Molecular and physiological study of water-deficit stress on selected Eragrostis species

Bibliography: leaves 60-73.Eragrostis nindensis and Eragrostis tef are wild and domestic grasses respectively that belong to the subfamily Eragrostideae. E. nindensis is desiccation tolerant while E. tef is desiccation sensitive. The responses of these plants to water-deficit stress were studied using molecular and physiological approaches. A cDNA library of E. nindensis was screened to identify differentially expressed genes during dehydration. Physiological studies included monitoring changes in photosynthesis, respiration, ultrastructure and membrane integrity of plants during dehydration and rehydration. The differential screening of the cDNA library, using a radio-labelled cDNA from hydrated and dehydrated leaves respectively, revealed two genes, referred as Nin-19 and Nin-44, that were differentially expressed in dehydrated leaves of E. nindensis. These genes were sequenced and partially characterized. Nin-19 did not show considerable identity with any known genes and was not studied any further. Nin-44 was identified as a dehydrin-like gene with approximately 99 % identity to seven water-deficit stress responsive genes on a section of about 60 bp near the 3' end. As its sequence was found to represent a partial insert size, two forward and reverse primers were designed to find the full length through RT-PCR. Despite repeated attempts, no products that could be used in subsequent procedures were achieved using this technique. Hence, further characterization of this gene also could not be performed and different approaches were suggested. The physiological studies showed that E. nindensis is desiccation tolerant but E. tefis not, the latter dying below RWC of about 33 %. Difference among plants in physiological responses became evident after 6 days of dehydration treatment, which resulted in a decline of RWC to 65%, 39% and 33% in E. nindensis, E.tif(R) (red-seeded) and E. tef (W) (white-seeded) plants respectively. A significant decrease in photosynthesis, transpiration, stomatal conductance, and an increase in electrolyte leakage occurred in all species after 6 days of dehydration, but leaves of E. tef (W) did not recover from this level of dehydration when watered. Instead, new leaves were observed to re-grow from the stem nodes. The leaves of red-seeded variety of E. tef did recover fully from RWC of 39 %. After a further 3 days dehydration both varieties of E. tef died. On the other hand, E. nindensis was found to survive extreme water-deficit (-10 % RWC tested here) and recovered full physiological activity when watered. The electrolyte leakage study on these plants indicated major injury on E. tef(W), being intermediate in E. tef(R) and very low in E. nindensis, which coincided with the trend of declining in RWC and other metabolic activities measured. The ultrastructural study on E. tef varieties also showed evidence of the damage caused by dehydration, but the difference among these species was not significant enough to indicate the level of susceptibility of the plants to dehydration damage. The study demonstrated that E. tef varieties are not drought tolerant and showed a considerable difference in their responses to water-deficit stress with each other and with respect to E. nindensis. However, E. tef (R) seems to have a better control over transpiration and some form of repair mechanism operational at least until dehydration to 39 % RWC. This is proposed to be a better performing cereal in conditions of water stress. On the other hand E. nindnsis did not suffer major injury from the dehydration treatment and confirmed to be desiccation tolerant

Physiological response of selected eragrostis species to water-deficit stress

Plant water-deficit stress is considered to be one of the greatest threats to world agriculture. In an effort to identify a better performing crop variety of Eragrostis tef under this stress, the responses of two varieties, white (W) and brown (B) seeded, were compared with those of the resurrection grass Eragrostis nindensis during a dehydration/rehydration cycle. After 6 days of dehydration, relative water content (RWC) dropped to 33%, 43% and 39% in En, Et(B) and Et(W) plants, respectively. This water loss was accompanied by a decline in transpiration and increased electrolyte leakage in the Et varieties. Et(W) did not recover from this level of drying when watered. Et(B) on the other hand recovered fully from 43% RWC, but lost viability after 9 days dehydration, RWC < 30%. En showed full metabolic recovery from drying to 10% RWC. Loss of viability in the Et varieties was accompanied by an increase in electrolyte leakage and irreversible decline in photosynthesis and transpiration. Ultrastructural study also indicated a drying-induced damage to membranes and organelles of Et tissues dehydrated for 6 days which was reversed in Et(B), 43% RWC, on rehydration. However, after 9 days of dehydration, (< 30% RWC), severe irreversible damage occurred to the entire subcellular organization of both Et varieties and was accompanied by loss of viability. This study shows that En is a true resurrection plant and both Et varieties are desiccation sensitive but drought tolerant to varying degrees. Et(B) is more drought tolerant and hence a better choice crop in drought prone areas. Comparative study of closely related plant species might be a better approach in finding adaptive characters in crop plants with respect to environmental stresses.Key words: Eragrostis tef, eragrostis nindensis, dehydration, desiccation, gas exchange, leakage, ultrastructure

Characterization of two, desiccation linked, Group 1 LEA proteins from the resurrection plant Xerophyta humilis

Studies on resurrection plants and other anhydrobiotic organisms show expression of Late Embryogenesis Abundant (LEA) proteins associated with desiccation tolerance. However, the precise role of these proteins has not been described. This study was undertaken to investigate expression, structure and function of XhLEA1-4S1 and XhLEA1-1S2, Group 1 LEA proteins from Xerophyta humilis, in order to shed light on their role in desiccation tolerance. Complementary DNA (cDNA) of these XhLEAs were cloned into bacterial expression vectors and the recombinant proteins expressed in E. coli. Antibodies were generated and used in determination of expression conditions and immunolocalization studies

Patient derived colonoids as drug testing platforms - Critical importance of oxygen concentration

Treatment of inflammatory bowel disease (IBD) is challenging, with a series of available drugs each helping only a fraction of patients. Patients may face time-consuming drug trials while the disease is active, thus there is an unmet need for biomarkers and assays to predict drug effect. It is well known that the intestinal epithelium is an important factor in disease pathogenesis, exhibiting physical, biochemical and immunologic driven barrier dysfunctions. One promising test system to study effects of existing or emerging IBD treatments targeting intestinal epithelial cells (IECs) is intestinal organoids (“mini-guts”). However, the fact that healthy intestinal epithelium is in a physiologically hypoxic state has largely been neglected, and studies with intestinal organoids are mainly performed at oxygen concentration of 20%. We hypothesized that lowering the incubator oxygen level from 20% to 2% would recapitulate better the in vivo physiological environment of colonic epithelial cells and enhance the translational value of intestinal organoids as a drug testing platform. In the present study we examine the effects of the key IBD cytokines and drug targets TNF/IL17 on human colonic organoids (colonoids) under atmospheric (20%) or reduced (2%) O2. We show that colonoids derived from both healthy controls and IBD-patients are viable and responsive to IBD-relevant cytokines at 2% oxygen. Because chemokine release is one of the important immunoregulatory traits of the epithelium that may be fine-tuned by IBD-drugs, we also examined chemokine expression and release at different oxygen concentrations. We show that chemokine responses to TNF/IL17 in organoids display similarities to inflamed epithelium in IBD-patients. However, inflammation-associated genes induced by TNF/IL17 were attenuated at low oxygen concentration. We detected substantial oxygen-dependent differences in gene expression in untreated as well as TNF/IL17 treated colonoids in all donors. Further, for some of the IBD-relevant cytokines differences between colonoids from healthy controls and IBD patients were more pronounced in 2% O2 than 20% O2. Our results strongly indicate that an oxygen concentration similar to the in vivo epithelial cell environment is of essence in experimental pharmacology

Effect of HIV on the frequency and number of Mycobacterium tuberculosis-specific CD4+ T cells in blood and the airways in latent tuberculosis infection

HIV-1 infection substantially increases the risk of developing tuberculosis (TB). There is extensive depletion of Mycobacterium tuberculosis (M.tuberculosis)-specific CD4+ T cells in blood in early HIV infection, but little is known about responses in the lungs at this stage. Given that mucosal organs are a principal target for HIV-mediated CD4 destruction, we investigated M.tuberculosis-specific responses in bronchoalveolar lavage (BAL), in persons with latent TB infection and untreated HIV-1 co-infection with preserved CD4 counts. M.tuberculosis-specific CD4+ cytokine responses (IFN-, TNF- and IL-2) were discordant in frequency and function between BAL and blood. Responses in BAL were 15-fold lower in HIV-infected compared to uninfected persons (p=0.048), whilst blood responses were 2-fold lower (p=0.006). However, an increase in T cells in the airways in HIV-infected persons resulted in the overall number of M.tuberculosis-specific CD4+ cells in BAL being similar. Our study highlights the important insights gained from studying TB immunity at the site of disease during HIV infection

Genetic diversity in tef [Eragrostis tef (Zucc.) Trotter]

Tef [Eragrostis tef (Zucc.) Trotter] is a cereal crop resilient to adverse climatic and soil conditions, and possessing desirable storage properties. Although tef provides high quality food and grows under marginal conditions unsuitable for other cereals, it is considered to be an orphan crop because it has benefited little from genetic improvement. Hence, unlike other cereals such as maize and wheat, the productivity of tef is extremely low. In spite of the low productivity, tef is widely cultivated by over six million small-scale farmers in Ethiopia where it is annually grown on more than three million hectares of land, accounting for over 30% of the total cereal acreage. Tef, a tetraploid with 40 chromosomes (2n = 4x = 40), belongs to the family Poaceae and, together with finger millet (Eleusine coracana Gaerth.), to the subfamily Chloridoideae. It was originated and domesticated in Ethiopia. There are about 350 Eragrostis species of which E. tef is the only species cultivated for human consumption. At the present time, the gene bank in Ethiopia holds over five thousand tef accessions collected from geographical regions diverse in terms of climate and elevation. These germplasm accessions appear to have huge variability with regard to key agronomic and nutritional traits. In order to properly utilize the variability in developing new tef cultivars, various techniques have been implemented to catalog the extent and unravel the patterns of genetic diversity. In this review, we show some recent initiatives investigating the diversity of tef using genomics, transcriptomics and proteomics and discuss the prospect of these efforts in providing molecular resources that can aid modern tef breeding

Selective reduction of IFN-γ single positive mycobacteria-specific CD4+ T cells in HIV-1 infected individuals with latent tuberculosis infection

HIV-1 is recognized to increase the risk for tuberculosis even before CD4+ T cell deficiency is profound. To better understand how HIV-1 alters immunity to latent tuberculosis, we compared the magnitude and functional profile of mycobacteria-specific CD4+ T cells between HIV-uninfected and HIV-infected individuals, using flow cytometry. In HIV-1 infection, IFN-γ single positive mycobacteria-specific CD4+ T cells were decreased, while the frequency of polyfunctional cells (IFN-γ+IL-2+TNF-α+) remained unchanged. Moreover, the proportion of IFN-γ single positive cells correlated inversely with viral replication. Our results suggest that HIV-1 affects mycobacteria-specific cells differentially, depending on their functional capacity

Genome-wide Phenotypic Profiling Identifies and Categorizes Genes Required for Mycobacterial Low Iron Fitness

Iron is vital for nearly all living organisms, but during infection, not readily available to pathogens. Infectious bacteria therefore depend on specialized mechanisms to survive when iron is limited. These mechanisms make attractive targets for new drugs. Here, by genome-wide phenotypic profiling, we identify and categorize mycobacterial genes required for low iron fitness. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), can scavenge host-sequestered iron by high-affinity iron chelators called siderophores. We take advantage of siderophore redundancy within the non-pathogenic mycobacterial model organism M. smegmatis (Msmeg), to identify genes required for siderophore dependent and independent fitness when iron is low. In addition to genes with a potential function in recognition, transport or utilization of mycobacterial siderophores, we identify novel putative low iron survival strategies that are separate from siderophore systems. We also identify the Msmeg in vitro essential gene set, and find that 96% of all growth-required Msmeg genes have a mutual ortholog in Mtb. Of these again, nearly 90% are defined as required for growth in Mtb as well. Finally, we show that a novel, putative ferric iron ABC transporter contributes to low iron fitness in Msmeg, in a siderophore independent manner

Dysregulation of the immune environment in the airways during HIV infection

HIV-1 increases susceptibility to pulmonary infection and disease, suggesting pathogenesis in the lung. However, the lung immune environment during HIV infection remains poorly characterized. This study examined T cell activation and the cytokine milieu in paired bronchoalveolar lavage (BAL) and blood from 36 HIV-uninfected and 32 HIV-infected participants. Concentrations of 27 cytokines were measured by Luminex, and T cells were phenotyped by flow cytometry. Blood and BAL had distinct cytokine profiles (p=0.001). In plasma, concentrations of inflammatory cytokines like IFN-γ (p=0.004) and TNF-α (p=0.004) were elevated during HIV infection, as expected. Conversely, BAL cytokine concentrations were similar in HIV-infected and uninfected individuals, despite high BAL viral loads (VL; median 48,000 copies/ml epithelial lining fluid). HIV-infected individuals had greater numbers of T cells in BAL compared to uninfected individuals (p=0.007); and BAL VL positively associated with CD4+ and CD8+ T cell numbers (p=0.006 and p=0.0002, respectively) and CXCL10 concentrations (p=0.02). BAL T cells were highly activated in HIV-infected individuals, with nearly 2-3 fold greater frequencies of CD4+CD38+ (1.8-fold; p=0.007), CD4+CD38+HLA-DR+ (1.9-fold; p=0.0006), CD8+CD38+ (2.8-fold; p=0.0006), CD8+HLA-DR+ (2-fold; p=0.022) and CD8+CD38+HLA-DR+ (3.6-fold; p<0.0001) cells compared to HIV-uninfected individuals. Overall, this study demonstrates a clear disruption of the pulmonary immune environment during HIV infection, with readily detectable virus and activated T lymphocytes, which may be driven to accumulate by local chemokines