Early-responsive molecular signatures associated with halophytic adaptation in Sesuvium portulacastrum (L.).

Sesuvium portulacastrum (L.) is a halophyte, adapted to grow naturally under saline environments. The ability to use Na and K interchangeably indicated its facultative halophyte nature. No significant growth reduction occurs in seedlings up to 250 mM NaCl, except for curling of the youngest leaf. Within 8 h of salt treatment, seedlings accumulate proline, glycine betaine and other amino acids in both root and shoot. Despite a continued increase of tissue Na content, the number of differentially expressed genes (DEGs) decreases between 8 and 24 h of salt exposure, indicating transcriptional restoration after the initial osmotic challenge. At 8 h, upregulated genes mainly encode transporters and transcription factors, while genes in growth-related pathways such as photosynthesis and ribosome-associated biogenesis are suppressed. Overexpression of SpRAB18 (an ABA-responsive dehydrin), one of the most strongly induced DEGs, in soybean was found to increase biomass in control conditions and the growth benefit was maintained when plants were grown in 100 mM NaCl, indicating conservation of function in halophyte and glycophyte. An open-access transcriptome database "SesuviumKB" (https://cb.imsc.res.in/sesuviumkb/) was developed to involve the scientific community in wide-scale functional studies of S. portulacastrum genes, that could pave the way to engineer salt tolerance in crops.

Co-expression of SpSOS1 and SpAHA1 in transgenic Arabidopsis plants improves salinity tolerance.

BACKGROUND: Na+ extrusion from cells is important for plant growth in high saline environments. SOS1 (salt overly sensitive 1), an Na+/H+ antiporter located in the plasma membrane (PM), functions in toxic Na+ extrusion from cells using energy from an electrochemical proton gradient produced by a PM-localized H+-ATPase (AHA). Therefore, SOS1 and AHA are involved in plant adaption to salt stress. RESULTS: In this study, the genes encoding SOS1 and AHA from the halophyte Sesuvium portulacastrum (SpSOS1 and SpAHA1, respectively) were introduced together or singly into Arabidopsis plants. The results indicated that either SpSOS1 or SpAHA1 conferred salt tolerance to transgenic plants and, as expected, Arabidopsis plants expressing both SpSOS1 and SpAHA1 grew better under salt stress than plants expressing only SpSOS1 or SpAHA1. In response to NaCl treatment, Na+ and H+ in the roots of plants transformed with SpSOS1 or SpAHA1 effluxed faster than wild-type (WT) plant roots. Furthermore, roots co-expressing SpSOS1 and SpAHA1 had higher Na+ and H+ efflux rates than single SpSOS1/SpAHA1-expressing transgenic plants, resulting in the former amassing less Na+ than the latter. As seen from comparative analyses of plants exposed to salinity stress, the malondialdehyde (MDA) content was lowest in the co-transgenic SpSOS1 and SpAHA1 plants, but the K+ level was the highest. CONCLUSION: These results suggest SpSOS1 and SpAHA1 coordinate to alleviate salt toxicity by increasing the efficiency of Na+ extrusion to maintain K+ homeostasis and protect the PM from oxidative damage induced by salt stress.

Decoding ice plants: challenges associated with barcoding and phylogenetics in the diverse succulent family Aizoaceae.

Aizoaceae is the largest succulent plant family in the world, including in excess of 1800 species. Despite its richness, a large proportion of its taxa are listed as data deficient and as such, has been identified as the top priority for taxonomic research in South Africa. Limitations to accurate taxonomic identification of taxa in the family may be partly attributed to the degree of technical knowledge required to identify taxa in the Aizoaceae. DNA barcoding may provide an alternative method of identification; however, the suitability of commonly used gene regions has not been tested in the family. Here, we analyse variable and parsimony informative characters (PIC), as well as the barcoding gap, in commonly used plastid regions (atpB-rbcL, matK, psbA-trnH, psbJ-petA, rpl16, rps16, trnD-trnT, trnL-trnF, trnQ-rps16, and trnS-trnG) and the nuclear region ITS (for Aizooideae only) across two subfamilies and two expanded clades within the Aizoaceae. The relative percentage of PIC was much greater in subfamilies Aizooideae and Mesembryanthemoideae than in Ruschioideae. Although nrITS had the highest percentage of PIC, barcoding gap analyses identified neither ITS nor any chloroplast region as suitable for barcoding of the family. From the results, it is evident that novel barcoding regions need to be explored within the Aizoaceae.

Complete chloroplast genome of Tetragonia tetragonioides: Molecular phylogenetic relationships and evolution in Caryophyllales.

The chloroplast genome of Tetragonia tetragonioides (Aizoaceae; Caryophyllales) was sequenced to provide information for studies on phylogeny and evolution within Caryophyllales. The chloroplast genome of Tetragonia tetragonioides is 149,506 bp in length and includes a pair of inverted repeats (IRs) of 24,769 bp that separate a large single copy (LSC) region of 82,780 bp and a small single copy (SSC) region of 17,188 bp. Comparative analysis of the chloroplast genome showed that Caryphyllales species have lost many genes. In particular, the rpl2 intron and infA gene were not found in T. tetragonioides, and core Caryophyllales lack the rpl2 intron. Phylogenetic analyses were conducted using 55 genes in 16 complete chloroplast genomes. Caryophyllales was found to divide into two clades; core Caryophyllales and noncore Caryophyllales. The genus Tetragonia is closely related to Mesembryanthemum. Comparisons of the synonymous (Ks), nonsynonymous (Ka), and Ka/Ks substitution rates revealed that nonsynonymous substitution rates were lower than synonymous substitution rates and that Ka/Ks rates were less than 1. The findings of the present study suggest that most genes are a purified selection.

Out of southern Africa: Origin, biogeography and age of the Aizooideae (Aizoaceae).

The Aizooideae is an early-diverging lineage within the Aizoaceae. It is most diverse in southern Africa, but also has endemic species in Australasia, Eurasia and South America. We derived a phylogenetic hypothesis from Bayesian and Maximum Likelihood analyses of plastid DNA-sequences. We find that one of the seven genera, the fynbos-endemic Acrosanthes, does not belong to the Aizooideae, but is an ancient sister-lineage to the subfamilies Mesembryanthemoideae & Ruschioideae. Galenia and Plinthus are embedded inside Aizoon and Aizoanthemum is polyphyletic. The Namibian endemic Tetragonia schenckii is sister to Tribulocarpus of the Sesuvioideae. For the Aizooideae, we explored their possible age by means of relaxed Bayesian dating and used Bayesian Binary MCMC reconstruction of ancestral areas to investigate their area of origin. Early diversification occurred in southern Africa in the Eocene-Oligocene, with a split into a mainly African lineage and an Eurasian-Australasian-African-South American lineage. These subsequently radiated in the early Miocene. For Tetragonia, colonisation of Australasia via long-distance dispersal from Eurasia gave rise to the Australasian lineage from which there were subsequent dispersals to South America and Southern Africa. Despite the relatively old age of the Aizooideae, more than half the species have radiated since the Pleiocene, coinciding with the large and rapid diversification of the Ruschioideae. The lineage made up of Tetragonia schenckii &Tribulocarpus split from the remainder of the Sesuvioideae already in the mid Oligocene and its disjunct distribution between Namibia and north-east Africa may be the result of a previously wider distribution within an early Arid African flora. Our reconstruction of ancestral character-states indicates that the expanding keels giving rise to hygrochastic fruits originated only once, i.e. after the split of the Sesuvioideae from the remainder of the Aizoaceae and that they were subsequently lost many times. Variously winged and spiky fruits, adapted to dispersal by wind and animals, have evolved independently in the Aizooideae and the Sesuvioideae. There is then a greater diversity of dispersal systems in the earlier lineages than in the Mesembryanthemoideae and Ruschioideae, where dispersal is mainly achieved by rain.

Microbial community dynamics in the rhizosphere of a cadmium hyper-accumulator.

Phytoextraction is influenced by the indigenous soil microbial communities during the remediation of heavy metal contaminated soils. Soil microbial communities can affect plant growth, metal availability and the performance of phytoextraction-assisting inocula. Understanding the basic ecology of indigenous soil communities associated with the phytoextraction process, including the interplay between selective pressures upon the communities, is an important step towards phytoextraction optimization. This study investigated the impact of cadmium (Cd), and the presence of a Cd-accumulating plant, Carpobrotus rossii (Haw.) Schwantes, on the structure of soil-bacterial and fungal communities using automated ribosomal intergenic spacer analysis (ARISA) and quantitative PCR (qPCR). Whilst Cd had no detectable influence upon fungal communities, bacterial communities underwent significant structural changes with no reduction in 16S rRNA copy number. The presence of C. rossii influenced the structure of all communities and increased ITS copy number. Suites of operational taxonomic units (OTUs) changed in abundance in response to either Cd or C. rossii, however we found little evidence to suggest that the two selective pressures were acting synergistically. The Cd-induced turnover in bacterial OTUs suggests that Cd alters competition dynamics within the community. Further work to understand how competition is altered could provide a deeper understanding of the microbiome-plant-environment and aid phytoextraction optimization.

An aquaporin gene from halophyte Sesuvium portulacastrum, SpAQP1, increases salt tolerance in transgenic tobacco.

KEY MESSAGE: SpAQP1 was strongly induced by salt in an ABA-independent way, promoted seed germination and root growth in transgenic tobaccos and increased salt tolerance by increasing the activities of antioxidative enzymes. Aquaporin (AQP) plays crucial roles in the responses of plant to abiotic stresses such as drought, salt and cold. Compared to glycophytes, halophytes often have excellent salt and drought tolerances. To uncover the molecular mechanism of halophyte Sesuvium portulacastrum tolerance to salt, in this study, an AQP gene, SpAQP1, from S. portulacastrum was isolated and characterized. The amino acid sequence of SpAQP1 shared high homology with that of plant plasma membrane intrinsic proteins (PIPs) and contained the distinct molecular features of PIPs. In the phylogenic tree, SpAQP1 was evidently classified as the PIP2 subfamily. SpAQP1 is expressed in roots, stems and leaves, and was significantly induced by NaCl treatment and inhibited by abscisic acid (ABA) treatment. When heterologously expressed in yeast and tobacco, SpAQP1 enhanced the salt tolerance of yeast strains and tobacco plants and promoted seed germination and root growth under salt stress in transgenic plants. The activity of antioxidative enzymes including superoxide dismutase, peroxidase and catalase was increased in transgenic plants overexpressing SpAQP1. Taken together, our studies suggested that SpAQP1 functioned in the responses of S. portulacastrum to salt stress and could increase salt tolerance by enhancing the antioxidative activity of plants.

SpBADH of the halophyte Sesuvium portulacastrum strongly confers drought tolerance through ROS scavenging in transgenic Arabidopsis.

Glycine betaine (GB) accumulation is involved in abiotic stress. However, it is not known whether BADH, the key enzyme of GB synthesis, utilizes the antioxidant system to confer drought stress tolerance. In this study, a novel member of the ALDH10 gene family, SpBADH, was isolated from Sesuvium portulacastrum. The expression of this gene was up-regulated by NaCl, PEG6000, H2O2, ABA and high temperature in S. portulacastrum. SpBADH overexpression in Arabidopsis resulted in higher BADH activity and GB content and might increase tolerance to drought/osmotic stresses, specifically strong tolerance to drought stress. Transgenic lines exhibited lower MDA and H2O2 contents but higher proline, POD, SOD and CAT contents than the wild type under drought and osmotic stresses. SpBADH overexpression in Arabidopsis also enhanced the expression of ROS-related genes including AtSOD, AtPOD, AtCAT, AtAPX and Atpsb under drought and osmotic stresses. Thus, SpBADH increases plant tolerance to drought or osmotic stresses by reducing H2O2, increasing proline, and activating antioxidative enzymes to improve ROS scavenging.

SpAHA1 and SpSOS1 Coordinate in Transgenic Yeast to Improve Salt Tolerance.

In plant cells, the plasma membrane Na+/H+ antiporter SOS1 (salt overly sensitive 1) mediates Na+ extrusion using the proton gradient generated by plasma membrane H+-ATPases, and these two proteins are key plant halotolerance factors. In the present study, two genes from Sesuvium portulacastrum, encoding plasma membrane Na+/H+ antiporter (SpSOS1) and H+-ATPase (SpAHA1), were cloned. Localization of each protein was studied in tobacco cells, and their functions were analyzed in yeast cells. Both SpSOS1 and SpAHA1 are plasma membrane-bound proteins. Real-time polymerase chain reaction (PCR) analyses showed that SpSOS1 and SpAHA1 were induced by salinity, and their expression patterns in roots under salinity were similar. Compared with untransformed yeast cells, SpSOS1 increased the salt tolerance of transgenic yeast by decreasing the Na+ content. The Na+/H+ exchange activity at plasma membrane vesicles was higher in SpSOS1-transgenic yeast than in the untransformed strain. No change was observed in the salt tolerance of yeast cells expressing SpAHA1 alone; however, in yeast transformed with both SpSOS1 and SpAHA1, SpAHA1 generated an increased proton gradient that stimulated the Na+/H+ exchange activity of SpSOS1. In this scenario, more Na+ ions were transported out of cells, and the yeast cells co-expressing SpSOS1 and SpAHA1 grew better than the cells transformed with only SpSOS1 or SpAHA1. These findings demonstrate that the plasma membrane Na+/H+ antiporter SpSOS1 and H+-ATPase SpAHA1 can function in coordination. These results provide a reference for developing more salt-tolerant crops via co-transformation with the plasma membrane Na+/H+ antiporter and H+-ATPase.

A phylogenetic hypothesis for the recently diversified Ruschieae (Aizoaceae) in southern Africa.

The Ruschieae is a large tribe of about 1600 species of succulent perennials. They form a major component of the arid parts of the Greater Cape Floristic Region, both in numbers of species and in their density of coverage. So far phylogenetic relationships within the tribe have been unresolved, largely through the paucity of variable molecular characters and this is ascribed to the tribe's recent and rapid radiation. Our phylogeny is based on 10 chloroplast gene regions and represents a nearly complete sampling of the 100 currently recognised genera of the Ruschieae. These chloroplast regions yielded relatively few phylogenetically informative characters, consequently providing only limited resolution in and poor support for many parts of the phylogeny. Nevertheless, for the first time, we provide well-supported evidence that taxa with mostly mesomorphic, often ephemeral leaves and weakly persistent fruits form a basal grade of lineages in the Ruschieae. These lineages subtend a large polytomy of taxa with almost exclusively xeromorphic, persistent leaves and strongly persisting fruits. Among the basal grade of lineages, those occurring within the winter-rainfall region typically shed their leaves or form (at least partly) a protective, dry sheath around the apical bud during the dry summer months, as a means of escaping the summer drought. This contrasts with taxa of the basal grade from outside the winter-rainfall region, in which the leaves persist. Our results show that, in both strongly and weakly persistent fruits, specialised characteristics of the fruit evolved repeatedly and so these structures are highly homoplasious. Perhaps as a consequence of repeated changes towards increased persistence and specialisation of leaves and fruits, several clades show little morphological cohesion. However, as in other groups in the Cape Flora, most clades in the Ruschieae represent regional groupings. Our analysis of sequences of the nuclear gene 'chloroplast-expressed glutamine synthetase' (ncpGS) revealed extensive paralogy within the Ruschieae, but found an intact reading frame in all its members. More data on the cytology of the Ruschieae is needed to evaluate whether the paralogy observed is due to gene duplication or polyploidy.

'Living stones' reveal alternative petal identity programs within the core eudicots.

Petals, defined as the showy laminar floral organs in the second floral whorl, have been shown to be under similar genetic control in distantly related core eudicot model organisms. On the basis of these findings, it is commonly assumed that the petal identity program regulated by B-class MADS-box gene homologs is invariant across the core eudicot clade. However, the core eudicots, which comprise >70% of angiosperm species, exhibit numerous instances of petal and sepal loss, transference of petal function between floral whorls, and recurrent petal evolution. In the face of these complex patterns of perianth evolution, the concept of a core eudicot petal identity program has not been tested. We therefore examined the petal identity program in the Caryophyllales, a core eudicot clade in which perianth differentiation into sepals and petals has evolved multiple times. Specifically, we analyzed the expression patterns of B- and C-class MADS-box homologs for evidence of a conserved petal identity program between sepal-derived and stamen-derived petaloid organs in the 'living stone' family Aizoaceae. We found that neither sepal-derived nor stamen-derived petaloid organs exhibit gene expression patterns consistent with the core eudicot petal identity program. B-class gene homologs are not expressed during the development of sepal-derived petals and are not implicated in petal identity in stamen-derived petals, as their transient expression coincides with early expression of the C-class homolog. We therefore provide evidence for petal development that is independent of B-class genes and suggest that different genetic control of petal identity has evolved within this lineage of core eudicots. These findings call for a more comprehensive understanding of perianth variation and its genetic causes within the core eudicots--an endeavor that will have broader implications for the interpretation of perianth evolution across angiosperms.

Unexpected morphological and karyological changes in invasive Carpobrotus (Aizoaceae) in Provence (S-E France) compared to native South African species.

Hybridization processes can lead to evolutionary changes, particularly in co-introduced congeneric plant species, such as Carpobrotus spp. which are recognized as invasive in Mediterranean climate regions. Morphological and karyological comparisons have therefore been made between native Carpobrotus edulis and C. acinaciformis in South Africa and their invasive counterparts in Provence (C. edulis and C. aff. acinaciformis). Morphological data exhibited the most significant differences in invasive C. aff. acinaciformis that forms a new phenotypic variant. Unexpected chromosomal restructuring has been highlighted for both taxa in Provence, with in particular a clear decrease in asymmetry, an increase in the intraspecific variability, and an interspecific convergence of karyotypes. These changes suggest a drift that has facilitated various crosses, and has been amplified through hybridization/introgression. Furthermore, several morphological and karyological transgressive characters have been found in the two invasive taxa. These results stress the important role and the rapidity of karyological changes in invasive processes.

Genetic differentiation in the genus Lithops L. (Ruschioideae, Aizoaceae) reveals a high level of convergent evolution and reflects geographic distribution.

Southern Africa is one of the hot spots for plant biodiversity, with ca. 80% of species endemic to this area. Rapid and recent radiations in Southern African plant genera were triggered by fine-scale differences in climate, topography and geology. The genus Lithops (Ruschioideae, Aizoaceae) contains 37 species and is widely distributed in Southern Africa. Species delimitation within the genus is challenging because the limited number of morphological characters in these reduced succulents varies intensely between populations, presumably as adaptations to local geological environments. We analysed phylogenetic relationships within Lithops using non-coding chloroplast DNA (trnS-trnG intergenic spacer), nuclear ribosomal internal transcribed spacer (nrITS) sequences and AFLP data. Genetic variability of the sequence data was very low, but AFLP data detected nine clades within Lithops that do not fit current morphology-based taxonomy. Two of these clades are separated by their distribution on the northern and eastern border of the distribution area, and four clades are found in the Gariep Centre in the estuary of the Orange River. Morphological similarities, especially colour of leaves, evolved repeatedly within the clades, thus we hypothesise that closely related species became adapted to different soil types in a mosaic-like geological environment. One-third of the species are found in the Gariep Centre, characterised by extremely diverse edaphic habitats.

Duplication of the Asymmetric Leaves1/Rough Sheath 2/Phantastica (ARP) gene precedes the explosive radiation of the Ruschioideae.

The Mesembryanthemoideae and Ruschioideae subfamilies are a major component of the Greater Cape Floristic Region in southern Africa. The Ruschioideae show an astonishing diversity of leaf shape and growth forms. Although 1,585 species are recognised within the morphologically diverse Ruschioideae, these species show minimal variation in plastid DNA sequence. We have investigated whether changes in selected leaf development transcription factors underpin the recent, rapid diversification of this large group of succulent plants. Degenerate primers designed to conserved regions of Asymmetric Leaves1/Rough Sheath 2/Phantastica (ARP) and the Class III HD-ZIP family of genes, were used to amplify sequences corresponding to these genes from several species within the Mesembryanthemoideae and Ruschioideae subfamilies. Two members of the Class III HD-ZIP family were identified in both the Mesembryanthemoideae and Ruschioideae, and were derived from an ancient gene duplication event that preceded the divergence of gymnosperms and angiosperms. While a single ARP orthologue was identified in the Mesembryanthemoideae, two paralogues, ARPa and ARPb, were identified in the Ruschioideae subfamily. ARPa was present in all species of Ruschioideae analysed in this study. ARPb has been lost from the Apatesieae and Dorotheantheae tribes, which form an early evolutionary branch from the Ruschieae tribe, as well as from selected species within the Ruschieae. The recent duplication and subsequent selected gene loss of the ARP transcription factor correlates with the rapid diversification of plant forms in the Ruschioideae.

Cloning and molecular characterization of fructose-1,6-bisphosphate aldolase gene regulated by high-salinity and drought in Sesuvium portulacastrum.

Sesuvium portulacastrum, a mangrove plant from seashore, is a halophyte species well adapted to salinity and drought. Some efforts have been made to describe its physiological and structural characteristics on salt and drought-tolerance, but the underlying molecular mechanism and key components have not yet been identified. Here, a fructose-1,6-bisphosphate aldolase gene, designated SpFBA, was isolated and characterized from S. portulacastrum roots in response to seawater. The SpFBA cDNA has a total length of 1452 bp with an open reading frame of 1071 bp, and is predicted to encode a precursor protein of 357 amino acid residues sharing high degree of homology with class I FBAs from other plants. Semi-quantitative RT-PCR analysis indicated that the SpFBA was more strongly expressed in roots than in leaves and stems, and the abiotic stimuli such as Seawater, NaCl, ABA, and PEG, could trigger a significant induction of SpFBA in S. portulacastrum roots within 2-12 h. Overproduction of Recombinant SpFBA resulted in an increased tolerance to salinity in transgenic Escherichia coli. All these results suggest that the SpFBA plays very important roles in responding to salt stress and related abiotic stimuli, and in improving the survival ability of S. portulacastrum under high salinity and drought.

Spatial scale of local adaptation and population genetic structure in a miniature succulent, Argyroderma pearsonii.

Explicit understanding of the spatial scale of evolutionary processes is required in order to set targets for their effective conservation. Here, we explore the spatial context of neutral and adaptive divergence in the species-rich Knersvlakte region of South Africa. Specifically, we aimed to assess the importance of erosional drainage basins as spatial units of evolutionary process. We used amplified fragment length polymorphism (AFLP) and reciprocal transplants to investigate genetic differentiation in Argyroderma pearsonii, sampled from sparse and dense quartz habitats within each of three drainage basins. This design allowed assessment of differentiation at two distinct spatial scales; between habitats within basins, and between basins. We found near-perfect concordance between genetic clusters and basin occupancy, suggesting restricted interbasin gene flow. In addition, transplants reveal adaptive divergence between basins on the dense quartz habitat. We have shown that neutral and adaptive differentiation occurs between basins, but not between habitats within basins, suggesting that conservation plans aimed at conserving multiple interconnected drainage basins will capture an important axis of evolutionary process on the Knersvlakte.

Evolutionary radiation of "stone plants" in the genus Argyroderma (Aizoaceae): unraveling the effects of landscape, habitat, and flowering time.

Recent phylogenetic evidence suggests that the extraordinary diversity of the Cape Floristic Kingdom in South Africa may be the result of widespread evolutionary radiation. Our understanding of the role of adaptive versus neutral processes in these radiations remains largely speculative. In this study we investigated factors involved in the diversification of Argyroderma, a genus within the most spectacular of the Cape radiations, that of the Ruschioid subfamily of the Aizoaceae. We used amplified fragment length polymorphisms and a suite of morphological traits to elucidate patterns of differentiation within and between species of Argyroderma across the range of the genus. We then used a matrix correlation approach to assess the influence of landscape structure, edaphic gradients, and flowering phenology on phenotypic and neutral genetic divergence in the system. We found evidence for strong spatial genetic isolation at all taxonomic levels. In addition, genetic differentiation occurs along a temporal axis, between sympatric species with divergent flowering times. Morphological differentiation, which previous studies suggest is adaptive, occurs along a habitat axis, between populations occupying different edaphic microenvironments. Morphological differentiation is in turn significantly associated with flowering time shifts. Thus we propose that diversification within Argyroderma has occurred through a process of adaptive speciation in allopatry. Spatially isolated populations diverge phenotypically in response to divergent habitat selection, which in turn leads to the evolution of reproductive isolation through divergence of flowering phenologies, perhaps as a correlated response to morphological divergence. Evidence suggests that diversification of the group has proceeded in two phases: the first involving divergence of allopatric taxa on varied microhabitats within a novel habitat type (the quartz gravel plains), and the second involving range expansion of an early flowering phenotype on the most extreme edaphic habitat and subsequent incomplete differentiation of allopatric populations of the early flowering group. These results point to adaptive speciation in allopatry as a likely model for the spectacular diversification of the ice-plant family in the dissected landscapes of the southern African winter rainfall deserts.

Genetic variation and phylogeographic analyses of two species of Carpobrotus and their hybrids in California.

Despite the commonality and study of hybridization in plants, there are few studies between invasive and noninvasive species that examine the genetic variability and gene flow of cytoplasmic DNA. We describe the phylogeographical structure of chloroplast DNA (cpDNA) variation within and among several interspecific populations of the putative native, Carpobrotus chilensis and the introduced, Carpobrotus edulis (Aizoaceae). These species co-occur throughout much of coastal California and form several 'geographical hybrid populations'. Two hundred and thirty-seven individuals were analysed for variation in an approximate 7.0 kb region of the chloroplast genome using PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism) data. Phylogenetic analyses and cpDNA population differentiation were conducted for all morphotypes. Historic geographical dispersion and the coefficient of ancestry of the haplotypes were determined using nested clade analyses. Two haplotypic groupings (I and II) were represented in C. chilensis and C. edulis, respectively. The variation in cpDNA data is in agreement with the previously reported allozyme and morphological data; this supports relatively limited variation and high population differentiation among C. chilensis and hybrids and more wide-ranging variation in C. edulis and C. edulis populations backcrossed with C. chilensis. C. chilensis disproportionately contributes to the creation of hybrids with the direction of gene flow from C. chilensis into C. edulis. The cpDNA data support C. chilensis as the maternal contributor to the hybrid populations.

Site-directed mutagenesis and protein 3D-homology modelling suggest a catalytic mechanism for UDP-glucose-dependent betanidin 5-O-glucosyltransferase from Dorotheanthus bellidiformis.

In livingstone daisy (Dorotheanthus bellidiformis), betanidin 5-O-glucosyltransferase (UGT73A5) is involved in the regiospecific glucosylation of betanidin and various flavonols. Based on sequence alignments several amino acid candidates which might be essential for catalysis were identified. The selected amino acids of the functionally expressed protein, suggested to be involved in substrate binding and turnover, were substituted via site-directed mutagenesis. The substitution of two highly conserved amino acids, Glu378, located in the proposed UDP-glucose binding site, and His22, located close to the N-terminus, led to the complete loss of enzyme activity. A 3D model of this regiospecific betanidin and flavonoid glucosyltransferase was constructed and the active site modelled. This model was based on the crystallographic structure of a bacterial UDP-glucose-dependent glucosyltransferase from Amycolatopsis orientalis used as a template and the generated null mutations. To explain the observed inversion in the configuration of the bound sugar, semiempirical calculations favour an SN-1 reaction, as one plausible alternative to the generally proposed SN-2 mechanism discussed for plant natural product glucosyltransferases. The calculated structural data do not only explain the abstraction of a proton from the acceptor betanidin, but further imply that the reaction mechanism might also involve a catalytic triad, with similarities described for the serine protease family.

Unmatched tempo of evolution in Southern African semi-desert ice plants.

The Succulent Karoo is an arid region, situated along the west coast of southern Africa. Floristically this region is part of the Greater Cape Flora and is considered one of the Earth's 25 biodiversity hotspots. Of about 5,000 species occurring in this region, more than 40% are endemic. Aizoaceae (ice plants) dominate the Succulent Karoo both in terms of species numbers (1,750 species in 127 genera) and density of coverage. Here we show that a well-supported clade within the Aizoaceae, representing 1,563 species almost exclusively endemic to southern Africa, has diversified very recently and very rapidly. The estimated age for this radiation lies between 3.8 and 8.7 million years (Myr) ago, yielding a per-lineage diversification rate of 0.77-1.75 per million years. Both the number of species involved and the tempo of evolution far surpass those of any previously postulated continental or island plant radiation. Diversification of the group is closely associated with the origin of several morphological features and one anatomical feature. Because species-poor clades lacking these features occur over a very similar distribution area, we propose that these characteristics are key innovations that facilitated this radiation.