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Premise Cleomaceae is emerging as a promising family to investigate a wide range of phenomena, such as C4 photosynthesis and floral diversity. However, functional techniques are lacking for elucidating this diversity. Herein, we establish virus-induced gene silencing (VIGS) as a method of generating functional data for Cleome violacea, bolstering Cleomaceae as a model system. Methods We leveraged the sister relationship of Cleomaceae and Brassicaceae by using constructs readily available for Arabidopsis thaliana to provide initial information about the feasibility of VIGS in C. violacea. We then developed endogenous constructs to optimize VIGS efficiency and viability for fruit development. Results PHYTOENE DESATURASE was successfully downregulated in C. violacea using both heterologous and endogenous constructs. The endogenous construct had the highest degree of downregulation, with many plants displaying strong photobleaching. FRUITFULL-treated plants were also successfully downregulated, with a high rate of survival but less effective silencing; only a small percentage of survivors showed a strong phenotype. Discussion Our optimized VIGS protocol in C. violacea enables functional gene analyses at different developmental stages. Additionally, C. violacea is amenable to heterologous knockdown, which suggests that a first pass using non-endogenous constructs is a possible route to test additional species of Cleomaceae.

Summary Unlike most ancient microRNAs, which conservatively target homologous genes across species, microRNA827 (miR827) targets two different types of SPX (SYG1/PHO81/XPR1)-domain-containing genes, NITROGEN LIMITATION ADAPTATION (NLA) and PHOSPHATE TRANSPORTER 5 (PHT5), in Arabidopsis thaliana and Oryza sativa to regulate phosphate (Pi) transport and storage, respectively. However, how miR827 shifted its target preference and its evolutionary history are unknown. Based on target prediction analysis, we found that in most angiosperms, miR827 conservatively targets PHT5 homologs, but in Brassicaceae and Cleomaceae it preferentially targets NLA homologs, and we provide evidence for the transition of target preference during Brassicales evolution. Intriguingly, we found a lineage-specific loss of the miR827-regulatory module in legumes. Analysis of miR827-mediated cleavage efficiency and the expression of PHT5 in A. thaliana indicated that accumulation of mutations in the target site and the exclusion of the target site by alternative transcriptional initiation eliminated PHT5 targeting by miR827. Here, we identified a transition of miR827 target preference during plant evolution and revealed the uniqueness of miR827-mediated regulation among conserved plant miRNAs. Despite the change in its target preference, upregulation of miR827 by Pi starvation and its role in regulating cellular Pi homeostasis were retained.

PREMISE OF THE STUDY: Glucosinolates (GS) are a class of plant secondary metabolites that provide defense against herbivores and may play an important role in pollinator attraction. Through coevolution with plant-interacting organisms, glucosinolates have diversifi ed into a variety of chemotypes through gene sub- and neofunctionalization. Polyploidy has been of major importance in the evolutionary history of these gene families and the development of chemically separate GS types. Here we study the eff ects of polyploidy in Tarenaya hassleriana (Cleomaceae) on the genes underlying GS biosynthesis. METHODS: We established putative orthologs of all gene families involved in GS biosynthesis through sequence comparison and their duplication method through calculation of synonymous substitution ratios, phylogenetic gene trees, and synteny comparison. We drew expression data from previously published work of the identifi ed genes and compared expression in several tissues. KEY RESULTS: We show that the majority of gene family expansion in T. hassleriana has taken place through the retention of polyploid duplicates, together with tandem and transpositional duplicates. We also show that the large majority (>75%) is actively expressed either globally or in specifi c tissues. We show that MAM and CYP83 gene families, which are crucial to GS diversifi cation in Brassicaceae, are also recruited into specifi c tissue expression pathways in Cleomaceae. CONCLUSIONS: Many GS genes have expanded through polyploidy, gene transposition duplication, and tandem duplication in Cleomaceae. Duplicate retention through these mechanisms is similar to A. thaliana, but based on the expression of GS genes, Cleomaceae-specifi c diversifi cation of GS genes has taken place.

Cleomaceae is a small pantropical family that is emerging as a promising system to investigate C4 photosynthesis, floral evolution, and comparative genomics. However, our understanding of these phenomena is hindered by a lack of a strong phylogenetic hypothesis, despite a number of previous studies. We reconstructed the phylogeny of the family using data from all three genomes, including three cpDNA (ndhF, matK, ycf1), one mtDNA (rps3), and one nrDNA (ITS) regions. Analyses strongly supported 15 clades: (1) Clade 1, which includes two Old World species, Cleome khorassanica and C. turkmena; (2) Cleome s.str., which includes the type C. ornithopodioides and Old World species; (3) Droserifolia, corresponding to three Old World species, C. droserifolia, C. fimbriata, C. quinquenervia; (4) Polanisia, equivalent to this New World genus; (5) Angustifolia, which includes four Old World species; (6) North American cleomoids, which includes four genera, Cleomella, Peritoma, Oxystylis, and Wislizenia; (7) Australian, which includes Old world species and worldwide weed Arivela viscosa; (8) Gynandropsis, equivalent to this monotypic genus; (9) Clade 6, which includes Old World species of Cleome and Dipterygium; (10) Dactylaena, corresponding to this genus and Physostemon; (11) African, which includes species distributed in Old World; (12) Andean, which includes Podandrogyne and tropical New World species of Cleome; (13) Melidiscus, which includes New World tropical species; (14) Cleoserrata, which includes New World tropical species; and (15) Tarenaya, a large New World clade. Major relationships amongst the clades are strongly supported for the first time, including North American cleomoids sister to all remaining Cleom aceae. While five genera are confirmed or newly identified here to be non-monophyletic (Cleome, Cleomella, Hemiscola, Peritoma, Tarenaya), six are supported (Cleoserrata, Dactylaena, Melidiscus, Physostemon, Podandrogyne, Polanisia). Thus, there are many taxonomic and evolutionary implications to our revised phylogenetic hypothesis.

The Brassicaceae family is of great scientific interest because it contains the plant model organism Arabidopsis thaliana. Currently, contemporary plant research activities expand to other Brassicaceae taxa. Despite that, the phylogeny of this family is only partly understood. The present study deepens our understanding of a family-wide phylogeny by using two new approaches in phylogenetic family-wide research. We used a molecular marker from the mitochondrial genome and utilised a relaxed molecular dating method. Our data generally confirms a recent tribal alignment of Brassicaceae. We present for the first time a biogeographical scenario for the broad-scale Brassicaceae evolution. We suggest that Brassicaceae most likely evolved some 19 mya in or near the eastern Mediterranean region from a common ancestor of its sister family Cleomaceae. The early Brassicaceae formed a lineage adapted to more open/drier habitats than its capparoid progenitors. The early Brassicaceae evolution was very rapid and the radiation was most likely driven by climatic changes that created open habitats and the well-documented expansion of open grass-dominated ecosystems. Moreover, our dating suggests that the radiation events correlate with an ancient genome duplication in the early history of the family, which is evidenced by recent genomic studies.

The summer-blooming annual forbs Cleome lutea and Cleome serrulata (Cleomaceae) are native across the US Intermountain West and Rocky Mountains, respectively. Their farmed seed is sought to help rehabilitate western rangelands in those regions. This study of the reproductive biologies and pollinator faunas of C. lutea and C. serrulata is the first for this cosmopolitan family, the sister family to the Brassicaceae. Unlike the S-allele self-incompatibility systems of some Brassicaceae, both species of Cleome were found to be self-fertile and capable of some autogamy. Compared with selfing, outcrossing did not enhance seed set, seed viability or seedling vigor for either species (in fact, selfed progeny were more robust). Large, openly visited plants yielded >20 000 seeds each. Like several species of the sister family Capparaceae, flowers of both species first shed their pollen, secreted nectar and became receptive nocturnally. Although no nocturnal visitors were found, both Cleome species attracted a diverse array of diurnal native bees, wasps and butterflies. Among the many floral generalist bees that work Cleome flowers for pollen and nectar are two managed agricultural pollinators, Apis mellifera and Megachile rotundata. These observations bode well for pollinating C. lutea and C. serrulata in small commercial seed fields. It appears that diverse wild bees would benefit from the addition of native Cleome to restoration seed mixes, with the objective of sustaining native pollinator faunas during the first few years of postfire plant community rehabilitation.

Capparaceae and Brassicaceae have long been known to be closely related families, with the monophyly of Capparaceae more recently questioned. To elucidate the relationship between Brassicaceae and Capparaceae as well as to address infrafamilial relationships within Capparaceae, we analyzed sequence variation for a large sampling, especially of Capparaceae, of these two families using two chloroplast regions, trnL-trnF and ndhF. Results of parsimony and likelihood analyses strongly support the monophyly of Brassicaceae plus Capparaceae, excluding Forchhammeria, which is clearly placed outside the Brassicaceae and Capparaceae clade and suggest the recognition of three primary clades-Capparaceae subfamily (subf.) Capparoideae, subf. Cleomoideae, and Brassicaceae. Capparaceae monophyly is strongly contradicted with Cleomoideae appearing as sister to Brassicaceae. Two traditionally recognized subfamilies of Capparaceae, Dipterygioideae and Podandrogynoideae, are embedded within Cleomoideae. Whereas habit and some fruit characteristics demarcate the three major clades, floral symmetry, stamen number, leaf type, and fruit type all show homoplasy. Clades within Capparoideae show a biogeographical pattern based on this sampling. These results are consistent with several alternative classification schemes.

The present work embodies epidermal structure, structure and ontogeny of stomata in five genera embracing sixteen species of the Capparidaceae namely Cleome (8 species) Capparis (5 species), Cadaba (1 species), Crataeva (1 species) and Maerua (1 species). The epidermal cells are polygonal, isodiametric or elongated arranged irregularly, with evenly or unevenly thickened, sinuous, straight or arched anticlinal walls. Two main types of trichomes: glandular (four types) and eglandular (five types) are noticed. The stomatal types include cyclocytic, triacytic, staurocytic, tetracytic, anomocytic, anisocytic, paracytic and with a single subsidiary cell. The ontogeny of stomata with a single subsidiary cell is perigenous or mesoperigenous, of paracytic mesoperigenous or mesogenous, of anisocytic is mesoperigenous or mesogenous, while that of the other types is perigenous. Abnormalities observed are: single guard cell; aborted guard cells; complete or incomplete division of guard cells; contiguous stomata; giant stomata and cytoplasmic connections. The present observations do not support the separation of Cleomaceae from the Capparidaceae. Die vorliegende Arbeit umfast die Epidermis-Struktur sowie Struktur und Ontogenie der Stomata von 5 Gattungen mit 16 Arten der Capparidaceae, namlich Cleoma (8 Arten), Capparis (5 Arten), Cadaba (1 Art), Crataeva (1 Art) und Maerua (1 Art). Die Epidermiszellen sind polygonal, isodiametrisch oder verlangert unregelmasig angeordnet mit gleichmasig oder ungleichmasig verdickten, gekrummten, geraden oder antiklinal gewolbten Wanden. Zwei Hauptformen von Trichomen wurden beobachtet, und zwar vier Formen drusiger und funf Formen nicht drusiger Trichome. Die Stomata gehoren zu folgenden Typen: cyclocytisch, triacytisch, staurocytisch, tetracytisch, anomocytisch, anisocytisch, paracytisch sowie solche mit einer einzigen Nebenzelle. Die Ontogenie der Schlieszellen mit einer einzelnen Nebenzelle verlauft perigen oder mesoperigen, die der paracytischen und anisocytischen Schlieszellen mesoperigen oder mesogen, wahrend die Ontogenie der ubrigen Typen perigen verlauft. Als Abnormitaten wurden beobachtet: nur eine einzelne Schlieszelle; verkummerte Schlieszellen; vollstandige oder unvollstandige Teilung der Schlieszellen; nebeneinander liegende Stomata; Riesen-Stomata sowie Zytoplasma-Verbindungen. Die vorliegenden Ergebnisse befurworten nicht eine Abtrennung der Cleomaceae von den Capparidaceae.