Suppression of LX ribonuclease in tomato results in a delay of leaf senescence and abscission

Although present in different organisms and conserved in their protein sequence, the biological functions of T2 ribonucleases (RNase) are generally unknown. Tomato (Lycopersicon esculentum) LX is a T2/S-like RNase and its expression is known to be associated with phosphate starvation, ethylene responses, and senescence and programmed cell death. In this study, LX function was investigated using antisense tomato plants in which the LX protein level was reduced. LX protein levels normally become elevated when leaves senesce and antisense inhibition of LX retarded the progression of senescence. Moreover, we observed a marked delay of leaf abscission in LX-deficient plants. This correlated with specific induction of LX protein in the tomato mature abscission zone tissue. LX RNase gene regulation and the consequences of antisense inhibition indicate that LX has an important functional role in both abscission and senescence.     

Overexpression of AtMHX in tobacco causes increased sensitivity to Mg<Superscript>2+</Superscript>, Zn<Superscript>2+</Superscript>, and Cd<Superscript>2+</Superscript> ions,induction of V-ATPase expression,and a reduction in plant size

AtMHX is a vacuolar transporter encoded by a single gene in Arabidopsis. Electrophysiological analysis showed that it exchanges protons with Mg²⁺, Zn²⁺, and Fe²⁺ ions. The physiological impact of AtMHX was examined so far only in tissue-culture grown seedlings of tobacco plants overexpressing this transporter. Here we investigated the impact of AtMHX on growth, response to different metals, and metal accumulation of mature tobacco plants, as well as Arabidopsis plants in which we overexpressed this transporter. The analyses were carried out in hydroponic growth-systems, in which the mineral composition could be effectively controlled, and the metal content of roots could be examined. Transformed tobacco plants showed necrotic lesions and apical burnings upon growth with increased levels of Mg²⁺, Zn²⁺, and Cd²⁺ ions. This suggested that AtMHX can carry in planta not only Mg²⁺ and Zn²⁺ ions, as previously deduced based on observations in tissue-culture, but also Cd²⁺ ions. Transformed plants of both tobacco and Arabidopsis showed a reduction in plant size. However, the overall response of Arabidopsis to AtMHX overexpression was minor. No change was detected in the mineral content of any organ of the transgenic tobacco or Arabidopsis plants. The necrotic lesions in tobacco resembled those seen in plants with perturbed proton balancing, raising the assumption that AtMHX can affect the proton homeostasis of cells. In agreement with this assumption, the transformed tobacco plants had increased expression and activity of the vacuolar H⁺-ATPase. The relative significance of AtMHX for metal and proton homeostasis still has to be elucidated.     

Evaluating automated parameter constraining procedures of neuron models by experimental and surrogate data

Neuron models, in particular conductance-based compartmental models, often have numerous parameters that cannot be directly determined experimentally and must be constrained by an optimization procedure. A common practice in evaluating the utility of such procedures is using a previously developed model to generate surrogate data (e.g., traces of spikes following step current pulses) and then challenging the algorithm to recover the original parameters (e.g., the value of maximal ion channel conductances) that were used to generate the data. In this fashion, the success or failure of the model fitting procedure to find the original parameters can be easily determined. Here we show that some model fitting procedures that provide an excellent fit in the case of such model-to-model comparisons provide ill-balanced results when applied to experimental data. The main reason is that surrogate and experimental data test different aspects of the algorithm’s function. When considering model-generated surrogate data, the algorithm is required to locate a perfect solution that is known to exist. In contrast, when considering experimental target data, there is no guarantee that a perfect solution is part of the search space. In this case, the optimization procedure must rank all imperfect approximations and ultimately select the best approximation. This aspect is not tested at all when considering surrogate data since at least one perfect solution is known to exist (the original parameters) making all approximations unnecessary. Furthermore, we demonstrate that distance functions based on extracting a set of features from the target data (such as time-to-first-spike, spike width, spike frequency, etc.)—rather than using the original data (e.g., the whole spike trace) as the target for fitting—are capable of finding imperfect solutions that are good approximations of the experimental data.     

Magnesium transport and function in plants: the tip of the iceberg

The maintenance of Mg²⁺ homeostasis in the plant is essential for viability. This review describes Mg²⁺ functions and balancing in plants, with special focus on the existing knowledge of the involved transport mechanisms. Mg²⁺ is essential for the function of many cellular enzymes and for the aggregation of ribosomes. Mg²⁺ concentrations also modulate ionic currents across the chloroplast and the vacuolar membranes, and might thus regulate ion balance in the cell and stomatal opening. The significance of Mg²⁺ homeostasis has been particularly established with regard to Mg²⁺'s role in photosynthesis. Mg²⁺ is the central atom of the chlorophyll molecule, and fluctuations in its levels in the chloroplast regulate the activity of key photosynthetic enzymes. Relatively little is known of the proteins mediating Mg²⁺ uptake and transport in plants. The plant vacuole seem to play a key role in Mg²⁺ homeostasis in plant cells. Physiological and molecular evidence indicate that Mg²⁺ entry to the vacuole is mediated by Mg²⁺/H⁺ exchangers. The Arabidopsis vacuolar Mg²⁺/H⁺ exchanger, AtMHX, is highly transcribed at the vascular tissue, apparently most abundantly at the xylem parenchyma. Inclusion of Mg²⁺ ions into the vacuoles of this tissue may determine their partitioning between the various plant organs. Impacts of Mg²⁺ imbalance are described with respect for both plant physiology and for its nutritional value to animal and human.     

Cloning and characterization of a novel Mg(2+)/H(+) exchanger.

Cellular functions require adequate homeostasis of several divalent metal cations, including Mg(2+) and Zn(2+). Mg(2+), the most abundant free divalent cytoplasmic cation, is essential for many enzymatic reactions, while Zn(2+) is a structural constituent of various enzymes. Multicellular organisms have to balance not only the intake of Mg(2+) and Zn(2+), but also the distribution of these ions to various organs. To date, genes encoding Mg(2+) transport proteins have not been cloned from any multicellular organism. We report here the cloning and characterization of an Arabidopsis thaliana transporter, designated AtMHX, which is localized in the vacuolar membrane and functions as an electrogenic exchanger of protons with Mg(2+) and Zn(2+) ions. Functional homologs of AtMHX have not been cloned from any organism. Ectopic overexpression of AtMHX in transgenic tobacco plants render them sensitive to growth on media containing elevated levels of Mg(2+) or Zn(2+), but does not affect the total amounts of these minerals in shoots of the transgenic plants. AtMHX mRNA is mainly found at the vascular cylinder, and a large proportion of the mRNA is localized in close association with the xylem tracheary elements. This localization suggests that AtMHX may control the partitioning of Mg(2+) and Zn(2+) between the various plant organs.     

Selection of O‐negative induced pluripotent stem cell clones for high‐density red blood cell production in a scalable perfusion bioreactor system

ObjectivesLarge‐scale generation of universal red blood cells (RBCs) from O‐negative (O‐ve) human induced pluripotent stem cells (hiPSCs) holds the potential to alleviate worldwide shortages of blood and provide a safe and secure year‐round supply. Mature RBCs and reticulocytes, the immature counterparts of RBCs generated during erythropoiesis, could also find important applications in research, for example in malaria parasite infection studies. However, one major challenge is the lack of a high‐density culture platform for large‐scale generation of RBCs in vitro.Materials and MethodsWe generated 10 O‐ve hiPSC clones and evaluated their potential for mesoderm formation and erythroid differentiation. We then used a perfusion bioreactor system to perform studies with high‐density cultures of erythroblasts in vitro.ResultsBased on their tri‐lineage (and specifically mesoderm) differentiation potential, we isolated six hiPSC clones capable of producing functional erythroblasts. Using the best performing clone, we demonstrated the small‐scale generation of high‐density cultures of erythroblasts in a perfusion bioreactor system. After process optimization, we were able to achieve a peak cell density of 34.7 million cells/ml with 92.2% viability in the stirred bioreactor. The cells expressed high levels of erythroblast markers, showed oxygen carrying capacity, and were able to undergo enucleation.ConclusionsThis study demonstrated a scalable platform for the production of functional RBCs from hiPSCs. The perfusion culture platform we describe here could pave the way for large volume‐controlled bioreactor culture for the industrial generation of high cell density erythroblasts and RBCs.

Human pluripotent stem cell‐derived red blood cells could help alleviate worldwide blood shortages and provide a secure year‐round supply. However, current generation methods lack the necessary scalability. Here, we present the selection of O‐negative hiPSC clones and demonstrate the small‐scale generation of functional erythroblasts in a high‐density perfusion bioreactor system.     

Plant-associated fungi support bacterial resilience following water limitation

Drought disrupts soil microbial activity and many biogeochemical processes. Although plant-associated fungi can support plant performance and nutrient cycling during drought, their effects on nearby drought-exposed soil microbial communities are not well resolved. We used H₂¹⁸O quantitative stable isotope probing (qSIP) and 16S rRNA gene profiling to investigate bacterial community dynamics following water limitation in the hyphospheres of two distinct fungal lineages (Rhizophagus irregularis and Serendipita bescii) grown with the bioenergy model grass Panicum hallii. In uninoculated soil, a history of water limitation resulted in significantly lower bacterial growth potential and growth efficiency, as well as lower diversity in the actively growing bacterial community. In contrast, both fungal lineages had a protective effect on hyphosphere bacterial communities exposed to water limitation: bacterial growth potential, growth efficiency, and the diversity of the actively growing bacterial community were not suppressed by a history of water limitation in soils inoculated with either fungus. Despite their similar effects at the community level, the two fungal lineages did elicit different taxon-specific responses, and bacterial growth potential was greater in R. irregularis compared to S. bescii-inoculated soils. Several of the bacterial taxa that responded positively to fungal inocula belong to lineages that are considered drought susceptible. Overall, H₂¹⁸O qSIP highlighted treatment effects on bacterial community structure that were less pronounced using traditional 16S rRNA gene profiling. Together, these results indicate that fungal–bacterial synergies may support bacterial resilience to moisture limitation.Subject terms: DNA sequencing, Fungal ecology, Stable isotope analysis, Climate-change ecology, Microbial ecology