Ectopic overexpression of a mungbean vacuolar Na+/H+ antiporter gene (VrNHX1) leads to increased salinity stress tolerance in transgenic Vigna unguiculata L. Walp

Salinity is a major threat to sustainable agriculture worldwide. Plant NHX exchangers play an important role in conferring salt tolerance under salinity stress. In this study, a vacuolar Na⁺/H⁺ antiporter gene VrNHX1 (Genbank Accession No. JN656211.1) from mungbean (Vigna radiata) was introduced into cowpea (Vigna unguiculata) by the Agrobacterium tumefaciens-mediated transformation method. Polymerase chain reaction and Southern blot hybridization confirmed the stable integration of VrNHX1 into the cowpea genome. Comparative expression analysis by semi-quantitative RT-PCR revealed higher expression of VrNHX1 in transgenic cowpea plants than wild-type. Under salt stress conditions, T2 transgenic 35S:VrNHX1 cowpea lines exhibited higher tolerance to 200 mM NaCl treatment than wild-type. Furthermore, T2 transgenic 35S:VrNHX1 lines maintained a higher K⁺/Na⁺ ratio in the aerial parts under salt stress and accumulated higher [Na⁺] in roots than wild-type. Physiological analysis revealed lower levels of lipid peroxidation, hydrogen peroxide and oxygen radical production but higher levels of relative water content and proline, ascorbate and chlorophyll contents in T2 transgenic 35S:VrNHX1 lines.     

Flow cytometric analysis of tobacco and cowpea protoplasts infected in vivo and in vitro with alfalfa mosaic virus

Immunofluorescence flow cytometry was used to study the distribution of viral antigen in protoplast populations. Protoplasts were isolated from healthy and alfalfa mosaic virus (AMV) infected tobacco leaves (designated in vivo infected). Furthermore isolated tobacco and cowpea protoplasts were infected in vitro with AMV. The FITC-conjugated antibodies could penetrate formaldehyde fixed protoplasts. The flow cytometric measurements were rapid and reproducible. Comparable immunofluorescence patterns were found for all infected samples (per sample 10⁴ protoplasts were measured). Infectious virus could only be detected in in vivo infected tobacco protoplasts and in in vitro infected cowpea protoplasts.     

Dysregulated phosphatidylinositol signaling promotes endoplasmic-reticulum-stress-mediated intestinal mucosal injury and inflammation in zebrafish

Dysregulated phosphatidylinositol (PI) signaling has been implicated in human gastrointestinal (GI) malignancies and inflammatory states, underlining the need to study pathophysiological roles of PI in an in vivo genetic model. Here, we study the significance of PI in GI pathophysiology using the zebrafish mutant cdipt^hi559, which lacks PI synthesis, and unravel a crucial role of PI in intestinal mucosal integrity and inflammation. The cdipt^hi559 mutants exhibit abnormal villous architecture and disorganized proliferation of intestinal epithelial cells (IECs), with pathologies reminiscent of inflammatory bowel disease (IBD), including apoptosis of goblet cells, abnormal mucosecretion, bacterial overgrowth and leukocyte infiltration. The mutant IECs exhibit vacuolation, microvillus atrophy and impaired proliferation. The cdipt^hi559 gene expression profile shows enrichment of acute phase response signaling, and the endoplasmic reticulum (ER) stress factors hspa5 and xbp1 are robustly activated in the mutant GI tissue. Temporal electron micrographic analyses reveal that PI-deficient IECs undergo sequential ER-Golgi disruption, mitochondrial depletion, macroautophagy and cell death, consistent with chronic ER-stress-mediated cytopathology. Furthermore, pharmacological induction of ER stress by inhibiting protein glycosylation or PI synthase inhibition in leukocyte-specific reporter lines replicates the cdipt^hi559 inflammatory phenotype, suggesting a fundamental role of PI metabolism and ER stress in mucosal inflammation. Antibiotics and anti-inflammatory drugs resolved the inflammation, but not the autophagic necroapoptosis of IECs, suggesting that bacterial overgrowth can exacerbate ER stress pathology, whereas persistent ER stress is sufficient to trigger inflammation. Interestingly, the intestinal phenotype was partially alleviated by chemical chaperones, suggesting their therapeutic potential. Using zebrafish genetic and pharmacological models, this study demonstrates a newly identified link between intracellular PI signaling and ER-stress-mediated mucosal inflammation. The zebrafish cdipt mutants provide a powerful tool for dissecting the fundamental mechanisms of ER-stress-mediated human GI diseases and a platform to develop molecularly targeted therapies.KEY WORDS: Cdipt, Phosphoinositides, IBD, UPR     

Adaptive resistance to PI3Kα-selective inhibitor CYH33 is mediated by genomic and transcriptomic alterations in ESCC cells

Phosphoinositide-3 kinase alpha-specific inhibitors (PI3Kαi) displayed promising potential for the treatment of esophageal squamous cell carcinoma (ESCC) with frequent activation in PI3K signaling. However, acquired resistance is likely to develop and limit the efficacy of PI3Kαi like other targeted therapies. To identify genomic adaptation to PI3Kαi, we applied whole-genome sequencing and detected gene mutation and amplification in four lines of ESCC cells established with adapted resistance to a novel PI3Kαi CYH33. Particularly, HRAS^G12S mutation was found in KYSE180C cells. Overexpression of HRAS^G12S in ESCC parental cells rendered resistance to CYH33. By contrast, down-regulation of HRAS^G12S restored the sensitivity of KYSE180C1 cells to CYH33, and combination of CYH33 and MEK162 displayed synergistic effect against KYSE180C1 cells and xenografts. Furthermore, elevated mTORC1, mitogen-activated protein kinase (MAPK), and c-Myc signaling pathways were found in resistant cells by RNA sequencing and combination of CYH33 and RAD001, MEK162, or OTX015 overcame the resistance to CYH33, which was accompanied with enhanced inhibition on S6, extracellular signal-regulated kinase 1 (ERK), or c-Myc, respectively. Overall, we characterized the adaptations to PI3Kαi in ESCC cells and identified combinatorial regimens that may circumvent resistance.Subject terms: Cancer therapeutic resistance, Oncogenes     

Dynamics of Meloidogyne incognita Virulence to Resistance Genes Rk and Rk2 in Cowpea

The virulence index of three Meloidogyne incognita field isolates to the resistance gene Rk in cowpea was 0%, 75%, and 120%, with the index measured as reproduction on resistant plants as a percentage of the reproduction on susceptible plants. Continuous culture of the 75% virulent isolate on susceptible tomato for more than 5 years (about 25 generations) resulted in virulence decline to about 4%. The rate of the decline in virulence was described by exponential decay, indicating the progressive loss of virulence on a susceptible host. The 120% virulent isolate declined to 90% virulence during five generations on susceptible cowpea. Following virulence decline, the two isolates were compared over 5 years in inoculated field microplots both separately and as a mixture on susceptible, gene Rk, and gene Rk² cowpea plants. At infestation of the plots, the two isolates were 1.2% and 92.0% virulent, respectively, to gene Rk and 0.2% and 8.1% virulent, respectively, to gene Rk². Virulence to gene Rk in the two isolates and in mixture increased under 5 years of continuous Rk cowpea plants to 129% to 172% and under Rk² cowpea plants to 113% to 139 % by year 5. Virulence to gene Rk² increased during continuous cropping with Rk cowpea plants to 42% to 47% and with Rk² cowpea plants to 22% to 48% by year 5. Selection of Rk²-virulence was slower in the isolate with low itt-virulence. The virulence to both genes Rk and Rk² in the mixed population was not different from that in the highly virulent isolate by year 5 of all cropping combinations. Selection of Rk²-virulence on plants with Rk, and vice versa, indicated at least partial overlap of gene specificity between Rk and Rk² with respect to selection of nematode virulence. This observation should be considered when resistance is used in cowpea rotations.     

Differential Responses of CO2 Assimilation,Carbohydrate Allocation and Gene Expression to NaCl Stress in Perennial Ryegrass with Different Salt Tolerance

Little is known about the effects of NaCl stress on perennial ryegrass (Lolium perenne L.) photosynthesis and carbohydrate flux. The objective of this study was to understand the carbohydrate metabolism and identify the gene expression affected by salinity stress. Seventy-four days old seedlings of two perennial ryegrass accessions (salt-sensitive ‘PI 538976’ and salt-tolerant ‘Overdrive’) were subjected to three levels of salinity stress for 5 days. Turf quality in all tissues (leaves, stems and roots) of both grass accessions negatively and significantly correlated with GFS (Glu+Fru+Suc) content, except for ‘Overdrive’ stems. Relative growth rate (RGR) in leaves negatively and significantly correlated with GFS content in ‘Overdrive’ (P<0.01) and ‘PI 538976’ (P<0.05) under salt stress. ‘Overdrive’ had higher CO₂ assimilation and F_v/F_m than ‘PI 538976’. Intercellular CO₂ concentration, however, was higher in ‘PI 538976’ treated with 400 mM NaCl relative to that with 200 mM NaCl. GFS content negatively and significantly correlated with RGR in ‘Overdrive’ and ‘PI 538976’ leaves and in ‘PI 538976’ stems and roots under salt stress. In leaves, carbohydrate allocation negatively and significantly correlated with RGR (r² = 0.83, P<0.01) and turf quality (r² = 0.88, P<0.01) in salt-tolerant ‘Overdrive’, however, the opposite trend for salt-sensitive ‘PI 538976’ (r² = 0.71, P<0.05 for RGR; r² = 0.62, P>0.05 for turf quality). A greater up-regulation in the expression of SPS, SS, SI, 6-SFT gene was observed in ‘Overdrive’ than ‘PI 538976’. A higher level of SPS and SS expression in leaves was found in ‘PI 538976’ relative to ‘Overdrive’. Accumulation of hexoses in roots, stems and leaves can induce a feedback repression to photosynthesis in salt-stressed perennial ryegrass and the salt tolerance may be changed with the carbohydrate allocation in leaves and stems.     

Cowpea N rhizodeposition and its below-ground transfer to a co-existing and to a subsequent millet crop on a sandy soil of the Sudano-Sahelian eco-zone

Nitrogen (N) rhizodeposition by cowpea (Vigna unguiculata (L.) Walp) is potentially a large N source in cropping systems of Sub-Saharan Africa. A field experiment was conducted to measure cowpea N rhizodeposition under the conditions of the Sudano-Sahelian zone using direct ¹⁵N labelling techniques to trace the amount of deposition and its transfer to associated and subsequent crops. Half of the total cowpea crop N was located below-ground at plant maturity, which exceeded 20 kg?N ha^?1 when intercropped with millet. Only 15% of the below-ground cowpea N was recovered in roots, while 85% was found in the rhizodeposited pools. The experiment demonstrated that direct below-ground N transfer occurred from cowpea to millet in intercrop at a rate of 2 kg?N ha^?1 over the growing season. Forty percent of the 25 kg below-ground N that the cowpea crop left at harvest were identifiable in the top 0.30 m soil in the beginning of the next planting season 7 months later; a pool still present at the end of that second season. Thus, the subsequent crop of millet (Pennisetum glaucum (L.) R. Br.) only recovered 2.5 kg?N ha^?1 from the below-ground cowpea pre-crop N during this growth season. The role and potential of cowpea as N provider has been underestimated in the past by ignoring the large proportion of N contained in its rhizodeposits. However, information is needed to determine how losses of the rhizodeposited N can be minimized to fully harness the potential of cowpea as N provider in agro-ecosystems of the region.     

Leaf traits variation in Sesleria nitida growing at different altitudes in the Central Apennines

Global climate change may act as a potent agent of natural selection within species with Mediterranean mountain ecosystems being particularly vulnerable. The aim of this research was to analyze whether the phenotypic plasticity of Sesleria nitida Ten. could be indicative of its future adaptive capability to global warming. Morphological, anatomical, and physiological leaf traits of two populations of S. nitida growing at different altitudes on Mount Terminillo (Italy) were analyzed. The results showed that leaf mass per unit leaf area, leaf tissue density, and total leaf thickness were 19, 3, and 31% higher in leaves from the population growing at 1,895 m a.s.l. (B site) than in leaves from the population growing at 1,100 m a.s.l. (A site), respectively. Net photosynthetic rate (P ^N) and respiration rate (R _D) peaked in June in both A and B leaves [9.4 ± 1.3 μmol(CO₂) m^?2 s^?1 and 2.9 ± 0.9 μmol(CO₂) m^?2 s^?1, respectively] when mean air temperature was 16 ± 2°C. R _D/P _N was higher in B than in A leaves (0.35 ± 0.07 and 0.21 ± 0.03, respectively, mean of the study period). The mean plasticity index (PI = 0.24, mean of morphological, anatomical, and physiological leaf traits) reflected S. nitida adaptability to the environmental stress conditions at different altitudes on Mount Terminillo. Moreover, the leaf key traits of the two populations can be used to monitor wild populations over a long term in response to global change.     

Introduction and Expression of Cowpea Trypsin Inhibitor (CpTI) Gene in Transgenic Tobacco

A trypsin/chymotrypsin inhibitor (CpTI) was purified from mature cowpea seeds. A full length cDNA clone encoding the trypsin/chymotrypsin inhibitor protein was isolated from a cDNA library that was constructed using poly(A⁺) RNA from developing seeds. Tobacco protoplasts were transformed with a construct composed of CaMV 35S promoter, NPTII gene, CpTI gene, and NOS terminator, using polyethylene glycol (PEG)-mediated direct gene transfer. The CpTI gene has been integrated into the plant genome, and the expressed CpTI protein from transgenic plants was catalytically active.     

1H NMR Spectroscopic and Histopathological Studies on Propyleneimine-Induced Renal Papillary Necrosis in the Rat and the Multimammate Desert Mouse (Mastomys natalensis)

The renal papillary toxin, propyleneimine (PI), was administered at 20 or 30 μl/kg i.p. to male Sprague Dawley (SD) rats (n = 5), Fischer 344 (F344) rats (n = 4), and to multimammate desert mice (Mastomys natalensis, n = 4). Urine was collected at time points up to 4 days p.d. and the toxicological response of the different animal models to PI compared using ¹H NMR spectroscopy of urine, renal histopathology, and urinary assays for alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and γ-glutamyl transpeptidase (γGT). The renal papillae of both F344 and SD rats showed extensive necrotic lesions 4 days post-dosing and in some cases sloughing of the papilla. However, only slight renal papillary necrosis (RPN) was observed in Mastomys treated with 20 μl/kg PI and, although slight to moderate damage was observed at 30 μl/kg, PI-treated Mastomys showed substantially less RPN than either group of PI-treated rats. ¹H NMR urinalysis showed that PI treatment caused a decrease in the urinary concentrations of succinate (0–24 hr p.d.) and citrate (24–48 hr p.d.) and an increase in creatine (0–48 hr p.d.) in all animal models. Trimethylamine-N-oxide (24–48 hr) and 2-oxoglutarate concentrations decreased initially following the administration of PI and then rose above control levels. The ¹H NMR-detected urinary biochemical effects of PI in all three models were similar. However, taurine concentrations were elevated in the urine of Mastomys following PI treatment, perhaps indicating a degree of liver damage, whereas taurinuria was not seen in either SD or F344 rats. These observations are discussed in relation to the potential mechanism of PI-toxicity.     

Symbiotic functioning and bradyrhizobial biodiversity of cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis>L. Walp.) in Africa

Background  

Cowpea is the most important food grain legume in Sub-Saharan Africa. However, no study has so far assessed rhizobial biodiversity and/or nodule functioning in relation to strain IGS types at the continent level. In this study, 9 cowpea genotypes were planted in field experiments in Botswana, South Africa and Ghana with the aim of i) trapping indigenous cowpea root-nodule bacteria (cowpea "rhizobia") in the 3 countries for isolation, molecular characterisation using PCR-RFLP analysis, and sequencing of the 16S - 23S rDNA IGS gene, ii) quantifying N-fixed in the cowpea genotypes using the ¹⁵N natural abundance technique, and iii) relating the levels of nodule functioning (i.e. N-fixed) to the IGS types found inside nodules.     

(h)GR,beans and drought stress

Reactive oxygen species (ROS) are commonly found in plants as natural by-products of the metabolism but their production is greatly enhanced under abiotic stresses. Particular metabolites and enzymes belonging to the ascorbate-glutathione cycle are able to scavenge these deleterious molecules and modulate the cellular redox-status. In the March issue of Journal of Plant Physiology, we have shown that drought stress induces a raise in glutathione reductase (GR) activity and gene expression that could be related to the intensity of the drought treatment and the drought susceptibility of the bean cultivar (cowpea and/or common bean). In the present addendum we show new data on GR specific activity during progressive drought stress and recovery of the drought-susceptible bean cultivar which can be related to the previously found dual-targeted GR gene expression. Furthermore, since in leguminous plants homoglutathione (hGSH) is generally the most abundant low molecular weight thiol form, we discuss on the occurrence of a (homo)glutathione reductase activity in beans.Key words: common bean, cowpea, drought stress, (homo)glutathione, (homo)glutathione reductase, legumes, Phaseolus vulgaris, recovery, Vigna unguiculataDrought stress is the most common form of abiotic stress and plants are likely to encounter periods of water shortage at least once in their lifecycle. One of the inevitable consequences of drought stress is enhanced reactive oxygen species (ROS) production which will imbalance the cellular redox-status. This shift in the steady-state cellular redox-status is currently believed to have an initial signaling effect, triggering adaptive/defense responses (reviewed in ref. ¹). However, in order to avoid oxidative stress, enhanced ROS production must be kept under tight control by the cellular antioxidant machinery. Glutathione reductase (GR; EC 1.6.4.2) is a major cellular antioxidant enzyme. It belongs to the ascorbate-glutathione cycle and it is ubiquitously found in all cellular compartments.² Using several bean plants (common bean, Phaseolus vulgaris and cowpea, Vigna unguiculata) as a model system to study drought responses and relate them to the degree of drought tolerance and/or susceptibility, we have shown that severe drought stress leads to an enhanced cellular GR activity related to the drought susceptibility of the cultivar.^3,4 Similar results have also been found in a wheat system composed of drought-tolerant and susceptible cultivars.⁵ Regarding the more susceptible cultivar of our bean system (P.v. Carioca) and under severe drought stress (S3, Ψ_w = −2.0 MPa; RWC = 50.9%), total leaf GR activity was raised to approximately 200% when compared to control plants (C, Ψ_w = −0.5 MPa; RWC = 95.3%) (Fig. 1). This could translate a higher degree of oxidative stress due to enhanced ROS production in drought-susceptible cultivars than in drought-tolerant ones. In fact it has been shown that at the cellular level these drought-susceptible bean plants suffer a higher degree of membrane integrity loss when compared to the drought-tolerant.^6–8 This can be related to enhanced ROS production since proteins and lipids of cellular membranes are main targets of ROS peroxidation.⁹Open in a separate windowFigure 1GR-specific activity and relative water content (RWC%) in common bean (Phaseolus vulgaris) ‘Carioca’ leaves. GR-specific activity and RWC were measured in control, severely drought stressed and on rewatered plants. Values are means ± s.d. of three to five independent measurements. GR activity was assayed by following the oxidation of NADPH (decrease in absorbance at 340 nm) and expressed in nmoles min⁻¹ mg⁻¹ protein. RWC was measured according to Weatherley.²⁴ Control plants (C), Ψ_w = −0.5 MPa; severely droughted plants (S3), Ψ_w = −2.0 MPa; 24 h rehydrated plant (24R), Ψ_w = −0.5 MPa; 48 h rehydrated plant (48R), Ψ_w = −0.5 MPa.Considering the responses to drought at the whole-plant level, susceptible and tolerant beans also differ. In fact, drought-tolerant bean cultivars present a water-saving strategy by precocious control of stomatal opening which allows for photosynthetic activity to proceed at lower leaf Ψ_w.^10–12 The maintenance of stomatal opening and photosynthetic activity during drought stress results in lower ROS production by photorespiration and/or the Mehler reaction as opposed to complete stomatal closure where inhibition of CO₂ fixation occurs.¹ Indeed, in the drought-tolerant cowpea cultivar, total GR activity was found constant throughout the progressive drought treatment.³After 24 h rewatering (24R, Ψ_w = −0.5 MPa; RWC = 88.1%), from a moderate water stress: Ψ_w = −1.5 MPa; RWC = 69.2%, GR activity in the drought-susceptible bean cultivar was further raised by ∼270% as compared to control (Fig. 1). This enhanced GR activity can be directly related to the upregulation of the dual-targeted form of the bean GR gene (PvGRdt) (targeted to both chloroplasts and mitochondria) observed on rewatering of this drought-susceptible cultivar.⁴ In fact a significant upregulation of PvGRdt was detected as soon as 6 h after rewatering and the high expression levels were maintained up to 24 h after rewatering to then decrease at 48 h after rewatering.⁴ Hence in the drought-susceptible cultivar it seems that the dual-targeted form is more responsive to drought and rewatering than the cytosolic form. The same pattern was also seen on the less tolerant cowpea cultivar.³ Enhanced dual-targeted GR expression (and GR activity) under drought could be related to enhanced ROS production at those particular cellular compartments (mitochondria and chloroplasts). In fact, under a PEG-induced water deficit, drought-susceptible bean plants showed a higher number of disorganized chloroplasts when compared to the drought-tolerant,⁷ indicating that these organelles experienced oxidative stress during the treatment.The GR enzyme is responsible for the reduction of glutathione disulfide (GSSG) to glutathione (GSH) using NADPH, and not only it keeps glutathione in the reduced state but it is also responsible for the maintenance of the cellular GSH:GSSG ratio.^13,14 Interestingly, in leguminous plants such as the present bean plants, homoglutathione (hGSH) replaces completely or in part, glutathione (GSH). Homoglutathione has been shown to be the most abundant tripeptide in common bean and pea (Pisum sativum),¹⁵ in soybean (Glycine max),¹⁶ and in Lotus japonicus.¹⁷ The synthesis of hGSH proceeds through two ATP-dependent steps, the first step being common with GSH synthesis, the second step adding a β-Alanine instead of a Glycine to form the tripeptide. In the case of cowpea, which was up to now considered to be a non hGSH producing legume,¹⁵ we have recently detected the presence of a homoglutathione synthetase (hGSHS) mRNA and activity (MH Cruz de Carvalho, J Brunet, A Lameta, Y Zuily-Fodil and D Contour-Ansel, unpublished data).Besides the chemical difference of the two thiol tripeptides, many of the roles ascribed to GSH are also performed by hGSH,^18,19 particularly the control of the cellular redox status and ROS scavenging.²⁰ However, the presence of hGSH questions on the occurrence of a homoglutathione reductase (hGR). A role for hGR as a detoxifying enzyme of the ascorbate-glutathione cycle has been suggested, using hGSSG (oxidized homoglutathione) instead of GSSG (oxidized glutathione), thus maintaining the cellular homoglutathione pool in the reduced state and acting as an antioxidative molecule in these plants.^21,22 It can hence be suggested that in beans and other leguminous plants where both thiols co-exist (hGSH and GSH), the (h)GR enzyme will act as either a GR or a hGR in accordance to the thiol utilized in the ascorbate-glutathione cycle.     

Alkylsulfonamide-containing quinazoline derivatives as potent and orally bioavailable PI3Ks inhibitors

Phosphoinositide 3-kinases (PI3Ks) are regarded as promising targets for treatment of various cancers due to their roles in regulating cell proliferation, differentiation, migration, and survival. Here we report our efforts to develop potent and orally bioavailable PI3K inhibitors for the treatment of cancers. The alkylsulfonamide-containing quinazoline derivatives A1–A18 significantly inhibited PI3Kα, and cell proliferation among HCT-116, MCF-7 and SU-DHL-6 cell lines. The optimal compound A1 displayed potent inhibitory activity against PI3Kα (IC₅₀ = 4.5 nM), PI3Kβ (IC₅₀ = 4.5 nM), PI3Kγ (IC₅₀ = 4.5 nM), PI3Kδ (IC₅₀ = 4.5 nM) and significantly inhibited the growth of HCT-116, MCF-7 and SU-DHL-6 cell lines with IC₅₀ values of 0.82 µM, 0.99 µM and 0.19 µM, respectively. Western blot analysis demonstrated A1 significantly suppressed the phosphorylation of AKT^S473 in a dose-dependent manner. Furthermore, A1 could markedly inhibit cancer growth at the dose of 25 mg/kg in nude mouse HCT-116 xenograft model in vivo without causing significant weight loss or toxicity.     

An evolutionary approach uncovers a diverse response of tRNA 2-thiolation to elevated temperatures in yeast

Chemical modifications of transfer RNA (tRNA) molecules are evolutionarily well conserved and critical for translation and tRNA structure. Little is known how these nucleoside modifications respond to physiological stress. Using mass spectrometry and complementary methods, we defined tRNA modification levels in six yeast species in response to elevated temperatures. We show that 2-thiolation of uridine at position 34 (s²U₃₄) is impaired at temperatures exceeding 30°C in the commonly used Saccharomyces cerevisiae laboratory strains S288C and W303, and in Saccharomyces bayanus. Upon stress relief, thiolation levels recover and we find no evidence that modified tRNA or s²U₃₄ nucleosides are actively removed. Our results suggest that loss of 2-thiolation follows accumulation of newly synthesized tRNA that lack s²U₃₄ modification due to temperature sensitivity of the URM1 pathway in S. cerevisiae and S. bayanus. Furthermore, our analysis of the tRNA modification pattern in selected yeast species revealed two alternative phenotypes. Most strains moderately increase their tRNA modification levels in response to heat, possibly constituting a common adaptation to high temperatures. However, an overall reduction of nucleoside modifications was observed exclusively in S288C. This surprising finding emphasizes the importance of studies that utilize the power of evolutionary biology, and highlights the need for future systematic studies on tRNA modifications in additional model organisms.     

Novel cell line models to study mechanisms and overcoming strategies of proteasome inhibitor resistance in multiple myeloma

Experimental data on resistance mechanisms of multiple myeloma (MM) to ixazomib (IXA), a second-generation proteasome inhibitor (PI), are currently lacking. We generated MM cell lines with a 10-fold higher resistance to IXA as their sensitive counterparts, and observed cross-resistance towards the PIs carfilzomib (CFZ) and bortezomib (BTZ). Analyses of the IXA-binding proteasome subunits PSMB5 and PSMB1 show increased PSMB5 expression and activity in all IXA-resistant MM cells, and upregulated PSMB1 expression in IXA-resistant AMO1 cells. In addition, sequence analysis of PSMB5 revealed a p.Thr21Ala mutation in IXA-resistant MM1.S cells, and a p.Ala50Val mutation in IXA-resistant L363 cells, whereas IXA-resistant AMO1 cells lack PSMB5 mutations. IXA-resistant cells retain their sensitivity to therapeutic agents that mediate cytotoxic effects via induction of proteotoxic stress. Induction of ER stress and apoptosis by the p97 inhibitor CB-5083 was strongly enhanced in combination with the PI3Kα inhibitor BYL-719 or the HDAC inhibitor panobinostat suggesting potential therapeutic strategies to circumvent IXA resistance in MM. Taken together, our newly established IXA-resistant cell lines provide first insights into resistance mechanisms and overcoming treatment strategies, and represent suitable models to further study IXA resistance in MM.     

Metabolism and translocation of allantoin in ureide-producing grain legumes

Transfer of the nitrogen and carbon of allantoin to amino acids and protein of leaflets, stems and petioles, apices, peduncles, pods, and seeds of detached shoots of nodulated cowpea (Vigna unguiculata L. Walp. cv. Caloona) plants was demonstrated following supply of [2-¹⁴C], [1,3-¹⁵N]allantoin in the transpiration stream. Throughout vegetative and reproductive growth all plant organs showed significant ureolytic activity and readily metabolized [2-¹⁴C]allantoin to ¹⁴CO₂. A metabolic pathway for ureide nitrogen utilization via allantoic acid, urea, and ammonia was indicated. Levels of ureolytic activity in extracts from leaves and roots of nodulated cowpea were consistently maintained at higher levels than in non-nodulated, NO₃⁻ grown plants.

[¹⁴C]Ureides were recovered in extracts of aphids (Aphis craccivora and Macrosiphum euphorbieae) feeding at different sites on cowpea plants supplied with [2-¹⁴C]allantoin through the transpiration stream or to the upper surface of single leaflets. The data indicated that the ureides were effectively transferred from xylem or leaf mesophyll to phloem, and then translocated in phloem to fruits, apices, and roots.

     

Root to shoot communication and abscisic acid in calreticulin (CR) gene expression and salt-stress tolerance in grafted diploid potato clones

Potato is an important world crop but its cultivation is relatively limited by its sensitivity to salt-stress. Auto- and hetero-grafting was used to examine the effect of rootstock and abscisic acid (ABA) on expression of the Ca²⁺-storage protein calreticulin (CR) and salt-stress tolerance in potato. Sibling-selected diploid clones of potato (S. tuberosum) were utilized that are distinguished by differential root Na⁺ absorption; including type: late-maturing, LM and excluding type, early-maturing, EM under salt treatment; salt-stress sensitivity (S/T, sensitive or tolerant); and abscisic acid production (AD/AN, ABA-deficient or-normal sibling lines). CR expression, osmotic potential (OP) and leaf Ca²⁺ were measured at the end of a 5 days NaCl stress treatment applied at tuber initiation. Increased CR expression was induced by NaCl stress and associated with salt tolerance in early-maturing tolerant (EMT) and late-maturing tolerant (LMT) clones with higher levels of CR in LMT compared to the EMT clone. Early-maturing sensitive (EMS) clone salt tolerance increased when grafted onto LMT but not onto EMT rootstocks. EMS scions maintained less negative leaf OP when grafted onto LMT rootstocks than grafting onto the EMT rootstock. Exogenous ABA application induced a less negative upper leaf OP in the salt-stress sensitive AD clone but not in the AN clone. AD clones were characterized by low CR levels, which did not increase after stress. However, grafting the AD clone onto LMT increased CR expression in the AD portion of the graft combination. Salt-stress induced CR expression and is positively associated with the presence of ABA and the salt-stress tolerant phenotypes. Both, elevation in CR expression and salt tolerance in the tolerant rootstocks, were translocated to sensitive scions although highest permeation depended on the LM type. Calreticulin expression appears to be involved in ABA-induced salt tolerance and both salt-stress tolerance and CR expression appear to be regulated by the roots.     

Pathways of Nitrogen Assimilation in Cowpea Nodules Studied using N(2) and Allopurinol

In the presence of 0.5 millimolar allopurinol (4-hydroxypyrazolo [3,4-d]pyrimidine), an inhibitor of NAD:xanthine oxidoreductase (EC 1.2.3.2), intact attached nodules of cowpea (Vigna unguiculata L. Walp. cv Vita 3) formed [¹⁵N]xanthine from ¹⁵N₂ at rates equivalent to those of ureide synthesis, confirming the direct assimilation of fixed nitrogen into purines. Xanthine accumulated in nodules and was exported in increasing amounts in xylem of allopurinol-treated plants. Other intermediates of purine oxidation, de novo purine synthesis, and ammonia assimilation did not increase and, over the time course of experiments (4 hours), allopurinol had no effect on nitrogenase (EC 1.7.99.2) activity. Negligible ¹⁵N-labeling of asparagine from ¹⁵N₂ was observed, suggesting that the significant pool (up to 14 micromoles per gram of nodule fresh weight) of this amide in cowpea nodules was not formed directly from fixation but may have accumulated as a consequence of phloem delivery.     

Stress-induced Synthesis of Phosphatidylinositol 3-Phosphate in Mycobacteria

Phosphoinositides play key roles in regulating membrane dynamics and intracellular signaling in eukaryotic cells. However, comparable lipid-based signaling pathways have not been identified in bacteria. Here we show that Mycobacterium smegmatis and other Actinomycetes bacteria can synthesize the phosphoinositide, phosphatidylinositol 3-phosphate (PI3P). This lipid was transiently labeled with [³H]inositol. Sensitivity of the purified lipid to alkaline phosphatase, headgroup analysis by high-pressure liquid chromatography, and mass spectrometry demonstrated that it had the structure 1,2-[tuberculostearoyl, octadecenoyl]-sn-glycero 3-phosphoinositol 3-phosphate. Synthesis of PI3P was elevated by salt stress but not by exposure to high concentrations of non-ionic solutes. Synthesis of PI3P in a cell-free system was stimulated by the synthesis of CDP-diacylglycerol, a lipid substrate for phosphatidylinositol (PI) biosynthesis, suggesting that efficient cell-free PI3P synthesis is dependent on de novo PI synthesis. In vitro experiments further indicated that the rapid turnover of this lipid was mediated, at least in part, by a vanadate-sensitive phosphatase. This is the first example of de novo synthesis of PI3P in bacteria, and the transient synthesis in response to environmental stimuli suggests that some bacteria may have evolved similar lipid-mediated signaling pathways to those observed in eukaryotic cells.     

Characterization of Tn5-induced symbiotically defective mutants of cowpea rhizobia

Symbiotically defective mutants of cowpea rhizobia strain IRC256 were isolated by random Tn5 mutagenesis and characterized. One auxotroph (MS1) requiring adenine and thiamine was a non-nodulating mutant (Nod⁻) and three prototrophic mutants were Nod⁺ Fix⁻ which formed small and ineffective nodules on cowpeas (Vigna unguiculata). Acetylene reduction activity of the Nod⁺ Fix⁻ mutants was reduced to 80–94% of that of the wild-type strain. The non-nodulating mutant (MS1) induced root-hair curling but did not show any nodule initiation or nodule development. Ultrastructural examination of nodules formed by Fix⁻ mutants showed that these contained few bacteroids, indicating either early senescence or a reduction in bacterial release into the cytoplasm of the host cell. DNA hybridization of total DNAs from a representative number of Tn5 mutants showed that each of them had one copy of the transposon Tn5 which was randomly inserted into the genome of cowpea rhizobia.