An abrupt increase in foliage ABA levels on incipient leaf death occurs across vascular plants.

Forest mortality during drought has been attributed to hydraulic failure, which can be challenging to measure. A limited number of alternative proxies for incipient leaf death exist. Here we investigate whether a terminal increase in abscisic acid (ABA) levels in leaves occurs across vascular land plants and is an indicator of imminent leaf death. For different species across vascular plants, we monitored ABA levels during lethal drought as well as leaf embolism resistance, across the canopy as leaves die following senescence, or when leaves are exposed to a heavy, lethal frost late in the growing season. We observed a considerable increase in foliage ABA levels once leaves showed signs of incipient leaf death. This increase in ABA levels upon incipient leaf death, could be induced by embolism during drought, by freezing or as leaves age naturally, and was observed in species spanning the phylogeny of vascular land plants as well as in an ABA biosynthetic mutant plant. A considerable increase in foliage ABA levels may act as an indicator of impending leaf death.

Stem and leaf xylem of angiosperm trees experiences minimal embolism in temperate forests during two consecutive summers with moderate drought.

Drought events may increase the likelihood that the plant water transport system becomes interrupted by embolism. Yet our knowledge about the temporal frequency of xylem embolism in the field is frequently lacking, as it requires detailed, long-term measurements. We measured xylem embolism resistance and midday xylem water potentials during the consecutive summers of 2019 and 2020 to estimate maximum levels of embolism in leaf and stem xylem of ten temperate angiosperm tree species. We also studied vessel and pit membrane characteristics based on light and electron microscopy to corroborate potential differences in embolism resistance between leaves and stems. Apart from A. pseudoplatanus and Q. petraea, eight species experienced minimum xylem water potentials that were close to or below those required to initiate embolism. Water potentials corresponding to ca. 12% loss of hydraulic conductivity (PLC) could occur in six species, while considerable levels of embolism around 50% PLC were limited to B. pendula and C. avellana. There was a general agreement in embolism resistance between stems and leaves, with leaves being equally or more resistant than stems. Also, xylem embolism resistance was significantly correlated to intervessel pit membrane thickness (TPM ) for stems, but not to vessel diameter and total intervessel pit membrane surface area of a vessel. Our data indicate that low amounts of embolism occur in most species during moderate summer drought, and that considerable levels of embolism are uncommon. Moreover, our experimental and TPM data show that leaf xylem is generally no more vulnerable than stem xylem.

Advanced vascular function discovered in a widespread moss.

The evolution of terrestrial plants capable of growing upwards into the dry atmosphere profoundly transformed the Earth. A transition from small, 'non-vascular' bryophytes to arborescent vascular plants during the Devonian period is partially attributed to the evolutionary innovation of an internal vascular system capable of functioning under the substantial water tension associated with vascular water transport. Here, we show that vascular function in one of the most widespread living bryophytes (Polytrichum commune) exhibits strong functional parallels with the vascular systems of higher plants. These parallels include vascular conduits in Polytrichum that resist buckling while transporting water under tension, and leaves capable of regulating transpiration, permitting photosynthetic gas exchange without cavitation inside the vascular system. The advanced vascular function discovered in this tallest bryophyte family contrasts with the highly inefficient water use found in their leaves, emphasizing the importance of stomatal evolution enabling photosynthesis far above the soil surface.

Stomatal (mis)behaviour.

When stomata first evolved they initiated the greening of terrestrial earth, and now more than 400 million years later these simple bi-cellular valves in the leaf surface regulate global fluxes of water and carbon. Despite their importance and superficial simplicity, the behaviour of stomata remains a great challenge to understand. Different approaches to studying stomatal control have yielded rather disparate models for how stomata respond to environmental stimuli. Much of this discord arises from the diversity of mechanisms apparently involved in changing guard cell turgor and hence the aperture of the stomatal pore. On the one hand, the physical tension produced by dragging water from the soil through the xylem to the leaves directly influences leaf and guard cell turgor, while on the other hand, phytohormone levels (most importantly abscisic acid), light, photosynthesis and atmospheric gases induce active changes in guard cell turgor by triggering ionic pumping. Each stomatal control mechanism has its own champion and no model has ever successfully integrated all components. In such an environment there is great value in examining how different parts of the stomatal control network interact, particularly the competition between 'hydraulic' signals related to leaf water content and 'metabolic' signals related to ambient photosynthetic conditions.

Staining of celiac disease-relevant T cells by peptide-DQ2 multimers.

Gluten-specific T cells in the small intestinal mucosa are thought to play a central role in the pathogenesis of celiac disease (CD). The vast majority of these T cells recognize gluten peptides when presented by HLA-DQ2 (DQA1*05/DQB1*02), a molecule which immunogenetic studies have identified as conferring susceptibility to CD. We have previously identified and characterized three DQ2-restricted gluten epitopes that are recognized by intestinal T cells isolated from CD patients, two of which are immunodominant. Because almost all of the gluten epitopes are restricted by DQ2, and because we have detailed knowledge of several of these epitopes, we chose to develop peptide-DQ2 tetramers as a reagent to further investigate the role of these T cells in CD. In the present study, stable soluble DQ2 was produced such that it contained leucine zipper dimerization motif and a covalently coupled peptide. We have made four different peptide-DQ2 staining reagents, three containing the gluten epitopes and one containing a DQ2-binding self-peptide that provides a negative control for staining. We show in this study that peptide-DQ2 when adhered to plastic specifically stimulates T cell clones and that multimers comprising these molecules specifically stain peptide-specific T cell clones and lines. Interestingly, T cell activation caused severe reduction in staining intensities obtained with the multimers and an Ab to the TCR. The problem of TCR down-modulation must be taken into consideration when using class II multimers to stain T cells that may have been recently activated in vivo.

Genes and environment in celiac disease.

Celiac disease is an intestinal disorder that develops as a result of interplay between genetic and environmental factors. HLA genes along with non-HLA genes predispose to the disease. Linkage studies have failed to identify chromosomal regions other than the HLA region which have major effects, indicating the existence of multiple non-HLA predisposing genes with modest effects. Association studies have shown that CTLA4 or a closely located gene is one of these genes. The primary HLA association in the majority of celiac disease patients is with DQ2 (DQA1*05/DQB1*02) and in the minority of patients with DQ8 (DQA1*0301/DQB1*0302). Gluten reactive CD4+ T cells can be isolated from small intestinal biopsies of celiac patients but not from controls. DQ2 or DQ8, but not other HLA molecules carried by patients, present peptides to these T cells. A number of distinct T cell gluten epitopes exist, most of them posttranslationally modified by deamidation. DQ2 and DQ8 bind the epitopes such that the glutamic acid residues created by deamidation are accommodated in pockets that have a preference for negatively charged side chains. There is evidence that deamidation in vivo is mediated by the enzyme tissue transglutaminase (tTG). Overall, the results point to control of the immune response to gluten by intestinal T cells restricted by the DQ2 or DQ8 molecules. This is likely to be a critical checkpoint for the development of celiac disease and could explain the dominant genetic role of HLA in this disorder. The products of the other predisposing genes may participate in pathway(s) that lead(s) to lesion formation. The minor genetic effects of the non-HLA genes could indicate a lack of critical checkpoints along these pathways, or that there are several pathways leading to the lesion formation.

T cells from celiac disease lesions recognize gliadin epitopes deamidated in situ by endogenous tissue transglutaminase.

Celiac disease is an HLA-DQ2-associated disorder characterized by intestinal T cell responses to ingested wheat gliadins. Initial studies used gliadin that had been subjected to non-enzymatic deamidation during pepsin/trypsin digestion to enrich for the gliadin-specific T cells in small intestinal celiac biopsies. These T cells recognized synthetic gliadin peptides only after their deamidation in vitro by purified tissue transglutaminase (tTG). However, as these studies used a deamidated antigen for re-stimulation prior to testing for antigen specificity, this raised the possibility that T cells specific for native epitopes had not been expanded in vitro and had thus been overlooked. To address this possibility and to look for more direct evidence that endogenous tTG mediates deamidation of gluten in the celiac lesions, we have here used a minimally deamidated chymotrypsin-digest of gliadin to challenge biopsies and then investigated the specificity of the T cell lines derived from them. Interestingly, these T cell lines only barely responded to the chymotrypsin-digested gliadins, but efficiently recognized the in vitro tTG-treated variants of the same gliadins. Moreover, the addition of a tTG-inhibitor during the gliadin challenge often resulted in T cell lines with abolished or reduced responses to deamidated gliadin. These data demonstrate that DQ2-restricted T cells within adult celiac lesions predominantly recognize deamidated gliadin epitopes that are formed in situ by endogenous tTG.

T cell recognition of the dominant I-A(k)-restricted hen egg lysozyme epitope: critical role for asparagine deamidation.

Type-B T cells raised against the immunodominant peptide in hen egg lysozyme (HEL(48-62)) do not respond to whole lysozyme, and this has been thought to indicate that peptide can bind to l-A(k) in different conformations. Here we demonstrate that such T cells recognize a deamidated form of the HEL peptide and not the native peptide. The sequence of the HEL epitope facilitates rapid and spontaneous deamidation when present as a free peptide or within a flexible domain. However, this deamidated epitope is not created within intact lysozyme, most likely because it resides in a highly structured part of the protein. These findings argue against the existence of multiple conformations of the same peptide-MHC complex and have important implications for the design of peptide-based vaccines. Furthermore, as the type-B T cells are known to selectively evade induction of tolerance when HEL is expressed as a transgene, these results suggest that recognition of posttranslationally modified self-antigen may play a role in autoimmunity.

Signal transduction through Vav-2 participates in humoral immune responses and B cell maturation.

B and T lymphocytes develop normally in mice lacking the guanine nucleotide exchange factor Vav-2. However, the immune responses to type II thymus-independent antigen as well as the primary response to thymus-dependent (TD) antigen are defective. Vav-2-deficient mice are also defective in their ability to switch immunoglobulin class, form germinal centers and generate secondary immune responses to TD antigens. Mice lacking both Vav-1 and Vav-2 contain reduced numbers of B lymphocytes and display a maturational block in the development of mature B cells. B cells from Vav-1(-/-)Vav-2(-/-) mice respond poorly to antigen receptor triggering, both in terms of proliferation and calcium release. These studies show the importance of Vav-2 in humoral immune responses and B cell maturation.

Structural organization of the mouse phosphatidylinositol 3-kinase p110d gene.

Phosphatidylinositol 3-kinases are a family of dual specificity lipid/protein kinases. The products of PI3K's, phosphatidylinositol(3,4,5) triphosphate and phosphatidylinositol(3,4) bisphosphate, act as second messengers connecting activated transmembrane receptors to signaling pathways that control gene transcription, proliferation, transformation, programmed cell death, adhesion, migration and vesicular transport. There is evidence that different isoforms of PI3K's activate specific signaling pathways and are thus responsible for integrating cellular responses. The elucidation of the genomic structure of the catalytic subunits is a necessary step for the investigation of the function of PI3K isoforms by inactivation of the gene in vivo. The structural organization of p110alpha, beta, and gamma genes has been previously reported. Here we report the cloning, sequencing, and structural organization of the mouse p110delta gene from a murine 129/Sv genomic library. The p110delta gene consists of 22 exons and spans over 13 kb. Comparison of the genomic structure with that of p110alpha, beta, and gamma demonstrates that the p110delta gene shares its exon structure with p110beta, the most closely related PI3K at the amino acid level.

Vav-2 controls NFAT-dependent transcription in B- but not T-lymphocytes.

We show here that Vav-2 is tyrosine phosphorylated following antigen receptor engagement in both B- and T-cells, but potentiates nuclear factor of activated T cells (NFAT)-dependent transcription only in B cells. Vav-2 function requires the N-terminus, as well as functional Dbl homology and SH2 domains. More over, the enhancement of NFAT-dependent transcription by Vav-2 can be inhibited by a number of dominant-negative GTPases. The ability of Vav-2 to potentiate NFAT-dependent transcription correlates with its ability to promote a sustained calcium flux. Thus, Vav-2 augments the calcium signal in B cells but not T cells, and a truncated form of Vav-2 can neither activate NFAT nor augment calcium signaling. The CD19 co-receptor physically interacts with Vav-2 and synergistically enhances Vav-2 phosphorylation induced by the B-cell receptor (BCR). In addition, we found that Vav-2 augments CD19-stimulated NFAT- dependent transcription, as well as transcription from the CD5 enhancer. These data suggest a role for Vav-2 in transducing BCR signals to the transcription factor NFAT and implicate Vav-2 in the integration of BCR and CD19 signaling.

Anthraquinone-oligodeoxynucleotide conjugates as inhibitors of gene transcription.

Several anthraquinone-oligodeoxynucleotide conjugates have been synthesized and their inhibition of transcription of the bla gene in E. coli has been determined. All conjugates at 10 microM inhibited transcription and three conjugates inhibited mRNA production by 40 to 50% for periods of up to 60 min.

A high incidence of Shigella-induced arthritis in a primate species: major histocompatibility complex class I molecules associated with resistance and susceptibility, and their relationship to HLA-B27.

The human major histocompatibility complex (MHC) class I gene, HLA-B27, is a strong risk factor for susceptibility to a group of disorders termed spondyloarthropathies. Rodents that express HLA-B27 develop spondyloarthropathies, implicating HLA-B27 in the etiology of these disorders. To determine whether an HLA-B27-like molecule was associated with spondyloarthropathies in nonhuman primates, we analyzed the MHC class I cDNAs expressed in a cohort of rhesus macaques that developed reactive arthritis after an outbreak of shigellosis. We identified several cDNAs with only limited sequence similarity to HLA-B27. Interestingly, one of these MHC molecules had a B pocket identical to that of HLA-B39. Pool sequencing of radiolabeled peptides bound by this molecule demonstrated that, like HLA-B27 and HLA-B39, it could bind peptides with arginine at the second position. However, extensive analysis of the MHC class I molecules in this cohort revealed no statistically significant association between any particular MHC class I allele and susceptibility to reactive arthritis. Furthermore, none of the rhesus MHC class I molecules bore a strong resemblance to HLA-B27, indicating that reactive arthritis can develop in this animal model in the absence of an HLA-B27-like molecule. Surprisingly, there was a statistically significant association between the rhesus macaque MHC A locus allele, Mamu-A*12, and the absence of reactive arthritis following Shigella infection.

The intestinal T cell response to alpha-gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase.

The great majority of patients that are intolerant of wheat gluten protein due to celiac disease (CD) are human histocompatibility leukocyte antigen (HLA)-DQ2(+), and the remaining few normally express HLA-DQ8. These two class II molecules are chiefly responsible for the presentation of gluten peptides to the gluten-specific T cells that are found only in the gut of CD patients but not of controls. Interestingly, tissue transglutaminase (tTG)-mediated deamidation of gliadin plays an important role in recognition of this food antigen by intestinal T cells. Here we have used recombinant antigens to demonstrate that the intestinal T cell response to alpha-gliadin in adult CD is focused on two immunodominant, DQ2-restricted peptides that overlap by a seven-residue fragment of gliadin. We show that tTG converts a glutamine residue within this fragment into glutamic acid and that this process is critical for T cell recognition. Gluten-specific T cell lines from 16 different adult patients all responded to one or both of these deamidated peptides, indicating that these epitopes are highly relevant to disease pathology. Binding studies showed that the deamidated peptides displayed an increased affinity for DQ2, a molecule known to preferentially bind peptides containing negatively charged residues. Interestingly, the modified glutamine is accommodated in different pockets of DQ2 for the different epitopes. These results suggest modifications of anchor residues that lead to an improved affinity for major histocompatibility complex (MHC), and altered conformation of the peptide-MHC complex may be a critical factor leading to T cell responses to gliadin and the oral intolerance of gluten found in CD.

Studies of gliadin-specific T-cells in celiac disease.

Celiac disease is an immune-mediated disorder that primarily affects the small intestinal mucosa. It is one of the few human disorders of which it is possible, and ethically acceptable, to obtain samples from the disease-affected tissue. This chapter describes how small intestinal biopsy specimens are utilized for studies of cell-mediated immune responses in celiac disease. The focus is mainly on practical procedures for isolation, growth under sterile conditions, and subsequent analyses of gliadin-specific T-cells derived from the small biopsy specimens. This chapter also provides guidelines for the preparation of different gliadin antigens suitable for T-cell analysis. Note that most of the T-cell assays described necessitate serological and/or genomic HLA typing of the celiac disease patients from whom the T-cells are derived.

Production of a panel of recombinant gliadins for the characterisation of T cell reactivity in coeliac disease.

BACKGROUND/AIMS: Coeliac disease is a chronic intestinal disorder most probably caused by an abnormal immune reaction to wheat gliadin. The identification of the HLA-DQ2 and HLA-DQ8 as the molecules responsible for the HLA association in coeliac disease strongly implicates a role for CD4 T cells in disease pathogenesis. Indeed, CD4 T cells specific for gliadin have been isolated from the small intestine of patients with coeliac disease. However, identification of T cell epitopes within gliadin has been hampered by the heterogeneous nature of the gliadin antigen. To aid the characterisation of gliadin T cell epitopes, multiple recombinant gliadins have been produced from a commercial Nordic wheat cultivar. METHODS: The alpha-gliadin and gamma-gliadin genes were amplified by polymerase chain reaction from cDNA and genomic DNA, cloned into a pET expression vector, and sequenced. Genes encoding mature gliadins were expressed in Escherichia coli and tested for recognition by T cells. RESULTS: In total, 16 alpha-gliadin genes with complete open reading frames were sequenced. These genes encoded 11 distinct gliadin proteins, only one of which was found in the Swiss-Prot database. Expression of these gliadin genes produced a panel of recombinant alpha-gliadin proteins of purity suitable for use as an antigen for T cell stimulation. CONCLUSION: This study provides an insight into the complexity of the gliadin antigen present in a wheat strain and has defined a panel of pure gliadin antigens that should prove invaluable for the future mapping of epitopes recognised by intestinal T cells in coeliac disease.

HLA binding and T cell recognition of a tissue transglutaminase-modified gliadin epitope.

DQ2 confers susceptibility to celiac disease (CD) and intestinal CD4(+) T cells of DQ2(+) CD patients preferentially recognize deamidated gliadin peptides. This modification can be mediated by tissue transglutaminase (tTG). We have investigated what role the tTG-modified residues play in DQ2 binding and T cell presentation using a model gamma-gliadin peptide (residues 134 - 153). Treatment of this peptide with tTG resulted in deamidation of Gln residues at positions 140, 148 and 150. Two of these residues act as DQ2 anchors at position P7 (148) and P9 (150) and increased the affinity of the modified peptide for DQ2 50-fold. Testing of a mutant DQ2 molecule demonstrated that the Lys residue at beta71 of DQ2 is important for binding of the deamidated peptide. A variant DQ2 molecule (with the same beta-chain but different alpha-chain) that does not confer susceptibility to CD was capable of presenting the gliadin peptide, but not pepsin/trypsin-digested gliadin, equally well to a T cell. This suggests that processing events might be involved in the preferential presentation of the gliadin peptide by the DQ2 molecule. Substitution of Gln with Glu in some positions not targeted by tTG, but in positions likely to be deamidated via non-enzymatic mechanisms, disrupted T cell recognition. This provides additional evidence that tTG is responsible for modification of gliadin in vivo.