Land plants acquired active stomatal control early in their evolutionary history

Stomata are pores that regulate plant gas exchange [1]. They evolved more than 400 million years ago [2, 3], but the origin of their active physiological responses to endogenous and environmental cues is unclear [2-6]. Recent research suggests that the stomata of lycophytes and ferns lack pore closure responses to abscisic acid (ABA) and CO(2). This evidence led to the hypothesis that a fundamental transition from passive to active control of plant water balance occurred after the divergence of ferns 360 million years ago [7, 8]. Here we show that stomatal responses of the lycophyte Selaginella [9] to ABA and CO(2) are directly comparable to those of the flowering plant Arabidopsis [10]. Furthermore, we show that the underlying intracellular signaling pathways responsible for stomatal aperture control are similar in both basal and modern vascular plant lineages. Our evidence challenges the hypothesis that acquisition of active stomatal control of plant carbon and water balance represents a critical turning point in land plant evolution [7, 8]. Instead, we suggest that the critical evolutionary development is represented by the innovation of stomata themselves and that physiologically active stomatal control originated at least as far back as the emergence of the lycophytes (circa 420 million years ago) [11].     

Development of the signalling pathways associated with sperm capacitation during epididymal maturation

As spermatozoa mature within the epididymis they acquire the potential for capacitation and ultimately fertilization. In biochemical terms, the former is reflected in the progressive activation of a signal transduction pathway characterized by cAMP-mediated induction of phosphotyrosine expression on the sperm tail. In this study, we have examined the cellular mechanisms controlling this maturational event. Caput epididymal spermatozoa exhibited tyrosine phosphorylation on the sperm head that was largely unresponsive to cAMP and not significantly impaired by removal of extracellular HCO(3) (-). In contrast, caudal epididymal spermatozoa exhibited low levels of phosphorylation on the sperm head, yet responded dramatically to cAMP by phosphorylating a new set of proteins on the sperm tail via mechanisms that were highly dependent on extracellular HCO(3) (-). The impact of extracellular HCO(3) (-) depletion on caudal cells was not associated with a significant change in the redox regulation of cAMP but could be fully reversed by buffering the intracellular pH with N-Tris[Hydroxymethyl]methyl-3-amino-propanesulfonic acid (TAPS). The pattern of tyrosine phosphorylation was also profoundly influenced by the presence or absence of added extracellular calcium. In the presence of this cation, only caudal spermatozoa could respond to increased extracellular cAMP with tyrosine phosphorylation of the sperm tail. However, in calcium-depleted medium, this difference completely disappeared. Under these conditions, caput and caudal spermatozoa were equally competent to exhibit phosphotyrosine expression on the sperm tail in response to cAMP. These results emphasize the pivotal role played by calcium and HCO(3) (-) in modulating the changes in tyrosine phosphorylation observed during epididymal maturation.     

POCUS: mining genomic sequence annotation to predict disease genes

Here we present POCUS (prioritization of candidate genes using statistics), a novel computational approach to prioritize candidate disease genes that is based on over-representation of functional annotation between loci for the same disease. We show that POCUS can provide high (up to 81-fold) enrichment of real disease genes in the candidate-gene shortlists it produces compared with the original large sets of positional candidates. In contrast to existing methods, POCUS can also suggest counterintuitive candidates.     

A novel disorder caused by defective biosynthesis of N-linked oligosaccharides due to glucosidase I deficiency

Glucosidase I is an important enzyme in N-linked glycoprotein processing, removing specifically distal alpha-1,2-linked glucose from the Glc3Man9GlcNAc2 precursor after its en bloc transfer from dolichyl diphosphate to a nascent polypeptide chain in the endoplasmic reticulum. We have identified a glucosidase I defect in a neonate with severe generalized hypotonia and dysmorphic features. The clinical course was progressive and was characterized by the occurrence of hepatomegaly, hypoventilation, feeding problems, seizures, and fatal outcome at age 74 d. The accumulation of the tetrasaccharide Glc(alpha1-2)Glc(alpha1-3)Glc(alpha1-3)Man in the patient's urine indicated a glycosylation disorder. Enzymological studies on liver tissue and cultured skin fibroblasts revealed a severe glucosidase I deficiency. The residual activity was <3% of that of controls. Glucosidase I activities in cultured skin fibroblasts from both parents were found to be 50% of those of controls. Tissues from the patient subjected to SDS-PAGE followed by immunoblotting revealed strongly decreased amounts of glucosidase I protein in the homogenate of the liver, and a less-severe decrease in cultured skin fibroblasts. Molecular studies showed that the patient was a compound heterozygote for two missense mutations in the glucosidase I gene: (1) one allele harbored a G-->C transition at nucleotide (nt) 1587, resulting in the substitution of Arg at position 486 by Thr (R486T), and (2) on the other allele a T-->C transition at nt 2085 resulted in the substitution of Phe at position 652 by Leu (F652L). The mother was heterozygous for the G-->C transition, whereas the father was heterozygous for the T-->C transition. These base changes were not seen in 100 control DNA samples. A causal relationship between the alpha-glucosidase I deficiency and the disease is postulated.     

The absorption, translocation and distribution of the herbicide glyphosate in maize expressing the CP-4 transgene

Maize (Zea mays L. var. Bonnie) transformed with a gene encoding a 5-enolpyruvylshikimate 3-phosphate synthase with altered sensitivity showed over 100-fold greater resistance to the herbicide glyphosate (N-[phosphonomethyl]glycine) in comparison with its non-transformed progenitor (parental control) at the third-leaf stage. Studies with [¹⁴C]-glyphosate at a dosage lethal to the parental control, but sublethal to the transgenic, revealed that a maximum of 45-65% of the applied dose was absorbed, with greater absorption occurring in transgenic plants. Translocation of glyphosate was closely related to its absorption (r value 0.956) with approximately 15% more of the applied dose being mobilized in transgenic plants than the parental controls. Analysis of electronic autoradiograms along the treated leaf lamina found discrete internal regions of glyphosate accumulation closely associated with the site of application. These regions contained lower amounts of glyphosate present in the treated leaf lamina was almost completely translocated in transgenic plants, while in the parental controls more remained and the leaf became necrotic. In both types of maize there was a small accumulation of herbicide in the tip region of the leaf which was not mobilized. Younger shoot tissues and roots were major sinks for translocated glyphosate accumulating approximately 25-40% of the applied dose depending upon treatment. In the parental control, equal amounts of glyphosate were found distributed between young shoot tissues and roots; while in transgenic plants, the young shoot tissue accumulated around three times more glyphosate than the roots. In both plant types, glyphosate was localized in the meristems and young, actively growing leaves. Specific glyphosate activity (the amount of glyphosate per unit dry weight of tissue) in the major sinks of the transgenic declined towards the end of the treatment period but remained relatively constant in the parental control. In conclusion, enhancing glyphosate resistance by genetic transformation influenced the absorption, translocation and distribution of this herbicide in whole plants.Keywords: Zea mays, glyphosate (N-[phosphonomethyl]-glycine), transgenic, absorption, translocation, source-sink.      

Population genetics of the wild yeast Saccharomyces paradoxus

Saccharomyces paradoxus is the closest known relative of the well-known S. cerevisiae and an attractive model organism for population genetic and genomic studies. Here we characterize a set of 28 wild isolates from a 10-km(2) sampling area in southern England. All 28 isolates are homothallic (capable of mating-type switching) and wild type with respect to nutrient requirements. Nine wild isolates and two lab strains of S. paradoxus were surveyed for sequence variation at six loci totaling 7 kb, and all 28 wild isolates were then genotyped at seven polymorphic loci. These data were used to calculate nucleotide diversity and number of segregating sites in S. paradoxus and to investigate geographic differentiation, population structure, and linkage disequilibrium. Synonymous site diversity is approximately 0.3%. Extensive incompatibilities between gene genealogies indicate frequent recombination between unlinked loci, but there is no evidence of recombination within genes. Some localized clonal growth is apparent. The frequency of outcrossing relative to inbreeding is estimated at 1.1% on the basis of heterozygosity. Thus, all three modes of reproduction known in the lab (clonal replication, inbreeding, and outcrossing) have been important in molding genetic variation in this species.     

Seeing 'cool' and 'hot'--infrared thermography as a tool for non-invasive, high-throughput screening of Arabidopsis guard cell signalling mutants

The use of Arabidopsis mutants defective in abscisic acid (ABA) perception has been instrumental in the understanding of stomatal function, in particular, ABA signalling in guard cells. The considerable attention devoted to ABA signalling in guard cells is due in part to (1) the fundamental role of ABA in drought stress and (2) the use of a screening protocol based on the sensitivity of seed germination to ABA. Such a screen has facilitated the isolation of ABA signalling mutants with genetic lesions that exert pleiotropic effects at the whole plant level. As such, there is a requirement for new approaches to complement the seed germination screen. The recent advances made in the use of infrared thermography as a non-invasive, high-throughput tool are reviewed here and the versatility of this technique for screening Arabidopsis defective in stomatal regulation is highlighted.     

A computational investigation of kinetoplastid trans-splicing

Trans-splicing is an unusual process in which two separate RNA strands are spliced together to yield a mature mRNA. We present a novel computational approach which has an overall accuracy of 82% and can predict 92% of known trans-splicing sites. We have applied our method to chromosomes 1 and 3 of Leishmania major, with high-confidence predictions for 85% and 88% of annotated genes respectively. We suggest some extensions of our method to other systems.