed phylogeny indicated that the populations should be classified into three clades corresponding to geographical regions: the Japan (Honshu+Shikoku) clade, the Taiwan-Iriomote clade, and the Southeast China clade. Lineage divergence between the Japan clade and the Taiwan-Iriomote and Southeast China clades occured 1.14 MYA (95% HPD: 0.82-3.86), followed by divergence between the Taiwan-Iriomote and Southeast China clades approximately 0.75 MYA (95% HPD: 0.45-1.3). Furthermore, corolla traits (floral lobe length to tube length ratios) correlated with geographical distributions. Moreover, restricted gene flow was detected among clades. Lastly, the lack of potential dispersal routes across an exposed ECS seafloor during the last glacial maximum suggests that migration among the Conandron clades was unlikely. In summary, the extant Conandron exhibits a disjunct distribution pattern as a result of vicariance rather than long-distance dispersal. We propose that allopatric divergence has occurred in C. ramondioides since the Pleistocene. Our findings highlight the critical influence of species' biological characteristics on shaping lineage diversification of East Asian relic herb species during climate oscillations since the Quaternary.

Insight into the Phylogeny and Binding Ability of WRKY Transcription Factors.

WRKY transcription factors (TFs), which make up one of the largest families of TFs in the plant kingdom, are key players in modulating gene expression relating to embryogenesis, senescence, pathogen resistance, and abiotic stress responses. However, the phylogeny and grouping of WRKY TFs and how their binding ability is affected by the flanking regions of W-box sequences remain unclear. In this study, we reconstructed the phylogeny of WRKY across the plant kingdom and characterized the DNA-binding profile of Arabidopsis thaliana WRKY (WRKY54) based on its W-box recognition sequence. We found that WRKY TFs could be separated into five clades, and that the functional zinc-finger motif at the C-terminal of WRKY appeared after several nucleotide substitutions had occurred at the 3'-end of the zinc-finger region in chlorophytes. In addition, we found that W-box flanking regions affect the binding ability of WRKY54 based on the results of a fluorescence-based electrophoretic mobility shift assay (fEMSA) and quartz crystal microbalance (QCM) analysis. The great abundance of WRKY TFs in plants implicates their involvement in diverse molecular regulatory networks, and the flanking regions of W-box sequences may contribute to their molecular recognition mechanism. This phylogeny and our findings on the molecular recognition mechanism of WRKY TFs should be helpful for further research in this area.

Stress associated proteins coordinate the activation of comprehensive antiviral immunity in Phalaenopsis orchids.

Viruses cause severe damage on crops, and identification of key gene(s) that can comprehensively activate antiviral immunity will provide insights for designing effective antiviral strategies. Salicylic acid (SA)-mediated antiviral immunity and RNA interference (RNAi) are two independently discovered antiviral pathways. Previously, we identified the orchid stress-associated protein (SAP), Pha13, which serves as a hub in SA-mediated antiviral immunity. As SAPs exist as a protein family, whether duplicated SAPs have redundant or distinctive functions in antiviral immunity remains elusive. We performed functional assays on orchid Pha21, a homolog of Pha13, using transient and transgenic approaches on orchid, Arabidopsis and Nicotiana benthamiana to overexpress and/or silence Pha21. The SA treatment induced the expression of both Pha13 and Pha21, whereas Pha21 was found to play a key role in the initiation of the RNAi pathway in Phalaenopsis orchids. We demonstrated that Pha21-mediated antiviral immunity and enhancement of the RNAi pathway is conserved between dicotyledons and monocotyledons. We provide new insight that orchid SAPs confer distinctive functions to coordinate both SA-signaling and RNAi for comprehensive activation of antiviral immunity, and this information will help us develop antiviral strategies on crops.

Phylogenetic and Structural Analysis of NIN-Like Proteins With a Type I/II PB1 Domain That Regulates Oligomerization for Nitrate Response.

Absorption of macronutrients such as nitrogen is a critical process for land plants. There is little information available on the correlation between the root evolution of land plants and the protein regulation of nitrogen absorption and responses. NIN-like protein (NLP) transcription factors contain a Phox and Bem1 (PB1) domain, which may regulate nitrate-response genes and seem to be involved in the adaptation to growing on land in terms of plant root development. In this report, we reveal the NLP phylogeny in land plants and the origin of NLP genes that may be involved in the nitrate-signaling pathway. Our NLP phylogeny showed that duplication of NLP genes occurred before divergence of chlorophyte and land plants. Duplicated NLP genes may lost in most chlorophyte lineages. The NLP genes of bryophytes were initially monophyletic, but this was followed by divergence of lycophyte NLP genes and then angiosperm NLP genes. Among those identified NLP genes, PB1, a protein-protein interaction domain was identified across our phylogeny. To understand how protein-protein interaction mediate via PB1 domain, we examined the PB1 domain of Arabidopsis thaliana NLP7 (AtNLP7) in terms of its molecular oligomerization and function as representative. Based on the structure of the PB1 domain, determined using small-angle x-ray scattering (SAXS) and site-directed mutagenesis, we found that the NLP7 PB1 protein forms oligomers and that several key residues (K867 and D909/D911/E913/D922 in the OPCA motif) play a pivotal role in the oligomerization of NLP7 proteins. The fact that these residues are all conserved across land plant lineages means that this oligomerization may have evolved after the common ancestor of extant land plants colonized the land. It would then have rapidly become established across land-plant lineages in order to mediate protein-protein interactions in the nitrate-signaling pathway.

Gene duplication and relaxation from selective constraints of GCYC genes correlated with various floral symmetry patterns in Asiatic Gesneriaceae tribe Trichosporeae.

Floral bilateral symmetry is one of the most important acquisitions in flower shape evolution in angiosperms. Members of Gesneriaceae possess predominantly zygomorphic flowers yet natural reversal to actinomorphy have independently evolved multiple times. The development of floral bilateral symmetry relies greatly on the gene CYCLOIDEA (CYC). Our reconstructed GCYC phylogeny indicated at least five GCYC duplication events occurred over the evolutionary history of Gesneriaceae. However, the patterns of GCYC expression following the duplications and the role of natural selection on GCYC copies in relation to floral symmetry remained largely unstudied. The Asiatic tribe Trichosporeae contains most reversals to actinomorphy. We thus investigated shifts in GCYC gene expression among selected zygomorphic species (Hemiboea bicornuta and Lysionotus pauciflorus) and species with reversals to actinomorphy (Conandron ramondioides) by RT-PCR. In the actinomorphic C. ramondioides, none of the three copies of GCYC was found expressed in petals implying that the reversal was a loss-of-function event. On the other hand, both zygomorphic species retained one GCYC1 copy that was expressed in the dorsal petals but each species utilized a different copy (GCYC1C for H. bicornuta and GCYC1D for L. pauciflorus). Together with previously published data, it appeared that GCYC1C and GCYC1D copies diversified their expression in a distinct species-specific pattern. To detect whether the selection signal (ω) changed before and after the duplication of GCYC1 in Asiatic Trichosporeae, we reconstructed a GCYC phylogeny using maximum likelihood and Bayesian inference algorithms and examined selection signals using PAML. The PAML analysis detected relaxation from selection right after the GCYC1 duplication (ωpre-duplication = 0.2819, ωpost-duplication = 0.3985) among Asiatic Trichosporeae species. We propose that the selection relaxation after the GCYC1 duplication created an "evolutionary window of flexibility" in which multiple copies were retained with randomly diverged roles for dorsal-specific expressions in association with floral symmetry changes.

Expression shifts of floral symmetry genes correlate to flower actinomorphy in East Asia endemic Conandron ramondioides (Gesneriaceae).

BACKGROUND: Bilateral symmetry flower (zygomorphy) is the ancestral state for Gesneriaceae species. Yet independent reversions to actinomorphy have been parallelly evolved in several lineages. Conandron ramondioides is a natural radially symmetrical species survived in dense shade mountainous habitats where specialist pollinators are scarce. Whether the mutations in floral symmetry genes such as CYC, RAD and DIV genes, or their expression pattern shifts contribute to the reversion to actinomorphy in C. ramondioides thus facilitating shifts to generalist pollinators remain to be investigated. To address this, we isolated putative orthologues of these genes and relate their expressions to developmental stages of flower actinomorphy. RESULTS: Tissue specific RT-PCR found no dorsal identity genes CrCYCs and CrRADs expression in petal and stamen whorls, while the ventral identity gene CrDIV was expressed in all petals. Thus, ventralized actinomorphy is evolved in C. ramondioides. However, CrCYCs still persists their expression in sepal whorl. This is congruent with previous findings that CYC expression in sepals is an ancestral state common to both actinomorphic and zygomorphic core Eudicot species. CONCLUSIONS: The loss of dorsal identity genes CrCYCs and CrRADs expression in petal and stamen whorl without mutating these genes specifies that a novel regulation change, possibly on cis-elements of these genes, has evolved to switch zygomorphy to actinomorphy.

Genetic Analysis of Floral Symmetry Transition in African Violet Suggests the Involvement of Trans-acting Factor for CYCLOIDEA Expression Shifts.

With the growing demand for its ornamental uses, the African violet (Saintpaulia ionantha) has been popular owing to its variations in color, shape and its rapid responses to artificial selection. Wild type African violet (WT) is characterized by flowers with bilateral symmetry yet reversals showing radially symmetrical flowers such as dorsalized actinomorphic (DA) and ventralized actinomorphic (VA) peloria are common. Genetic crosses among WT, DA, and VA revealed that these floral symmetry transitions are likely to be controlled by three alleles at a single locus in which the levels of dominance are in a hierarchical fashion. To investigate whether the floral symmetry gene was responsible for these reversals, orthologs of CYCLOIDEA (CYC) were isolated and their expressions correlated to floral symmetry transitions. Quantitative RT-PCR and in situ results indicated that dorsal-specific CYCs expression in WT S. ionantha (SiCYC and SiCYC1B) shifted in DA with a heterotopically extended expression to all petals, but in VA, SiCYC1s' dorsally specific expressions were greatly reduced. Selection signature analysis revealed that the major high-expressed copy of SiCYC had been constrained under purifying selection, whereas the low-expressed helper SiCYC1B appeared to be relaxed under purifying selection after the duplication into SiCYC and SiCYC1B. Heterologous expression of SiCYC in Arabdiopsis showed petal growth retardation which was attributed to limited cell proliferation. While expression shifts of SiCYC and SiCYC1B correlate perfectly to the resulting symmetry phenotype transitions in F1s of WT and DA, there is no certain allelic combination of inherited SiCYC1s associated with specific symmetry phenotypes. This floral transition indicates that although the expression shifts of SiCYC/1B are responsible for the two contrasting actinomorphic reversals in African violet, they are likely to be controlled by upstream trans-acting factors or epigenetic regulations.