Natural diversity uncovers P5CS1 regulation and its role in drought stress tolerance and yield sustainability in barley.

Proline accumulation is one of the major responses of plants to many abiotic stresses. However, the significance of proline accumulation for drought stress tolerance remains enigmatic in crop plants. First, we examined the natural variation of the pyrolline-5-carboxylate synthase (P5CS1) among 49 barley genotypes. Allele mining identified a previously unknown allelic series that showed polymorphisms at 42 cis-elements across the P5CS1 promoter. Selected haplotypes had quantitative variation in P5CS1 gene expression and proline accumulation, putatively influenced by both  abscisic acid-dependent and independent pathways under drought stress. Next, we introgressed the P5CS1 allele from a high proline accumulating wild barley accession ISR42-8 into cultivar Scarlett developing a near-isogenic line (NIL-143). NIL-143 accumulated higher proline concentrations than Scarlett under drought stress at seedling and reproductive stages. Under drought stress, NIL-143 showed less pigment damage, sustained photosynthetic health, and higher drought stress recovery compared to Scarlett. Further, the drought-induced damage to yield-related traits, mainly thousand-grain weight, was lower in NIL-143 compared with Scarlett in field conditions. Our data uncovered new variants of the P5CS1 promoter and the significance of the increased proline accumulation regulated by the P5CS1 allele of ISR42-8 in drought stress tolerance and yield stability in barley.

Conditioned media from the renal cell carcinoma cell line 786.O drives human blood monocytes to a monocytic myeloid-derived suppressor cell phenotype.

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells critical in mediating immune suppression in cancer patients. To develop an in vitro assay system that functionally mimics the tumor microenvironment, we cultured human monocytes with conditioned media from several cancer cell lines. Conditioned media from five tumor cell lines induced survival and differentiation of monocytes into cells characteristically similar to macrophages and MDSCs. Notably, media from the 786.O renal cell carcinoma line induced monocytes to acquire a monocytic MDSC phenotype characterized by decreased HLA-DR expression, increased nitric oxide production, enhanced proliferation, and ability to suppress autologous CD3+ T cell proliferation. We further demonstrated that these in vitro MDSCs are phenotypically and functionally similar to patient-derived MDSCs. Inhibitors of STAT3, CK2, and GM-CSF resulted in partial reversal of the MDSC phenotype. MDSCs generated in vitro from 786.O tumor conditioned media represent a platform to identify potential therapeutics that inhibit MDSC activities.

Induction of electrophile-responsive element (EpRE)-mediated gene expression by tomato extracts in vitro.

The market for food products with additional health benefits is increasing rapidly and tools for identification of bio-functional characteristics of food items are essential. To facilitate the detection of beneficial effects of tomato on gene expression, methods to prepare tomato extracts suitable to test in the EpRE LUX assay and other cell-based reporter gene assays for health-related bioactivity mechanisms, were developed. An isoprenoid-containing chloroform extract of tomato fruit and most individual isoprenoids did not induce electrophile-responsive element (EpRE)-mediated gene expression. A semi-polar extract of tomato fruits, enzymatically hydrolysed to remove the glycosyl residues from the phenolic ingredients was able to induce EpRE-mediated luciferase expression at both mRNA and protein level, which might be partly due to the presence of quercetin, kaempferol, naringenin and naringenin chalcone. It was concluded that induction of EpRE-regulated genes, such as detoxifying phase II and antioxidant enzymes, may contribute to the beneficial health effects of tomato.

Seed-specific elevation of non-symbiotic hemoglobin AtHb1: beneficial effects and underlying molecular networks in Arabidopsis thaliana.

BACKGROUND: Seed metabolism is dynamically adjusted to oxygen availability. Processes underlying this auto-regulatory mechanism control the metabolic efficiency under changing environmental conditions/stress and thus, are of relevance for biotechnology. Non-symbiotic hemoglobins have been shown to be involved in scavenging of nitric oxide (NO) molecules, which play a key role in oxygen sensing/balancing in plants and animals. Steady state levels of NO are suggested to act as an integrator of energy and carbon metabolism and subsequently, influence energy-demanding growth processes in plants. RESULTS: We aimed to manipulate oxygen stress perception in Arabidopsis seeds by overexpression of the non-symbiotic hemoglobin AtHb1 under the control of the seed-specific LeB4 promoter. Seeds of transgenic AtHb1 plants did not accumulate NO under transient hypoxic stress treatment, showed higher respiratory activity and energy status compared to the wild type. Global transcript profiling of seeds/siliques from wild type and transgenic plants under transient hypoxic and standard conditions using Affymetrix ATH1 chips revealed a rearrangement of transcriptional networks by AtHb1 overexpression under non-stress conditions, which included the induction of transcripts related to ABA synthesis and signaling, receptor-like kinase- and MAP kinase-mediated signaling pathways, WRKY transcription factors and ROS metabolism. Overexpression of AtHb1 shifted seed metabolism to an energy-saving mode with the most prominent alterations occurring in cell wall metabolism. In combination with metabolite and physiological measurements, these data demonstrate that AtHb1 overexpression improves oxidative stress tolerance compared to the wild type where a strong transcriptional and metabolic reconfiguration was observed in the hypoxic response. CONCLUSIONS: AtHb1 overexpression mediates a pre-adaptation to hypoxic stress. Under transient stress conditions transgenic seeds were able to keep low levels of endogenous NO and to maintain a high energy status, in contrast to wild type. Higher weight of mature transgenic seeds demonstrated the beneficial effects of seed-specific overexpression of AtHb1.

Antisense inhibition of the plastidial glucose-6-phosphate/phosphate translocator in Vicia seeds shifts cellular differentiation and promotes protein storage.

The glucose-6-phosphate/phosphate translocator (GPT) acts as an importer of carbon into the plastid. Despite the potential importance of GPT for storage in crop seeds, its regulatory role in biosynthetic pathways that are active during seed development is poorly understood. We have isolated GPT1 from Vicia narbonensis and studied its role in seed development using a transgenic approach based on the seed-specific legumin promoter LeB4. GPT1 is highly expressed in vegetative sink tissues, flowers and young seeds. In the embryo, localized upregulation of GPT1 at the onset of storage coincides with the onset of starch accumulation. Embryos of transgenic plants expressing antisense GPT1 showed a significant reduction (up to 55%) in the specific transport rate of glucose-6-phosphate as determined using proteoliposomes prepared from embryos. Furthermore, amyloplasts developed later and were smaller in size, while the expression of genes encoding plastid-specific translocators and proteins involved in starch biosynthesis was decreased. Metabolite analysis and stable isotope labelling demonstrated that starch biosynthesis was also reduced, although storage protein biosynthesis increased. This metabolic shift was characterized by upregulation of genes related to nitrogen uptake and protein storage, morphological variation of the protein-storing vacuoles, and a crude protein content of mature seeds of transgenics that was up to 30% higher than in wild-type. These findings provide evidence that (1) the prevailing level of GPT1 abundance/activity is rate-limiting for the synthesis of starch in developing seeds, (2) GPT1 exerts a controlling function on assimilate partitioning into storage protein, and (3) GPT1 is essential for the differentiation of embryonic plastids and seed maturation.

In vivo confocal microscopy: increased conjunctival or episcleral leukocyte adhesion in patients who wear contact lenses with lower oxygen permeability (Dk) values.

PURPOSE: Contact lens wear is known to threaten the health of the ocular surface. In vivo confocal microscopy (IVCM) to visualize leukocyte rolling and extravasation in inflammation was recently described. We tested the hypothesis that contact lens wear is associated with measurable inflammation in superficial vessels. METHODS: Leukocyte rolling and sticking (hallmarks of the inflammatory process) were recorded by IVCM. IVCM was performed on conjunctival or episcleral blood vessels bilaterally on 55 contact lens wearers (15 male, 40 female) and 22 non-contact lens wearers (8 male, 14 female). Data were analyzed in 2 ways. Considering each vessel as an independent variable resulted in 132 analyzable vessel segments (13 daily disposable contact lenses, 67 traditional contact lenses, 14 rigid gas-permeable lenses, and 38 controls). Considering each subject as an independent variable resulted in analyzable data for 47 subjects (5 daily disposable contact lens wearers, 22 traditional contact lens wearers, 5 rigid contact lens wearers, and 15 control patients). Free-flowing, sticking, and rolling cells were counted in the vessels. Multiple parameters including mean flow velocity, shear rate, rolling cells/mm/min, and sticking cells/mm were calculated. RESULTS: We found no significant difference in leukocyte adhesion between control patients and patients wearing daily disposable, traditional disposable, or rigid gas-permeable lenses in both types of statistical analyses. However, the data regarding vessel segments as an independent variable show that there were more rolling cells in patients who wore contact lenses with oxygen permeability values (Dk) less than 10 as compared to those who wore contact lenses with oxygen permeability values greater than 16 (P < 0.01) or compared to controls (P < 0.01). CONCLUSION: IVCM is a novel, powerful technique to recognize a critical but subclinical component of inflammation. Although our data indicate that contact lens wear does not markedly increase rolling and sticking of leukocytes in conjunctival or episcleral vessels, there may be subclinical inflammation in association with lenses with the lowest oxygen permeability.