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1.

Key message

A rapid and efficient Agrobacterium -mediated transformation system in sorghum has been developed employing standard binary vectors and bar gene as a selectable marker.

Abstract

Sorghum (Sorghum bicolor) is an important food and biofuel crop worldwide, for which improvements in genetic transformation are needed to study its biology and facilitate agronomic and commercial improvement. Here, we report optimization of regeneration and transformation of public sorghum genotype P898012 using standard binary vectors and bar gene as a selectable marker. The tissue culture regeneration time frame has been reduced to 7–12 weeks with a yield of over 18 plants per callus, and the optimized transformation system employing Agrobacterium tumefaciens strain AGL1 and the bar with a MAS promoter achieved an average frequency over 14 %. Of randomly analyzed independent transgenic events, 40–50 % carry single copy of integrated T-DNA. Some independent transgenic events were derived from the same embryogenic callus lines, but a 3:1 Mendelian segregation ratio was found in all transgenic events with single copy as estimated by Southern blots. The system described here should facilitate studies of sorghum biology and agronomic improvement.
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2.

Objective

To produce (S)-3-hydroxy-1-(3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-4-(2,4,5-trifluorophenyl)butan-1-one (S)-1 from 4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro [1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)-1-(2,4,5-trifluorophenyl)butan-2-one (2) by microbial bioreduction.

Results

A new isolate of Pseudomonas pseudoalcaligenes reduced enantioselectively prochiral ketone 2 to chiral alcohol (S)-1. Whole cells of the bacterium were tolerant towards 20 % (v/v) DMSO and 10 g 2/l. Under the optimal conditions, the preparative-scale bioreduction yielded (S)-1 at 90 % yield and >99 % ee. Cells could be re-used with the yield and ee of product being 45 % and >99 %, respectively, after five cycles.

Conclusion

Bioreduction using whole cells of P. pseudoalcaligenes is an attractive approach to produce (S)-1, as a chiral intermediate of the anti-diabetic drug, sitagliptin.
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3.

Introduction

Actinomycetes produce the majority of the antibiotics currently in clinical use. The efficiency of antibiotic production is affected by multiple factors such as nutrients, pH, temperature and growth phase. Finding the optimal harvesting time is crucial for successful isolation of the desired bioactive metabolites from actinomycetes, but for this conventional chemical analysis has limitations due to the metabolic complexity.

Objectives

This study explores the utility of NMR-based metabolomics for (1) optimizing fermentation time for the production of known and/or unknown bioactive compounds produced by actinomycetes; (2) elucidating the biosynthetic pathway for microbial natural products; and (3) facilitating the biotransformation of nature-abundant chemicals.

Method

The aqueous culture broth of actinomycete Streptomyces sp. MBT76 was harvested every 24 h for 5 days and each broth was extracted by ethyl acetate. The extracts were analyzed by 1H NMR spectroscopy and the data were compared with principal component analysis (PCA) and orthogonal projection to latent structures (OPLS) analysis. Antimicrobial test were performed by agar diffusion assay.

Results

The secondary metabolites production by Streptomyces sp. MBT76 was growth phase-dependent. Isocoumarins (19), undecylprodiginine (10), streptorubin B (11), 1H-pyrrole-2-carboxamide (12), acetyltryptamine (13), and fervenulin (14) were identified, and their optimal production time was determined in crude extracts without tedious chromatographic fractionation. Of these compounds, 5,6,7,8-tetramethoxyl-3-methyl-isocoumarin (9) is as a novel compound, which was most likely synthesized by a type I iterative polyketide synthase (PKS) encoded by the icm gene cluster. Multivariate data analysis of the 1H NMR spectra showed that acetyltryptamine (13) and tri-methoxylated isocoumarins (7 and 8) were the major determinants of antibiotic activity during later time points. The methoxylation was exploited to allow bioconversion of exogenously added genistein into a suite of methoxylated isoflavones (1518). Methoxylation increased the antimicrobial efficacy of isocoumarins, but decreased that of the isoflavones.

Conclusion

Our results show the applicability of NMR-based metabolic profiling to streamline microbial biotransformation and to determine the optimal harvesting time of actinomycetes for antibiotic production.
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4.

Key message

Striking hydro-climatic differences of 2 years (wet; dry) dramatically control the increment pattern of L. huasango in varying extent, even causing a “growth collapse” during the La Niña drought 2010/2011.

Abstract

We present the first multi-year long time series of local climate data in the seasonally dry tropical forest in Southern Ecuador and related growth dynamics of Loxopterygium huasango, a deciduous tree species. Local climate was investigated by installing an automatically weather station in 2007 and the daily tree growth variability was measured with high-resolution point dendrometers. The climatic impact on growth behaviour was evaluated. Hydro-climatic variables, like precipitation and relative humidity, were the most important factors for controlling tree growth. Changes in rainwater input affected radial increment rates and daily amplitudes of stem diameter variations within the study period from 2009 to 2013. El Niño Southern Oscillation (ENSO) related variations of tropical Pacific Ocean sea surface temperatures influenced the trees’ increment rates. Average radial increments showed high inter-annual (up to 7.89 mm) and inter-individual (up to 3.88 mm) variations. Daily amplitudes of stem diameter variations differed strongly between the two extreme years 2009 (wet) and 2011 (dry). Contrary to 2009, the La Niña drought in 2011 caused a rapid reduction of the daily amplitudes, indicating a total cessation (‘growth collapse’) of stem increment under ENSO-related drought conditions and demonstrating the high impact of climatic extreme events on carbon sequestration of the dry tropical forest ecosystem.
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5.

Key message

Storage changed carbohydrate and protein levels, which can be related to embryo viability. The fatty acid profile was constant, and the embryo composition was similar to the mesocarp, not the endosperm.

Abstract

Macaw palm fruits have a diverse biochemical constitution, and there is significant commercial interest in this species among food, pharmaceutical, cosmetics, and bioenergy industries. We evaluated changes in the reserve compounds of macaw palm mesocarp and seeds from fruits stored for 1 year under three different conditions. Protein and carbohydrate levels were highest in the embryo than in the endosperm. Fatty acid profiles were very similar over time under all storage conditions and in each structure evaluated, with the embryo composition being very similar to the mesocarp. Macaw palm oil remained well preserved under all storage conditions tested, but seed reserves and seed viability are best maintained at room temperatures. The endosperm contained higher levels of saturated fatty acids than either the embryo or mesocarp, making seeds more resistant to oxidative deterioration than the mesocarp. The results showed that the composition of the mesocarp oil promises the production of high-quality biodiesel from this structure, and changes in carbohydrate and protein levels show that laboratory conditions are the most efficient for maintaining seed quality during storage.
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6.

Keymessage

The temporal gradations of the investigated phenolics in Norway spruce bark after bark beetle (Ips typographus) attack followed the general eco-physiological concept. Treatment with salicylic acid inhibits bark beetle colonisation, alleviates the phenolic responses and activates the synthesis of condensed tannins on later sampling dates.

Abstract

Conifer bark is the target of numerous organisms due to its assimilated transport and nutrient storage functions. In the presented study, 100 mM salicylic acid (SA) was applied onto Norway spruce stems prior to being infested with bark beetles (Ips typographus L.), to study the temporal gradation of changes in condensed tannins (CT) and total phenolics (tPH) and their significance for mediating stress-tolerance. A significant accumulation of CT was monitored in untreated trees in response to progressive bark beetle infestation occurring from May onwards. In SA-treated infested trees, the CT values remained at control levels until May, but after the re-treatment of infested trees in June, the concentrations of CT rose significantly in comparison to the controls. The tPH values dropped 16 days after SA-treatment, independent of infestation, and later on remained at control level until July. In contrast, tPH contents accumulated in untreated infested trees in May, eased in June and increased again in July, when the trees were affected by the second generation of bark beetles. To sum up, in May and July when the highest beetle-flight activity was monitored the metabolic shift of phenolics within untreated infested trees differed significantly from the response of SA-treated trees. In addition, on SA-treated trees less entrance holes were monitored over the whole period of sampling when compared to untreated infested trees. These results provide evidence that SA-treatment alleviates the phenolic responses, activates the synthesis of condensed tannins and inhibits bark beetle colonisation.
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7.

Introduction

Aqueous–methanol mixtures have successfully been applied to extract a broad range of metabolites from plant tissue. However, a certain amount of material remains insoluble.

Objectives

To enlarge the metabolic compendium, two ionic liquids were selected to extract the methanol insoluble part of trunk from Betula pendula.

Methods

The extracted compounds were analyzed by LC/MS and GC/MS.

Results

The results show that 1-butyl-3-methylimidazolium acetate (IL-Ac) predominantly resulted in fatty acids, whereas 1-ethyl-3-methylimidazolium tosylate (IL-Tos) mostly yielded phenolic structures. Interestingly, bark yielded more ionic liquid soluble metabolites compared to interior wood.

Conclusion

From this one can conclude that the application of ionic liquids may expand the metabolic snapshot.
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8.

Key message

In black spruce stands on permafrost, trees and understory plants showed higher biomass allocation especially to ‘thin’ fine roots (diam. < 0.5 mm) when growing on shallower permafrost table.

Abstract

Black spruce (Picea mariana) forests in interior Alaska are located on permafrost and show greater below-ground biomass allocation than non-permafrost forests. However, information on fine roots (roots <2 mm in diameter), which have a key role in nutrient uptake and below-ground carbon flux, is still limited especially for effects of different permafrost conditions. In this study, we examined fine root biomass in two black spruce stands with different depths to the permafrost table. In the shallow permafrost (SP) plot, fine root biomass of black spruce trees was 70 % of that in the deep permafrost (DP) plot. In contrast, ratio of the fine root biomass to above-ground biomass was greater in the SP plot than in the DP plot. Understory plants, on the other hand, showed larger fine root biomass in the SP plot than in the DP plot, whereas their above-ground biomass was similar between the two plots. In addition, biomass proportion of ‘thin’ fine roots (diam. <0.5 mm) in total fine roots was greater in the SP plot than in the DP plot. These results suggest that black spruce trees and understory plants could increase biomass allocation to fine roots for efficient below-ground resource acquisition from colder environments with shallower permafrost table. In the SP plot, fine roots of understory plants accounted for 30 % of the stand fine root biomass, suggesting that understory plants such as Ledum and Vaccinium spp. would have significant contribution to below-ground carbon dynamics in permafrost forests.
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9.

Key message

Tall birch trees allocate extra resource due to aboveground temperature elevation to bud and male flower production rather than to plant growth. Saplings increased only plant growth under warming. Size-dependent response should be considered.

Abstract

We experimentally heated canopy organs of tall birch trees (Betula ermanii Cham.; 18–20 m high) growing at a high latitude to determine how leaf phenology, plant growth, and bud and male flower production might shift in response to increases in aboveground temperature during global climate change. We warmed the canopies with infrared heat lamps fixed to steel pipe scaffolds built around the trees. The temperature of the warmed canopies increased by approximately 1 °C. Warming extended the length of the growing season of canopy leaves (by accelerating leaf flush and delaying leaf fall), and significantly increased the numbers of buds and male flowers per shoot. Bud production and shoot length were positively correlated in both warmed and control branches. However, warming did not increase canopy shoot lengths. The intercept value of the positive regression slope between bud production and shoot length for warmed branches was higher than that for control branches. Thus, canopy warming had a direct positive effect on the bud production but had no indirect effect via increases in shoot length. Our experiment showed that tall birch trees allocated extra resources made available by increased aboveground temperature to bud and male flower production rather than to plant growth.
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10.

Key message

Sunlight is a key environmental factor in growth, flowering and shaping of the Dracaena draco tree. Unidirectional light deforms the tree and may cause it to tilt.

Abstract

Dracaena draco, a tree-like monocot, lives in cycles of vegetative growth and flowering. The cycles, as well as the tree growth form, are under genetic control. What controls their length has been unknown before. We propose that it is sunlight. Our trees of the same origin, growing for 20 years in the garden in varying sunlight conditions, started to flower when 9–12, 16 and 18–19 years old, for those growing in full sun, part shade and shade, respectively. In full sun, they grow shorter trunks than those in shade, catching overhead sun. Their branches also had shorter or longer growth and flowering cycles depending on sunlight availability. D. draco tree exhibited strong phototropic response and its crown was organized by the direction of growing tips. In full and in overhead sun, it had a regular form but asymmetrical in unidirectional, oblique sunlight. An asymmetrical crown and the absence of reaction wood may cause the D. draco tree tilting and progressive loss of balance.
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11.

Key message

Simultaneous RNAi silencing of the FAD2 and FAE1 genes in the wild species Lepidium campestre improved the oil quality with 80 % oleic acid content compared to 11 % in wildtype.

Abstract

Field cress (Lepidium campestre) is a wild biennial species within the Brassicaceae family with desirable agronomic traits, thus being a good candidate for domestication into a new oilseed and catch crop. However, it has agronomic traits that need to be improved before it can become an economically viable species. One of such traits is the seed oil composition, which is not desirable either for food use or for industrial applications. In this study, we have, through metabolic engineering, altered the seed oil composition in field cress into a premium oil for food processing, industrial, or chemical industrial applications. Through seed-specific RNAi silencing of the field cress fatty acid desaturase 2 (FAD2) and fatty acid elongase 1 (FAE1) genes, we have obtained transgenic lines with an oleic acid content increased from 11 % in the wildtype to over 80 %. Moreover, the oxidatively unstable linolenic acid was decreased from 40.4 to 2.6 %, and the unhealthy erucic acid was reduced from 20.3 to 0.1 %. The high oleic acid trait has been kept stable for three generations. This shows the possibility to use field cress as a platform for genetic engineering of oil compositions tailor-made for its end uses.
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12.

Key message

CHX had remarkable inhibition on P. edulis photosynthesis, and the reflectance indexes and F 685 / F 735 had the potential value for quantifying the effects of antibiotics on trees.

Abstract

To reveal the effects of antibiotics on photosynthesis and provide help for remote sensing the influence of antibiotics on trees, we investigated the effects of cycloheximide (CHX) on Phyllostachys edulis. In CHX treatment, the photosynthetic pigment content in P. edulis was decreased markedly, which led to the increase in the reflectance spectra in visible region. CHX reduced the donor side and acceptor side of photosystem II (PSII), density of reaction centers, quantum production and electron transport in PSII, and raised the dissipation of absorbed light energy. Besides the dissipation, the absorbed light energy can be emitted as fluorescence with two main peaks in the red (685 nm) and far-red (735 nm) region, respectively. In 0.50 mM CHX treatment, a significant decline in the height and area of the peak at 685 nm might result from Chl loss reducing the light absorption and lower photochemical reaction in PSII. When fourth derivative analysis of fluorescence emission spectra was performed, the changes of the peaks at 718, 735 and 750 nm might result from the decline of absorbed solar radiation caused by the reduced pigment content and/or the damages to the PSI. In CHX treatment, a remarkable increase in intercellular CO2 concentrations and light compensation point and decrease in light saturation point demonstrated that the CO2 assimilation ability was decreased. Those results suggested that the photosynthesis in trees can be reduced after they were watered with wastewater containing CHX. The reflectance indexes and F 685/F 735 (H 685/H 735 and A 685/A 735) were markedly affected by CHX, demonstrating that they had the potential value for quantifying the effects of antibiotics on trees.
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13.

Key message

Water-soluble carbohydrate accumulation can be selected in wheat breeding programs with consideration of genetic × environmental interactions and relationships with other important characteristics such as relative maturity and nitrogen concentration, although the correlation between WSC traits and grain yield is low and inconsistent.

Abstract

The potential to increase the genetic capacity for water-soluble carbohydrate (WSC) accumulation is an opportunity to improve the drought tolerance capability of rainfed wheat varieties, particularly in environments where terminal drought is a significant constraint to wheat production. A population of elite breeding germplasm was characterized to investigate the potential for selection of improved WSC concentration and total amount in water deficit and well-watered environments. Accumulation of WSC involves complex interactions with other traits and the environment. For both WSC concentration (WSCC) and total WSC per area (WSCA), strong genotype × environment interactions were reflected in the clear grouping of experiments into well-watered and water deficit environment clusters. Genetic correlations between experiments were high within clusters. Heritability for WSCC was larger than for WSCA, and significant associations were observed in both well-watered and water deficit experiment clusters between the WSC traits and nitrogen concentration, tillering, grains per m2, and grain size. However, correlations between grain yield and WSCC or WSCA were weak and variable, suggesting that selection for these traits is not a better strategy for improving yield under drought than direct selection for yield.
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14.

Background

With the advances in the next-generation sequencing technologies, researchers can now rapidly examine the composition of samples from humans and their surroundings. To enhance the accuracy of taxonomy assignments in metagenomic samples, we developed a method that allows multiple mismatch probabilities from different genomes.

Results

We extended the algorithm of taxonomic assignment of metagenomic sequence reads (TAMER) by developing an improved method that can set a different mismatch probability for each genome rather than imposing a single parameter for all genomes, thereby obtaining a greater degree of accuracy. This method, which we call TADIP (Taxonomic Assignment of metagenomics based on DIfferent Probabilities), was comprehensively tested in simulated and real datasets. The results support that TADIP improved the performance of TAMER especially in large sample size datasets with high complexity.

Conclusions

TADIP was developed as a statistical model to improve the estimate accuracy of taxonomy assignments. Based on its varying mismatch probability setting and correlated variance matrix setting, its performance was enhanced for high complexity samples when compared with TAMER.
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15.

Background and Aims

It was previously demonstrated that stolons of Fragaria vesca respond to patches of varying nutrient quality; however, the mechanism of patch-detection remained unknown. Here we provide support for a process by which F. vesca perceives nutrient-rich patches, consistent with nutrient foraging prior to rooting.

Methods

Volatile organic compounds (VOCs) emitted from unsterilized and sterilized field substrates were collected and analyzed by stir-bar headspace extraction gas chromatography-mass spectrometry using a method modified for soil and litter systems. Selected compounds were chosen to represent unsterilized and sterilized field substrates. These synthetic volatile compound mixtures were then applied to neutral substrate to test the ability of F. vesca to choose between unsterilized versus sterilized substrates.

Results

Primary stolons exhibited chemotropism towards unsterilized (natural) substrates and grew away from the sterilized volatile substrates when the alternate choice was a negative control. We conclude that the presence of carboxylic acids tends to stimulate stolon elongation and chemotropism while aldehydes, ketones and monoterpenes tend to suppress it.

Conclusions

We provide evidence that developing stolons of F. vesca forage for nutrient-rich patches via volatile cues similar to those emitted from the soil through microflora activity.
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16.

Key message

A Brd2 allele suppresses heading date by altering the expression of heading date regulators such as OsMADS50 , and also negatively regulates chlorophyll biosynthesis.

Abstract

Heading date and plant height are important determinants of yield in rice (Oryza sativa L.). In this study, we characterized a late heading, dwarf mutant known as lhdd10 selected following ethyl methane sulfonate (EMS)-treatment of ssp. indica cultivar 93-11. lhdd10 showed late heading, dwarfness and slightly darker-green leaves than wild-type 93-11 under long-day and short-day conditions. We isolated lhdd10 by map-based cloning; it encoded a putative FAD-linked oxidoreductase protein (a brassinosteroid biosynthetic gene) that localized to the nucleus. LHDD10 was constitutively expressed in various tissues, but more so in shoot apices and panicles. Our data showed that lhdd10 influences heading date by controlling the expression of heading date regulators, such as OsMADS50 in both LD and SD conditions. lhdd10 also negatively regulated expression of chlorophyll biosynthetic genes to reduce the chlorophyll content. Our data indicated that BRs play important roles in regulating heading date and chlorophyll biosynthesis. This work provides material that will allow study of how BRs regulate heading date in rice.
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17.

Key message

We investigated a Frankia Alnus sieboldiana symbiosis, including the minimum inoculum dose for constant nodulation, the period of time to nodulation after inoculation, and the effects of N on nodulation.

Abstract

Frankia is a nitrogen-fixing actinomycete that forms root nodules in some dicotyledonous plants, which are called actinorhizal. We studied nodule formation in Alnus sieboldiana, an actinorhizal plant, after inoculation with a Frankia isolate to establish techniques for Frankia inoculation and the cultivation of inoculated plants. Root nodules formed on seedlings of A. sieboldiana by 2 weeks after inoculation, and N2 fixation measured by acetylene reduction activity started 3 weeks after inoculation. Nodulation was observed after inoculation with a Frankia isolate at 0.001 μL packed cell volume (pcv). The number of nodules formed on the seedlings inoculated with Frankia at more than 0.05 μL pcv was not significantly different. Nodule development and N2 fixation were reduced when inoculated seedlings were treated weekly with 15 mM NH4NO3-N. In contrast, treatment with 3.75 or 0.9375 mM NH4NO3-N did not inhibit nodule development or N2 fixation of inoculated seedlings by 15 weeks of N treatment.
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18.

Key message

This study demonstrates that Millettia stuhlmannii produces annual growth rings responsive to seasonal climate and should be useful for dendrochronology.

Abstract

Millettia stuhlmannii is a highly valuable and potentially overexploited timber species indigenous to southeastern Africa. It is of particular economic importance in Mozambique though relatively little is known about its growth rate or response to climate. This study investigates whether M. stuhlmannii is potentially useful for dendrochronology—that is whether this species forms annual growth rings that are responsive to external forcing such as climate. Five methods were used to determine whether M. stuhlmannii growth rings are indeed annual in nature, including analysis of ring anatomy, dating trees of known age, cambial wounding, classical cross-dating, and comparison of annual growth to climate variables. Growth rings of Millettia stuhlmannii are distinct and well formed, young trees from plantations of known age formed an appropriate number of distinct annual rings, trees showed distinct wood reaction to cambial wounding, adding exactly one complete ring in one calendar year, cross-dating within and between trees was somewhat successful, and annual growth is significantly correlated with wet season precipitation. Results of this study indicate that M. stuhlmannii is a potentially useful species for dendrochronology. These findings should allow a better understanding of this species’ growth dynamics and ecology, as well as its response to climate variability in the past and potentially to future climate change.
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19.

Background

Genetic variants of the Triggering Receptor Expressed on Myeloid Cells-2 (TREM2) confer increased risk of developing late-onset Alzheimer’s Disease (LOAD) and other neurodegenerative disorders. Recent studies provided insight into the multifaceted roles of TREM2 in regulating extracellular β-amyloid (Aβ) pathology, myeloid cell accumulation, and inflammation observed in AD, yet little is known regarding the role of TREM2 in regulating intracellular microtubule associated protein tau (MAPT; tau) pathology in neurodegenerative diseases and in AD, in particular.

Results

Here we report that TREM2 deficiency leads to accelerated and exacerbated hyperphosphorylation and aggregation of tau in a humanized mouse model of tauopathy. TREM2 deficiency also results, indirectly, in dramatic widespread dysregulation of neuronal stress kinase pathways.

Conclusions

Our results suggest that deficiency of microglial TREM2 leads to heightened tau pathology coupled with widespread increases in activated neuronal stress kinases. These findings offer new insight into the complex, multiple roles of TREM2 in regulating Aβ and tau pathologies.
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20.

Key message

Leaf relative water content, leaf area, leaf fresh weight, and SPAD chlorophyll meter readings along with Co - rbcL and Co - rbcS expression can be used for evaluating Camellia oleifera responses to combined drought and heat stress and subsequent recovery after rainfall events.

Abstract

Leaf characteristics, soluble protein and total soluble sugar contents as well as Rubisco-related gene expression in three cultivars of C. oleifera were measured during a combined drought and heat stress period and after subsequent rainfall events. Leaf relative water content (RWC) was significantly correlated with leaf area (LA), leaf fresh weight (FW), SPAD chlorophyll meter readings, and the levels of Co-rbcL and Co-rbcS expression. Results suggest that leaf RWC, LA, leaf FW, SPAD readings together with Co-rbcL and Co-rbcS expression can be used for evaluating responses of C. oleifera cultivars to combined drought and heat stress and subsequent recovery after rainfall events. Rubisco activase might be used for evaluating plant recovery after rainfall. This study identified cultivars differing in tolerance to the combined stress and recovery. Information derived from this study should be valuable for improving survivability and productivity of C. oleifera cultivars.
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