Insight into multi-site mechanisms of glycosyl transfer in (1-->4)beta-D-glycans provided by the cereal mixed-linkage (1-->3),(1-->4)beta-D-glucan synthase.

Synthases of cellulose, chitin, hyaluronan, and all other polymers containing (1-->4)beta-linked glucosyl, mannosyl and xylosyl units have overcome a substrate orientation problem in catalysis because the (1-->4)beta-linkage requires that each of these sugar units be inverted nearly 180 degrees with respect to its neighbors. We and others have proposed that this problem is solved by two modes of glycosyl transfer within a single catalytic subunit to generate disaccharide units, which, when linked processively, maintain the proper orientation without rotation or re-orientation of the synthetic machinery in 3-dimensional space. A variant of the strict (1-->4)beta-D-linkage structure is the mixed-linkage (1-->3),(1-->4)beta-D-glucan, a growth-specific cell wall polysaccharide found in grasses and cereals. beta-Glucan is composed primarily of cellotriosyl and cellotetraosyl units linked by single (1-->3)beta-D-linkages. In reactions in vitro at high substrate concentration, a polymer composed of almost entirely cellotriosyl and cellopentosyl units is made. These results support a model in which three modes of glycosyl transfer occur within the synthase complex instead of just two. The generation of odd numbered units demands that they are connected by (1-->3)beta-linkages and not (1-->4)beta-. In this short review of beta-glucan synthesis in maize, we show how such a model not only provides simple mechanisms of synthesis for all (1-->4)beta-D-glycans but also explains how the synthesis of callose, or strictly (1-->3)beta-D-glucans, occurs upon loss of the multiple modes of glycosyl transfer to a single one.     

A New Imaging Platform for Visualizing Biological Effects of Non-Invasive Radiofrequency Electric-Field Cancer Hyperthermia

Herein, we present a novel imaging platform to study the biological effects of non-invasive radiofrequency (RF) electric field cancer hyperthermia. This system allows for real-time in vivo intravital microscopy (IVM) imaging of radiofrequency-induced biological alterations such as changes in vessel structure and drug perfusion. Our results indicate that the IVM system is able to handle exposure to high-power electric-fields without inducing significant hardware damage or imaging artifacts. Furthermore, short durations of low-power (< 200 W) radiofrequency exposure increased transport and perfusion of fluorescent tracers into the tumors at temperatures below 41°C. Vessel deformations and blood coagulation were seen for tumor temperatures around 44°C. These results highlight the use of our integrated IVM-RF imaging platform as a powerful new tool to visualize the dynamics and interplay between radiofrequency energy and biological tissues, organs, and tumors.     

Crop phenotyping in a context of global change: What to measure and how to do it

High-throughput crop phenotyping, particularly under field conditions, is nowadays perceived as a key factor limiting crop genetic advance. Phenotyping not only facilitates conventional breeding, but it is necessary to fully exploit the capabilities of molecular breeding, and it can be exploited to predict breeding targets for the years ahead at the regional level through more advanced simulation models and decision support systems. In terms of phenotyping, it is necessary to determined which selection traits are relevant in each situation, and which phenotyping tools/methods are available to assess such traits. Remote sensing methodologies are currently the most popular approaches, even when lab-based analyses are still relevant in many circumstances. On top of that, data processing and automation, together with machine learning/deep learning are contributing to the wide range of applications for phenotyping. This review addresses spectral and red–green–blue sensing as the most popular remote sensing approaches, alongside stable isotope composition as an example of a lab-based tool, and root phenotyping, which represents one of the frontiers for field phenotyping. Further, we consider the two most promising forms of aerial platforms (unmanned aerial vehicle and satellites) and some of the emerging data-processing techniques. The review includes three Boxes that examine specific case studies.     

The nitrogen balance of vegetable crops irrigated with untreated effluent

In the agricultural areas near Santiago, Chile,ca. 780 kg N ha^?1 yr^?1 are added to vegetable cropsvia irrigation with untreated sewage effluent draining from the metropolitan area. Nitrate levels in surface wells in the area, from which drinking water is derived, often exceed established limits for human consumption. Of the 779 kg N ha^?1 added to crops in one year, 161–287 kg N ha^?1 yr^?1 were removed by crop harvest and much of the remainder apparently eventually leached to the 1–15 m deep water table.     

Synthesis of polyphosphoinositides in transverse tubule and sarcoplasmic reticulum membranes of frog skeletal muscle.

Synthesis of polyphosphoinositides has been studied in transverse (T-) tubule and sarcoplasmic reticulum (SR) membrane fractions of frog skeletal muscle, following 32P-labeling with [gamma-32P]ATP. Purified SR and T-tubule fractions respectively synthesize 9.4 +/- 0.8 and 71.9 +/- 9.8 pmol PtdInsP/mg per min, indicating nearly 8-fold higher activity of PtdIns kinase in the T-tubules than in the SR. The activity of this enzyme in both membrane systems is maximum at pH 7 and pCa 6. PtdInsP2 is synthesized from the endogenous PtdInsP, only in T-tubule membranes by the action of PtdInsP kinase. This lipid is the most intensely 32P-labeled phosphoinositide (181.7 +/- 9.2 pmol/mg per min) in these membranes. PtdIns kinase in the T-tubule and SR membranes, and PtdInsP kinase in the former are modulated by the free [Mg2+]. Loss of radiolabel from transiently maximal 32P-incorporation in polyphosphoinositides in T-tubule membranes, concomitant with a decrease in the ATP concentration in the incubation buffer, shows the occurrence of phosphoinositidases in these membranes. Under the conditions used, no such activities were evident in SR membranes. Compound 48/80, a mixture of condensation products of N-methyl-p-methoxyphenethylamine with formaldehyde, known to block phosphoinositidase C and phospholipase A2, causes a dose-dependent increase in the 32P-label of PtdInsP, in T-tubule membranes. The synthesis of lyso PtdInsP2, a deacylated form of PtdInsP2 which occurs in nearly equal quantities in both T-tubule and SR membranes, may result from a mechanism independent of phospholipase A2.     

Correlation between Hemichrome Stability and the Root Effect in Tetrameric Hemoglobins

Oxidation of Hbs leads to the formation of different forms of Fe(III) that are relevant to a range of biochemical and physiological functions. Here we report a combined EPR/x-ray crystallography study performed at acidic pH on six ferric tetrameric Hbs. Five of the Hbs were isolated from the high-Antarctic notothenioid fishes Trematomus bernacchii, Trematomus newnesi, and Gymnodraco acuticeps, and one was isolated from the sub-Antarctic notothenioid Cottoperca gobio. Our EPR analysis reveals that 1), in all of these Hbs, at acidic pH the aquomet form and two hemichromes coexist; and 2), only in the three Hbs that exhibit the Root effect is a significant amount of the pentacoordinate (5C) high-spin Fe(III) form found. The crystal structure at acidic pH of the ferric form of the Root-effect Hb from T. bernacchii is also reported at 1.7 Å resolution. This structure reveals a 5C state of the heme iron for both the α- and β-chains within a T quaternary structure. Altogether, the spectroscopic and crystallographic results indicate that the Root effect and hemichrome stability at acidic pH are correlated in tetrameric Hbs. Furthermore, Antarctic fish Hbs exhibit higher peroxidase activity than mammalian and temperate fish Hbs, suggesting that a partial hemichrome state in tetrameric Hbs, unlike in monomeric Hbs, does not remove the need for protection from peroxide attack, in contrast to previous results from monomeric Hbs.     

Mechanisms associated with tolerance to flooding during germination and early seedling growth in rice (Oryza sativa)

Background and Aims

Flooding slows seed germination, imposes fatalities and delays seedling establishment in direct-seeded rice. This study describes responses of contrasting rice genotypes subjected to flooding or low oxygen stress during germination and discusses the basis of tolerance shown by certain cultivars.

Methods

In one set of experiments, dry seeds were sown in soil and either watered normally or flooded with 10 cm of water. Seedling survival and shoot and root growth were assessed and seed portions of germinating seedlings were assayed for soluble sugars and starch concentrations. The whole germinating seedlings were assayed for amylase and peroxidase activities and for ethylene production. Activities of enzymes associated with anaerobic respiration were examined and gene expression was analysed separately with seeds germinating under different amounts of dissolved oxygen in dilute agar.

Key Results

Flooding during germination reduced survival but to a lesser extent in tolerant genotypes. Starch concentration in germinating seeds decreased while sugar concentration increased under flooding, but more so in tolerant genotypes. Amylase activity correlated positively with elongation (r = 0·85 for shoot and 0·83 for root length) and with plant survival (r = 0·92). Tolerant genotypes had higher amylase activity and higher RAmy3D gene expression. Ethylene was not detected in seeds within 2 d after sowing, but increased thereafter, with a greater increase in tolerant genotypes starting 3 d after sowing. Peroxidase activity was higher in germinating seeds of sensitive genotypes and correlated negatively with survival.

Conclusions

Under low oxygen stress, tolerant genotypes germinate, grow faster and more seedlings survive. They maintain their ability to use stored starch reserves through higher amylase activity and anaerobic respiration, have higher rates of ethylene production and lower peroxidase activity as germinating seeds and as seedlings. Relevance of these traits to tolerance of flooding during germination and early growth is discussed.Key words: Amylase, anoxia, crop establishment, direct-seeded rice, ethylene, flooding, germination, hypoxia, Oryza sativa