Distinctions betweenHordeum bulbosum andH. murinum s.l. in seed and herbarium collections

It is sometimes necessary to identify eitherH. bulbosum orH. murinum on the basis of the inflorescence or seeds alone. The majority of taxonomic keys use the presence of swollen basal culms for the former against the annual habit for the latter. Confusion is due to similarities in inflorescences and spikelet morphology. Lodicules which always persist and are present beside the fruit in a mature caryopsis, and other characters such as the awns of the lemmas of the lateral spikelets enable conclusive distinction.     

Stomatal responses to light in the facultative Crassulacean acid metabolism species, Portulacaria afra

Guard cell responses to light are mediated by guard cell chlorophyll and by a specific blue light photoreceptor. Gas exchange and epidermal peel techniques were employed to investigate these responses in the facultative Crassulacean acid metabolism (CAM) species, Portulacaria afra (L.) Jacq. In P. afra individuals performing C₃ metabolism, red light stimulated an increase in leaf conductance in intact leaves and stomatal opening in isolated epidermal peels, indicating the presence in guard cells of the chlorophyll-mediated response to light. Under a background of continuous red illumination, conductance exhibited transient increases following pulses of blue but not red light, indicating that the specific stomatal response to blue light was also operative. In contrast, in CAM individuals, conductance in gas exchange experiments and stomatal opening in epidermal peel experiments were not stimulated by red light. In CAM plants, conductance did not increase following blue light pulses administered over a range of temperatures, vapor pressure differences (VPD), ambient CO₂ concentrations and background red light intensities. These results indicate that P. afra does possess typical guard cell responses to light when performing C₃ metabolism. The metabolic pathways mediating these responses are either lost or inhibited when CAM is induced.     

Expression of the high capacity calcium-binding domain of calreticulin increases bioavailable calcium stores in plants

Modulation of cytosolic calcium levels in both plants and animals is achieved by a system of Ca²⁺-transport and storage pathways that include Ca²⁺ buffering proteins in the lumen of intracellular compartments. To date, most research has focused on the role of transporters in regulating cytosolic calcium. We used a reverse genetics approach to modulate calcium stores in the lumen of the endoplasmic reticulum. Our goals were two-fold: to use the low affinity, high capacity Ca²⁺ binding characteristics of the C-domain of calreticulin to selectively increase Ca²⁺ storage in the endoplasmic reticulum, and to determine if those alterations affected plant physiological responses to stress. The C-domain of calreticulin is a highly acidic region that binds 20–50 moles of Ca²⁺ per mole of protein and has been shown to be the major site of Ca²⁺ storage within the endoplasmic reticulum of plant cells. A 377-bp fragment encoding the C-domain and ER retention signal from the maize calreticulin gene was fused to a gene for the green fluorescent protein and expressed in Arabidopsis under the control of a heat shock promoter. Following induction on normal medium, the C-domain transformants showed delayed loss of chlorophyll after transfer to calcium depleted medium when compared to seedlings transformed with green fluorescent protein alone. Total calcium measurements showed a 9–35% increase for induced C-domain transformants compared to controls. The data suggest that ectopic expression of the calreticulin C-domain increases Ca²⁺ stores, and that this Ca²⁺ reserve can be used by the plant in times of stress.     

Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), and huanglongbing disease do not exist in the Stapleton Station area of the Northern Territory of Australia

Abstract  A series of specimens of the Asian citrus psyllid, Diaphorina citri , collected from the Northern Territory (NT) in 1915 was recently rediscovered in the Natural History Museum, London. Surveys were conducted in 2002 on suitable hosts in the locality of the 1915 collections to see if the infestation had persisted. These failed to detect either D. citri or the bacterium that it transmits and that causes huanglongbing disease in citrus. It is presumed that D. citri was eradicated fortuitously by the removal of all citrus plants above latitude 19°S during an eradication program for citrus canker in the NT from 1916 until 1922.     

Cloning, expression and characterization of a UDP-galactose 4-epimerase from Escherichia coli

The gene galE encoding UDP-galactose 4-epimerase was cloned into E. coli BL21(DE3) from the chromosomal DNA of E. coli strain K-12. High expression of the soluble recombinant epimerase was achieved in the cell lysate. In order to evaluate the use of this epimerase in enzymatic synthesis of important -Gal epitopes (oligosaccharides with a terminal Gal1,3Gal sequence), a new radioactivity assay (1,3-galactosyltransferase coupled assay) was established to characterize its activity in producing UDP-galactose from UDP-glucose. Approximately 2700 units (100 mg) enzyme with a specific activity of 27 U mg^–1 protein could be obtained from one liter of bacterial culture. The epimerase was active in a wide pH range with an optimum at pH 7.0. This expression system established a viable route to the enzymatic production of -Gal oligosaccharides to support xenotransplantation research.     

Ants as egg predators of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in Australian cotton crops

Abstract Helicoverpa armigera is a major pest of Australian cotton crops. To assess the impact of ant predation on H. armigera populations, the behaviour of four common ant taxa was observed in cotton crops in northern New South Wales over the 1999−2000 and 2001−02 seasons. Areas of cotton were artificially stocked with H. armigera eggs prior to observation. Pheidole spp. were the most frequently observed ants within the crop canopy in 1999−2000 and took the most H. armigera eggs. Iridomyrmex spp. were more frequently observed than Pheidole spp. in 2001−02 and also took some H. armigera eggs. Neither Paratrechina spp. nor Rhytidoponera metallica (Smith) took any H. armigera eggs, although both were seen in the crop canopy. Irrigation, cultivation and insecticide application disrupted foraging ants and limited their impact on H. armigera populations.     

Characterization of a cryptic plasmid from a Greenland ice core Arthrobacter isolate and construction of a shuttle vector that replicates in psychrophilic high G+C Gram-positive recipients

Over 60 Greenland glacial isolates were screened for plasmids and antibiotic resistance/sensitivity as the first step in establishing a genetic system. Sequence analysis of a small, cryptic, 1,950 bp plasmid, p54, from isolate GIC54, related to Arthrobacter agilis, showed a region similar to that found in theta replicating Rhodococcus plasmids. A 6,002 bp shuttle vector, pSVJ21, was constructed by ligating p54 and pUC18 and inserting a chloramphenicol acetyl transferase (CAT) cassette conferring chloramphenicol resistance. Candidate Gram-positive recipients were chosen among glacial isolates based on phylogenetic relatedness, relatively short doubling times at low temperatures, sensitivity to antibiotics, and absence of indigenous plasmids. We developed an electroporation protocol and transformed seven isolates related to members of the Arthrobacter, Microbacterium, Curtobacterium, and Rhodoglobus genera with pSVJ21. Plasmid stability was demonstrated by successive transformation into Escherichia coli and four Gram-positive isolates, growth without antibiotic, and plasmid re-isolation. This shuttle vector and our transformation protocol provide the basis for genetic experiments with different high G+C Gram-positive hosts to study cold adaptation and expression of cold-active enzymes at low temperatures.     

PHENOPSIS, an automated platform for reproducible phenotyping of plant responses to soil water deficit in Arabidopsis thaliana permitted the identification of an accession with low sensitivity to soil water deficit

The high-throughput phenotypic analysis of Arabidopsis thaliana collections requires methodological progress and automation. Methods to impose stable and reproducible soil water deficits are presented and were used to analyse plant responses to water stress. Several potential complications and methodological difficulties were identified, including the spatial and temporal variability of micrometeorological conditions within a growth chamber, the difference in soil water depletion rates between accessions and the differences in developmental stage of accessions the same time after sowing. Solutions were found. Nine accessions were grown in four experiments in a rigorously controlled growth-chamber equipped with an automated system to control soil water content and take pictures of individual plants. One accession, An1, was unaffected by water deficit in terms of leaf number, leaf area, root growth and transpiration rate per unit leaf area. Methods developed here will help identify quantitative trait loci and genes involved in plant tolerance to water deficit.     

The Origin of Primary Plastids: A Pas de Deux or a Ménage à Trois?

The idea of an endosymbiotic origin of plastids has become incontrovertible, but many important aspects of plastid origins remain obscured in the mists of more than a billion years of evolutionary history. This commentary provides a critical summary of a recent proposal that primary plastid endosymbiosis was facilitated by the secretion into the host cytosol of effector proteins from intracellular Chlamydiales pathogens that allowed the host to utilize carbohydrates exported from the incipient plastid. Although not without flaws, the model provides an explanation for why primary plastids have evolved so rarely and why Archaeplastida, among all phagotrophic eukaryotes, succeeded in establishing primary plastids.It was over a century ago in 1905 that Mereschkowsky proposed that plastids derived from engulfed cyanobacteria (Martin and Kowallik, 1999), and more than 40 years since this idea entered the biological mainstream, primarily through the work of Lynn Margulis (e.g., Sagan, 1967; Margulis, 1970, 1981). The basic idea of an endosymbiotic origin of plastids is supported by abundant lines of evidence, especially by phylogenetic analyses showing that plastid genomes represent a particular branch of the cyanobacteria. Nonetheless, many important aspects of plastid origins remain. Ball et al. (2013) have now contributed to our understanding of primary endosymbiosis, exploring the possible role of Chlamydieae, intracellular parasitic bacteria, in allowing for the integration of cyanobacterial and host carbohydrate metabolism.Looking across the entire tree of life, we see many instances in which a lineage has made a remarkable transition in its way of life, often resulting in a major adaptive radiation. In seeking to explain these major transitions, evolutionary biologists generally start by identifying the traits needed for survival in the new adaptive zone and documenting the order in which they were acquired. However, a full evolutionary narrative should also explain why, if an evolutionary adaptive zone transition is so beneficial, did it not occur more frequently? And also why, among all of life’s diversity, did one specific lineage make the leap? For example, the reason that among all the many lineages of multicellular algae it was just the charophytes that succeeded in invading land (as land plants) has been suggested to be due to specific preadaptations of these algae, such as being specialized for freshwater (Graham, 1993; Becker and Marin, 2009) and having particular developmental (Graham et al., 2000) or biochemical characteristics (Sørensen et al., 2011). Ball et al. (2013) attempt to provide such a narrative to help explain why one particular clade of eukaryotes, the Archaeplastida, came to host the endosymbiotic cyanobacteria that evolved into plastids.Phylogenies of plastid-encoded genes confirm that plastids form a single clade embedded within the Cyanobacteria, consistent with a single event of plastid origination. The data from the nuclear genome is also consistent with this inference: Viridiplantae (which includes green algae and land plants), rhodophytes, and glaucophytes, the three eukaryotic lineages that are inferred on anatomical grounds to have plastids that were derived directly from engulfed cyanobacteria (primary plastids), form a clade, Archaeplastida (Keeling, 2004, 2010; Price et al., 2012). Evolution of primary plastids has indeed been an evolutionary rarity of the highest degree.The lack of additional origins of primary plastids is not simply because photoendosymbiosis is difficult. At least seven independent eukaryotic lineages have taken up single-celled eukaryotic algae as plastids, including ancestors of such successful groups as euglenoids, brown algae/diatoms, and dinoflagellates (Keeling, 2010). By contrast, there is only one other instance where a heterotrophic eukaryote evolved a stable association with an endosymbiotic cyanobacterium, the cercozoan genus Paulinella (Marin et al., 2005; Nowack et al., 2008), and the status of this case as a true plastid has been subject to debate (Theissen and Martin, 2006). The obvious conclusion from the rarity of primary plastid origins is that it is more difficult to integrate a prokaryotic endosymbiont into the host’s metabolism than another eukaryote. But what might the problem be, and how might the ancestor of Archaeplastida have overcome that impediment? Ball et al. (2013) argue that one of the main challenges during plastid origin is meshing host and plastid carbohydrate metabolism and that Archaeplastida were uniquely able to accommodate a cyanobacterial endosymbiont because they were subject to coinfections by chlamydial endoparasites.The idea that Chlamydiae have been important in the evolutionary history of Archaeplastida was first hinted at by the discovery that a surprisingly large number of genes in the Chlamydia trachomatis genome have closely related genes in plants (Stephens et al., 1998). Subsequent analyses have suggested that this is the result of direct horizontal gene transfer from Chlamydiae into ancestral Archaeplastida (Greub and Raoult, 2003; Huang and Gogarten, 2007; Becker et al., 2008; Moustafa et al., 2008). Phylogenomic analyses have identified between 21 and 55 specific genes that appeared to have been acquired by plants from Chlamydiae (Huang and Gogarten, 2007; Becker et al., 2008; Moustafa et al., 2008). Ball et al. (2013) corroborate this conclusion with their own analysis of currently available sequence data. Even using rather stringent criteria, they show that there are many more cases of chlamydial genes being sister to archaeplastidial genes than is the case for other eukaryotic clades, even clades such as fungi and animals, which are overrepresented in sequence databases.The conclusion that chlamydial genes’ presence in plants is a result of direct horizontal gene transfer is not universally accepted. Some suspect that these genes are the result of more complex histories entailing gene transfers among different prokaryotic lineages, including the cyanobacterial ancestors of plastids, perhaps combined with gene transfers into Archaeplastida later in their evolution and/or transfers from Archaeplastida into Chlamydiae (Brinkman et al., 2002; Dagan and Martin, 2009; Martin et al., 2012). However, the relatively large number of chlamydial genes in plant genomes points to some history of stable association between Chlamydiae and Archaeplastida that allowed abundant gene transfer. This leaves open the possibility that Chlamydiae were present in the host cell at the time that primary plastids evolved (Huang and Gogarten, 2007). If that were so, might chlamydial genes have facilitated plastid evolution?Following on from the group’s prior work on carbohydrate metabolism in Archaeplastida (Deschamps et al., 2008), Ball et al. (2013) explore the possibility that key genes were acquired from Chlamydiae. They focused on genes that would have been needed for sugar nucleotides exported from the cyanobacterium to be converted into glycogen, the original storage polysaccharide, and those that would be needed to effectively use stored glycogen. Cyanobacteria are assumed to have exported ADP-Glc. This would have posed a problem, however, since eukaryotic glucan synthases use UDP-Glc as substrate, rather than ADP-Glc. Ball et al. (2013) argued that the archaeplastidial glucan synthases that are most likely to have had ADP-Glc activity in solution are members of the class III or class IV starch synthases. A phylogenetic analysis of glucan synthases identified a well-supported (98% bootstrap) clade composed only of archaeplastidial class III and class IV sequences and a number of bacterial sequences (see Supplemental Figure 1 online in Ball et al., 2013). As shown in an unrooted version of this subtree (Figure 1A), the chlamydial sequences would end up sister to Archaeplastida under plausible rooting scenarios. Notably, this tree rules out a cyanobacterial origin of the archaeplastidial genes. Similarly, Ball et al. (2013) argued that effective use of glycogen stores required direct debranching activity, which is provided in modern Archaeplastida by isoamylase, which is closely related to chlamydial GlgX proteins (Huang and Gogarten, 2007). Phylogenetic analysis of GlgX/isoamylase sequences yields an unrooted tree on which plausible roots imply, once again, that archaeplastidial genes have chlamydial ancestry (Figure 1B).Open in a separate windowFigure 1.Unrooted Phylogenetic Trees for Gene Families Involved in Carbohydrate Metabolism.Both trees are heavily pruned and have branches with bootstrap percentages below 50% collapsed. Branches are coded by taxon: green = Viridiplantae; red = red algae; brown = glaucophytes; pink = Chlamydiae; cyan = cyanobacteria; black = other bacteria.(A) The starch synthase III-IV tree based on Supplemental Figure 1 from Ball et al. (2013). In the original analysis, this subtree was rooted on the branch marked with an arrow, but a more plausible rooting would be on one of the black (bacterial) branches.(B) Glycogen direct debranching genes based on Figure 5 and Supplemental Figure 2 from Ball et al. (2013). This tree is unrooted, but the true root most likely resides on one of the black branches.However, the inference that genes needed for proper interactions between a eukaryotic host and a prokaryotic endosymbiont were acquired from Chlamydiae poses a problem. It is unlikely that these genes entered the archaeplastidial genome before plastid endosymbiosis because they would have had no function, and genes do not persist in genomes for long if they lack a function. But equally, if these enzymes were critical for the establishment of the plastid, then they had to be present at the time of endosymbiosis. Ball et al. (2013) propose a resolution of this paradox: Suitable glucan synthases (GlgA) and direct glycogen debranching enzymes (GlgX) were present in the cytoplasm of the host cell because they were being secreted into the cytoplasm by coinfecting Chlamydiae. In support of this hypothesis, Ball et al. showed that many extant chlamydial proteins involved in carbohydrate metabolism, including GlgX, have sequences consistent with being effector proteins released into the host cell by a type III secretion system. Furthermore, an in vivo assay using Shigella flexneri as a model confirmed that the N-terminal peptide of several of proteins, including GlgX and GlgA, allows secretion through the type III system, suggesting that they could act as effector proteins in Chlamydiae.Putting the pieces together, Ball et al. (2013) propose a narrative in which the ancestral Archaeplastida were subject to infection by Chlamydiae, which injected a suite of proteins into host cells. These effector proteins served to manipulate carbohydrate metabolism to the parasite’s advantage. The presence of these enzymes in the cytoplasm had the beneficial side effect of allowing the host and an incipient plastid to enter into a mutualistic association that would otherwise have been impossible because of the inability of the host to synthesize and use storage carbohydrates from ADP-Glc. Only later, after horizontal gene transfer introduced the enzyme-coding genes into the nucleus, could the mutualism persist without Chlamydiae. Only at that point would selection favor Archaeplastida that resisted chlamydial infection. Or, to put it simplistically, rather than viewing the endosymbiotic origin of plastids as a dance of two partners, Ball et al. suggest it was a more complex, ménage à trois.The Ball et al. model has some compelling features. It provides an explanation for why primary plastids have evolved so rarely and why Archaeplastida, among all phagotrophic eukaryotes, succeeded in establishing primary plastids. However, for all its appeal, the hypothesis is not without its flaws. For example, many groups of eukaryotes are or have been subject to infection by Chlamydiae, so the model does not fully explain why there were no other origins of primary plastids. Also, there remains a possibility that the 50 or so genes transferred from Chlamydiae to Viridiplantae were acquired at different times in the course of simple parasitism, with the apparent enrichment of carbohydrate metabolism genes in the list being coincidental. Nonetheless, by combining new insights into the evolution of carbohydrate metabolism in Archaeplastida with phylogenomics and experimental studies of potential metabolic effector proteins, Ball et al. (2013) have contributed to our understanding of one of the most important events in the origin of life on earth: the origin of plastids.