The PRK/Rubisco shunt strongly influences Arabidopsis seed metabolism and oil accumulation, affecting more than carbon recycling

The carbon efficiency of storage lipid biosynthesis from imported sucrose in green Brassicaceae seeds is proposed to be enhanced by the PRK/Rubisco shunt, in which ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) acts outside the context of the Calvin–Benson–Bassham cycle to recycle CO2 molecules released during fatty acid synthesis. This pathway utilizes metabolites generated by the nonoxidative steps of the pentose phosphate pathway. Photosynthesis provides energy for reactions such as the phosphorylation of ribulose 5-phosphate by phosphoribulokinase (PRK). Here, we show that loss of PRK in Arabidopsis thaliana (Arabidopsis) blocks photoautotrophic growth and is seedling-lethal. However, seeds containing prk embryos develop normally, allowing us to use genetics to assess the importance of the PRK/Rubisco shunt. Compared with nonmutant siblings, prk embryos produce one-third less lipids—a greater reduction than expected from simply blocking the proposed PRK/Rubisco shunt. However, developing prk seeds are also chlorotic and have elevated starch contents compared with their siblings, indicative of secondary effects. Overexpressing PRK did not increase embryo lipid content, but metabolite profiling suggested that Rubisco activity becomes limiting. Overall, our findings show that the PRK/Rubisco shunt is tightly integrated into the carbon metabolism of green Arabidopsis seeds, and that its manipulation affects seed glycolysis, starch metabolism, and photosynthesis.ISSN:1040-4651ISSN:1531-298XISSN:1532-298

The GPAT4/6/8 clade functions in Arabidopsis root suberization nonredundantly with the GPAT5/7 clade required for suberin lamellae.

Lipid polymers such as cutin and suberin strengthen the diffusion barrier properties of the cell wall in specific cell types and are essential for water relations, mineral nutrition, and stress protection in plants. Land plant-specific glycerol-3-phosphate acyltransferases (GPATs) of different clades are central players in cutin and suberin monomer biosynthesis. Here, we show that the GPAT4/6/8 clade in Arabidopsis thaliana, which is known to mediate cutin formation, is also required for developmentally regulated root suberization, in addition to the established roles of GPAT5/7 in suberization. The GPAT5/7 clade is mainly required for abscisic acid-regulated suberization. In addition, the GPAT5/7 clade is crucial for the formation of the typical lamellated suberin ultrastructure observed by transmission electron microscopy, as distinct amorphous globular polyester structures were deposited in the apoplast of the gpat5 gpat7 double mutant, in contrast to the thinner but still lamellated suberin deposition in the gpat4 gpat6 gpat8 triple mutant. Site-directed mutagenesis revealed that the intrinsic phosphatase activity of GPAT4, GPAT6, and GPAT8, which leads to monoacylglycerol biosynthesis, contributes to suberin formation. GPAT5/7 lack an active phosphatase domain and the amorphous globular polyester structure observed in the gpat5 gpat7 double mutant was partially reverted by treatment with a phosphatase inhibitor or the expression of phosphatase-dead variants of GPAT4/6/8. Thus, GPATs that lack an active phosphatase domain synthetize lysophosphatidic acids that might play a role in the formation of the lamellated structure of suberin. GPATs with active and nonactive phosphatase domains appear to have nonredundant functions and must cooperate to achieve the efficient biosynthesis of correctly structured suberin

Investigation of salinity tolerance mechanism in barley roots using Semi-Targeted lipidomics approach with high-resolution mass spectrometry techniques

Salinity in soils is one of the major factors that adversely affects agriculture by inhibiting plant growth, resulting in low crop productivity. Among cereal crops (eg. rice, wheat), barley (Hordeum vulgare L.) is rated as salt-tolerant, and exhibits a considerable variation in salt tolerance amongst its cultivars. Barley is a food and brewing crop, and as a glycophyte it suffers substantial yield loss when grown under saline conditions. Relatively little is currently understood of salt stress perception and responses in plant roots, which involve complex changes at the physiological, metabolic, molecular, transcriptional, and genetic levels. We aim to develop new tools to unravel how plants respond to the perception of salt stress. Evidence is accumulating that lipid signalling is an integral part of the complex regulatory networks that plants utilize to respond to salinity through modifications of membrane lipids. These occur through changes in activity of such enzymes as phospholipase D and diacylglycerol kinase that produce different classes of lipid and lipid-derived messengers. In addition, abiotic stress provokes enhanced production of reactive oxygen species, resulting in lipid modifications by the oxidation of lipid species. Lipidomics analysis using liquid chromatography mass spectrometry (LC-MS) revealed that roots from tolerant and sensitive cultivars respond differently to salt stress. To investigate the modifications of lipids leading to root responses to salinity, we are using a combination of multiple approaches, such as targeted and untargeted lipidomics of barley roots. Matrix Assisted Laser Desorption Ionisation Mass Spectrometry Imaging (MALDI-MSI) was also employed to examine the spatial distribution of lipids in barley roots grown under control and saline conditions. The combination of LC-MS and MALDI-MSI identified a large number of metabolites and lipids with a unique spatial distribution. MSI was capable of discriminating salt vs control treated roots, identifying major lipid changes under salt treatment in a spatial manner. Non-uniform spatial distribution of metabolites was observed among different barley cultivars. Major PC lipid species and carbohydrates were identified to change the most in salt treated roots compared to control. Given the lack of fundamental knowledge of the lipids involved in signalling and metabolism under saline stress, our results provide insight into novel mechanisms of how barley roots respond to salt stress

Metabolic priming in G6PDH isoenzyme-replaced tobacco lines improves stress tolerance and seed yields via altering assimilate partitioning

We investigated the basis for better performance of transgenic Nicotiana tabacum plants with G6PDH-isoenzyme replacement in the cytosol (Xanthi::cP2::cytRNAi, Scharte et al., 2009). After six generations of selfing, infiltration of Phytophthora nicotianae zoospores into source leaves confirmed that defence responses (ROS, callose) are accelerated, showing as fast cell death of the infected tissue. Yet, stress-related hormone profiles resembled susceptible Xanthi and not resistant cultivar SNN, hinting at mainly metabolic adjustments in the transgenic lines. Leaves of non-stressed plants contained twofold elevated fructose-2,6-bisphosphate (F2,6P2) levels, leading to partial sugar retention (soluble sugars, starch) and elevated hexose-to-sucrose ratios, but also more lipids. Above-ground biomass lay in between susceptible Xanthi and resistant SNN, with photo-assimilates preferentially allocated to inflorescences. Seeds were heavier with higher lipid-to-carbohydrate ratios, resulting in increased harvest yields - also under water limitation. Abiotic stress tolerance (salt, drought) was improved during germination, and in floated leaf disks of non-stressed plants. In leaves of salt-watered plants, proline accumulated to higher levels during illumination, concomitant with efficient NADP(H) use and recycling. Non-stressed plants showed enhanced PSII-induction kinetics (upon dark–light transition) with little differences at the stationary phase. Leaf exudates contained 10% less sucrose, similar amino acids, but more fatty acids – especially in the light. Export of specific fatty acids via the phloem may contribute to both, earlier flowering and higher seed yields of the Xanthi-cP2 lines. Apparently, metabolic priming by F2,6P2—combined with sustained NADP(H) turnover—bypasses the genetically fixed growth–defence trade-off, rendering tobacco plants more stress-resilient and productive

Transient birefringence and dichroism in ZnO studied with fs-time-resolved spectroscopic ellipsometry

The full transient dielectric-function (DF) tensor of ZnO after UV-laser excitation in the spectral range 1.4–3.6 eV is obtained by measuring an m-plane-oriented ZnO thin film with femtosecond (fs)-time-resolved spectroscopic ellipsometry. From the merits of the method, we can distinguish between changes in the real and the imaginary part of the DF as well as changes in birefringence and dichroism, respectively. We find pump-induced switching from positive to negative birefringence in almost the entire measured spectral range for about 1 ps. Simultaneously, weak dichroism in the spectral range below 3.0 eV hints at contributions of inter-valence-band transitions. Line-shape analysis of the DF above the band gap based on discrete exciton, exciton-continuum, and exciton-phonon-complex contributions shows a maximal dynamic increase in the transient exciton energy by 80 meV. The absorption coefficient below the band gap reveals an exponential line shape attributed to Urbach-rule absorption mediated by exciton–longitudinal-optic-phonon interaction. The transient DF is supported by first-principles calculations for 1020cm−3 excited electron-hole pairs in ideal bulk ZnO.Funding: project "Advanced research using high intensity laser produced photons and particles" (ADONIS) from the European Regional Development Fund [CZ.02.1.01/0.0/0.0/16 019/0000789]; project "Structural dynamics of biomolecular systems"(ELIBIO) from the European Regional Development Fund (EFRE) [CZ.02.1.01/0.0/0.0/15 003/0000447]; Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)German Research Foundation (DFG) [SFB 762, 31047526, FOR 1616 (SCHM2710/2)]; project I-COSIMA [SAB 100315366]; Ministry of Education, Youth and Sports from the National Programme of Sustainability II; National Science FoundationNational Science Foundation (NSF) [DMR-1555153, CBET-1437230, OCI-0725070, ACI-1238993]; state of Illinois; Universitat Leipzig within core research area Complex Matter; Leipzig University</p

Cell wall-localized BETA-XYLOSIDASE4 contributes to immunity of Arabidopsis against Botrytis cinerea.

Plant cell walls constitute physical barriers that restrict access of microbial pathogens to the contents of plant cells. The primary cell wall of multicellular plants predominantly consists of cellulose, hemicellulose, and pectin, and its composition can change upon stress. BETA-XYLOSIDASE4 (BXL4) belongs to a seven-member gene family in Arabidopsis (Arabidopsis thaliana), one of which encodes a protein (BXL1) involved in cell wall remodeling. We assayed the influence of BXL4 on plant immunity and investigated the subcellular localization and enzymatic activity of BXL4, making use of mutant and overexpression lines. BXL4 localized to the apoplast and was induced upon infection with the necrotrophic fungal pathogen Botrytis cinerea in a jasmonoyl isoleucine-dependent manner. The bxl4 mutants showed a reduced resistance to B. cinerea, while resistance was increased in conditional overexpression lines. Ectopic expression of BXL4 in Arabidopsis seed coat epidermal cells rescued a bxl1 mutant phenotype, suggesting that, like BXL1, BXL4 has both xylosidase and arabinosidase activity. We conclude that BXL4 is a xylosidase/arabinosidase that is secreted to the apoplast and its expression is upregulated under pathogen attack, contributing to immunity against B. cinerea, possibly by removal of arabinose and xylose side-chains of polysaccharides in the primary cell wall

The maize lipoxygenase, ZmLOX10, mediates green leaf volatile, jasmonate and herbivore-induced plant volatile production for defense against insect attack

Fatty acid derivatives are of central importance for plant immunity against insect herbivores; however, major regulatory genes and the signals that modulate these defense metabolites are vastly understudied, especially in important agro-economic monocot species. Here we show that products and signals derived from a single Zea mays (maize) lipoxygenase (LOX), ZmLOX10, are critical for both direct and indirect defenses to herbivory. We provide genetic evidence that two 13-LOXs, ZmLOX10 and ZmLOX8, specialize in providing substrate for the green leaf volatile (GLV) and jasmonate (JA) biosynthesis pathways, respectively. Supporting the specialization of these LOX isoforms, LOX8 and LOX10 are localized to two distinct cellular compartments, indicating that the JA and GLV biosynthesis pathways are physically separated in maize. Reduced expression of JA biosynthesis genes and diminished levels of JA in lox10 mutants indicate that LOX10-derived signaling is required for LOX8-mediated JA. The possible role of GLVs in JA signaling is supported by their ability to partially restore wound-induced JA levels in lox10 mutants. The impaired ability of lox10 mutants to produce GLVs and JA led to dramatic reductions in herbivore-induced plant volatiles (HIPVs) and attractiveness to parasitoid wasps. Because LOX10 is under circadian rhythm regulation, this study provides a mechanistic link to the diurnal regulation of GLVs and HIPVs. GLV-, JA- and HIPV-deficient lox10 mutants display compromised resistance to insect feeding, both under laboratory and field conditions, which is strong evidence that LOX10-dependent metabolites confer immunity against insect attack. Hence, this comprehensive gene to agro-ecosystem study reveals the broad implications of a single LOX isoform in herbivore defense. © 2013 Blackwell Publishing Ltd