Alternative Splicing, An Overlooked Defense Frontier of Plants with Respect to Bacterial Infection.

Disease represents a major problem in sustainable agricultural development. Plants interact closely with various microorganisms during their development and in response to the prevailing environment. In particular, pathogenic microorganisms can cause plant diseases, affecting the fertility, yield, and longevity of plants. During the long coevolution of plants and their pathogens, plants have evolved both molecular pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) signaling networks in order to regulate host cells in response to pathogen infestation. Additionally, in the postgenomic era, alternative splicing (AS) has become uncovered as one of the major drivers of proteome diversity, and abnormal RNA splicing is closely associated with bacterial infections. Currently, the complexity of host-bacteria interactions is a much studied area of research that has shown steady progress over the past decade. Although the development of high-throughput sequencing technologies and their application in transcriptomes have revolutionized our understanding of AS, many mechanisms related to host-bacteria interactions remain still unclear. To this end, this review summarizes the changes observed in AS during host-bacteria interactions and outlines potential therapeutics for bacterial diseases based on existing studies. In doing so, we hope to provide guidelines for plant disease management in agriculture.

Flocculation of oleaginous green algae with Mortierella alpina fungi.

Microalgae are promising sources of valuable bioproducts such as biofuels, food, and nutraceuticals. However, harvesting microalgae is challenging due to their small size and low biomass concentrations. To address this challenge, bio-flocculation of starchless mutants of Chlamydomonas reinhardtii (sta6/sta7) was investigated with Mortierella alpina, an oleaginous fungus with high concentrations of arachidonic acid (ARA). Triacylglycerides (TAG) reached 85 % of total lipids in sta6 and sta7 through a nitrogen regime. Scanning electron microscopy determined cell-wall attachment and extra polymeric substances (EPS) to be responsible for flocculation. An algal-fungal biomass ratio around 1:1 (three membranes) was optimal for bio-flocculation (80-85 % flocculation efficiency in 24 h). Nitrogen-deprived sta6/sta7 were flocculated with strains of M. alpina (NVP17b, NVP47, and NVP153) with aggregates exhibiting fatty acid profiles similar to C. reinhardtii, with ARA (3-10 % of total fatty acids). This study showcases M. alpina as a strong bio-flocculation candidate for microalgae and advances a mechanistic understanding of algal-fungal interaction.

Tuber rugosum, a new species from northeastern North America: Slug mycophagy aides in electron microscopy of ascospores.

Species in the genus Tuber are ascomycetous fungi that produce hypogeous fruiting bodies commonly called truffles. These fungi are ecologically relevant owing to the ectomycorrhizal symbiosis they establish with plants. One of the most speciose lineages within Tuber is the Rufum clade, which is widely distributed throughout Asia, Europe, and North America and is estimated to include more than 43 species. Most species in this clade have spiny spores, and many still have not been formally described. Here, we describe T. rugosum based on multigene phylogenetic analysis and its unique morphological characters. Tuber rugosum (previously designated in literature as Tuber sp. 69) has been collected throughout the Midwest, USA, and Quebec, Canada, and is an ectomycorrhizal symbiont of Quercus trees, as confirmed through morphological and molecular analyses of root tips presented here. We also present a novel method for preparing Tuber ascospores for scanning electron microscope imaging that includes feeding, digestion, and spore excretion by the slug Arion subfuscus. Following this method, spores become free from ascus and other mycelial debris that could obscure morphological traits during their passage through the snail gut while maintaining ornamentation. Finally, we report the fatty acid analysis, a fungicolous species association, and we provide an updated taxonomic key of the Rufum clade.

In vivo cadmium-assisted dilute acid pretreatment of the phytoremediation sweet sorghum for enzymatic hydrolysis and cadmium enrichment.

Phytoremediation with energy crops is considered an integrated technology that provides both environment and energy benefits. Herein, the sweet sorghum cultivated on Cd-contaminated farmland (1.21 mg/kg of Cd in the soil) showed promising phytoremediation potential, and the approach for utilizing sorghum stalks was explored. Sweet sorghum bagasse with Cd contamination was pretreated with dilute acid in order to improve enzymatic saccharification and achieve Cd recovery, resulting in harmless and value-added utilization. After pretreatment, hemicelluloses were dramatically degraded, and the lignocellulosic structures were partially deconstructed with xylan removal up to 98.1%. Under the optimal condition (0.75% H2SO4), the highest total sugar yield was 0.48 g/g of raw bagasse; and nearly 98% of Cd was enriched in the liquid phase. Compared with normal biomass, Cd reduced the biomass recalcitrance and further facilitated the deconstruction of biomass under super dilute acid conditions. This work provided an example for the subsequent valorization of Cd-containing biomass and Cd recovery, which will greatly facilitate the development of phytoremediation of heavy metal contaminated soil.

RICE ACYL-COA-BINDING PROTEIN6 Affects Acyl-CoA Homeostasis and Growth in Rice.

BACKGROUNDS: Acyl-coenzyme A (CoA) esters are important intermediates in lipid metabolism with regulatory properties. Acyl-CoA-binding proteins bind and transport acyl-CoAs to fulfill these functions. RICE ACYL-COA-BINDING PROTEIN6 (OsACBP6) is currently the only one peroxisome-localized plant ACBP that has been proposed to be involved in ß-oxidation in transgenic Arabidopsis. The role of the peroxisomal ACBP (OsACBP6) in rice (Oryza sativa) was investigated. RESULTS: Here, we report on the function of OsACBP6 in rice. The osacbp6 mutant showed diminished growth with reduction in root meristem activity and leaf growth. Acyl-CoA profiling and lipidomic analysis revealed an increase in acyl-CoA content and a slight triacylglycerol accumulation caused by the loss of OsACBP6. Comparative transcriptomic analysis discerned the biological processes arising from the loss of OsACBP6. Reduced response to oxidative stress was represented by a decline in gene expression of a group of peroxidases and peroxidase activities. An elevation in hydrogen peroxide was observed in both roots and shoots/leaves of osacbp6. Taken together, loss of OsACBP6 not only resulted in a disruption of the acyl-CoA homeostasis but also peroxidase-dependent reactive oxygen species (ROS) homeostasis. In contrast, osacbp6-complemented transgenic rice displayed similar phenotype to the wild type rice, supporting a role for OsACBP6 in the maintenance of the acyl-CoA pool and ROS homeostasis. Furthermore, quantification of plant hormones supported the findings observed in the transcriptome and an increase in jasmonic acid level occurred in osacbp6. CONCLUSIONS: In summary, OsACBP6 appears to be required for the efficient utilization of acyl-CoAs. Disruption of OsACBP6 compromises growth and led to provoked defense response, suggesting a correlation of enhanced acyl-CoAs content with defense responses.

Characterization and function of a sunflower (Helianthus annuus L.) Class II acyl-CoA-binding protein.

Acyl-CoA-binding proteins (ACBP) bind to long-chain acyl-CoA esters and phospholipids, enhancing the activity of different acyltransferases in animals and plants. Nevertheless, the role of these proteins in the synthesis of triacylglycerols (TAGs) remains unclear. Here, we cloned a cDNA encoding HaACBP1, a Class II ACBP from sunflower (Helianthus annuus), one of the world's most important oilseed crop plants. Transcriptome analysis of this gene revealed strong expression in developing seeds from 16 to 30 days after flowering. The recombinant protein (rHaACBP1) was expressed in Escherichia coli and purified to be studied by in vitro isothermal titration calorimetry and for phospholipid binding. Its high affinity for saturated palmitoyl-CoA (16:0-CoA; KD 0.11 µM) and stearoyl-CoA (18:0-CoA; KD 0.13 µM) esters suggests that rHaACBP1 could act in acyl-CoA transfer pathways that involve saturated acyl derivatives. Furthermore, rHaACBP1 also binds to both oleoyl-CoA (18:1-CoA; KD 6.4 µM) and linoleoyl-CoA (18:2-CoA; KD 21.4 µM) esters, the main acyl-CoA substrates used to synthesise the TAGs that accumulate in sunflower seeds. Interestingly, rHaACBP1 also appears to bind to different species of phosphatidylcholines (dioleoyl-PC and dilinoleoyl-PC), glycerolipids that are also involved in TAG synthesis, and while it interacts with dioleoyl-PA, this is less prominent than its binding to the PC derivative. Expression of rHaACBP in yeast alters its fatty acid composition, as well as the composition and size of the host acyl-CoA pool. These results suggest that HaACBP1 may potentially fulfil a role in the transport and trafficking of acyl-CoAs during sunflower seed development.

The Microalga Nannochloropsis during Transition from Quiescence to Autotrophy in Response to Nitrogen Availability.

The marine microalgae Nannochloropsis oceanica (CCMP1779) is a prolific producer of oil and is considered a viable and sustainable resource for biofuel feedstocks. Nitrogen (N) availability has a strong impact on the physiological status and metabolism of microalgal cells, but the exact nature of this response is poorly understood. To fill this gap we performed transcriptomic profiling combined with cellular and molecular analyses of N. oceanica CCMP1779 during the transition from quiescence to autotrophy. N deprivation-induced quiescence was accompanied by a strong reorganization of the photosynthetic apparatus and changes in the lipid homeostasis, leading to accumulation of triacylglycerol. Cell cycle activation and re-establishment of photosynthetic activity observed in response to resupply of the growth medium with N were accompanied by a rapid degradation of triacylglycerol stored in lipid droplets (LDs). Besides observing LD translocation into vacuoles, we also provide evidence for direct interaction between the LD surface protein (NoLDSP) and AUTOPHAGY-RELATED8 (NoATG8) protein and show a role of microlipophagy in LD turnover in N. oceanica CCMP1779. This knowledge is crucial not only for understanding the fundamental mechanisms controlling the cellular energy homeostasis in microalgal cells but also for development of efficient strategies to achieve higher algal biomass and better microalgal lipid productivity.

Experimental evolution of phytoplankton fatty acid thermal reaction norms.

Temperature effects on the fatty acid (FA) profiles of phytoplankton, major primary producers in the ocean, have been widely studied due to their importance as industrial feedstocks and to their indispensable role as global producers of long-chain, polyunsaturated FA (PUFA), including omega-3 (ω3) FA required by organisms at higher trophic levels. The latter is of global ecological concern for marine food webs, as some evidence suggests an ongoing decline in global marine-derived ω3 FA due to both a global decline in phytoplankton abundance and to a physiological reduction in ω3 production by phytoplankton as temperatures rise. Here, we examined both short-term (physiological) and long-term (evolutionary) responses of FA profiles to temperature by comparing FA thermal reaction norms of the marine diatom Thalassiosira pseudonana after ~500 generations (ca. 2.5 years) of experimental evolution at low (16°C) and high (31°C) temperatures. We showed that thermal reaction norms for some key FA classes evolved rapidly in response to temperature selection, often in ways contrary to our predictions based on prior research. Notably, 31°C-selected populations showed higher PUFA percentages (including ω3 FA) than 16°C-selected populations at the highest assay temperature (31°C, above T. pseudonana's optimum temperature for population growth), suggesting that high-temperature selection led to an evolved ability to sustain high PUFA production at high temperatures. Rapid evolution may therefore mitigate some of the decline in global phytoplankton-derived ω3 FA production predicted by recent studies. Beyond its implications for marine food webs, knowledge of the effects of temperature on fatty acid profiles is of fundamental importance to our understanding of the mechanistic causes and consequences of thermal adaptation.

Algal-fungal symbiosis leads to photosynthetic mycelium.

Mutualistic interactions between free-living algae and fungi are widespread in nature and are hypothesized to have facilitated the evolution of land plants and lichens. In all known algal-fungal mutualisms, including lichens, algal cells remain external to fungal cells. Here, we report on an algal-fungal interaction in which Nannochloropsis oceanica algal cells become internalized within the hyphae of the fungus Mortierella elongata. This apparent symbiosis begins with close physical contact and nutrient exchange, including carbon and nitrogen transfer between fungal and algal cells as demonstrated by isotope tracer experiments. This mutualism appears to be stable, as both partners remain physiologically active over months of co-cultivation, leading to the eventual internalization of photosynthetic algal cells, which persist to function, grow and divide within fungal hyphae. Nannochloropsis and Mortierella are biotechnologically important species for lipids and biofuel production, with available genomes and molecular tool kits. Based on the current observations, they provide unique opportunities for studying fungal-algal mutualisms including mechanisms leading to endosymbiosis.

Enhancing oil production and harvest by combining the marine alga Nannochloropsis oceanica and the oleaginous fungus Mortierella elongata.

BACKGROUND: Although microalgal biofuels have potential advantages over conventional fossil fuels, high production costs limit their application in the market. We developed bio-flocculation and incubation methods for the marine alga, Nannochloropsis oceanica CCMP1779, and the oleaginous fungus, Mortierella elongata AG77, resulting in increased oil productivity. RESULTS: By growing separately and then combining the cells, the M. elongata mycelium could efficiently capture N. oceanica due to an intricate cellular interaction between the two species leading to bio-flocculation. Use of a high-salt culture medium induced accumulation of triacylglycerol (TAG) and enhanced the contents of polyunsaturated fatty acids (PUFAs) including arachidonic acid and docosahexaenoic acid in M. elongata. To increase TAG productivity in the alga, we developed an effective, reduced nitrogen-supply regime based on ammonium in environmental photobioreactors. Under optimized conditions, N. oceanica produced high levels of TAG that could be indirectly monitored by following chlorophyll content. Combining N. oceanica and M. elongata to initiate bio-flocculation yielded high levels of TAG and total fatty acids, with ~ 15 and 22% of total dry weight (DW), respectively, as well as high levels of PUFAs. Genetic engineering of N. oceanica for higher TAG content in nutrient-replete medium was accomplished by overexpressing DGTT5, a gene encoding the type II acyl-CoA:diacylglycerol acyltransferase 5. Combined with bio-flocculation, this approach led to increased production of TAG under nutrient-replete conditions (~ 10% of DW) compared to the wild type (~ 6% of DW). CONCLUSIONS: The combined use of M. elongata and N. oceanica with available genomes and genetic engineering tools for both species opens up new avenues to improve biofuel productivity and allows for the engineering of polyunsaturated fatty acids.

Nontransgenic Marker-Free Gene Disruption by an Episomal CRISPR System in the Oleaginous Microalga, Nannochloropsis oceanica CCMP1779.

Utilization of microalgae has been hampered by limited tools for creating loss-of-function mutants. Furthermore, modified strains for deployment into the field must be free of antibiotic resistance genes and face fewer regulatory hurdles if they are transgene free. The oleaginous microalga, Nannochloropsis oceanica CCMP1779, is an emerging model for microalgal lipid metabolism. We present a one-vector episomal CRISPR/Cas9 system for N. oceanica that enables the generation of marker-free mutant lines. The CEN/ARS6 region from Saccharomyces cerevisiae was included in the vector to facilitate its maintenance as circular extrachromosal DNA. The vector utilizes a bidirectional promoter to produce both Cas9 and a ribozyme flanked sgRNA. This system efficiently generates targeted mutations, and allows the loss of episomal DNA after the removal of selection pressure, resulting in marker-free nontransgenic engineered lines. To test this system, we disrupted the nitrate reductase gene ( NR) and subsequently removed the CRISPR episome to generate nontransgenic marker-free nitrate reductase knockout lines (NR-KO).

Galactoglycerolipid Lipase PGD1 Is Involved in Thylakoid Membrane Remodeling in Response to Adverse Environmental Conditions in Chlamydomonas.

Photosynthesis occurs in the thylakoid membrane, where the predominant lipid is monogalactosyldiacylglycerol (MGDG). As environmental conditions change, photosynthetic membranes have to adjust. In this study, we used a loss-of-function Chlamydomonas reinhardtii mutant deficient in the MGDG-specific lipase PGD1 (PLASTID GALACTOGLYCEROLIPID DEGRADATION1) to investigate the link between MGDG turnover, chloroplast ultrastructure, and the production of reactive oxygen species (ROS) in response to different adverse environmental conditions. The pgd1 mutant showed altered MGDG abundance and acyl composition and altered abundance of photosynthesis complexes, with an increased PSII/PSI ratio. Transmission electron microscopy showed hyperstacking of the thylakoid grana in the pgd1 mutant. The mutant also exhibited increased ROS production during N deprivation and high light exposure. Supplementation with bicarbonate or treatment with the photosynthetic electron transport blocker DCMU protected the cells against oxidative stress in the light and reverted chlorosis of pgd1 cells during N deprivation. Furthermore, exposure to stress conditions such as cold and high osmolarity induced the expression of PGD1, and loss of PGD1 in the mutant led to increased ROS production and inhibited cell growth. These findings suggest that PGD1 plays essential roles in maintaining appropriate thylakoid membrane composition and structure, thereby affecting growth and stress tolerance when cells are challenged under adverse conditions.

Nannochloropsis, a rich source of diacylglycerol acyltransferases for engineering of triacylglycerol content in different hosts.

BACKGROUND: Photosynthetic microalgae are considered a viable and sustainable resource for biofuel feedstocks, because they can produce higher biomass per land area than plants and can be grown on non-arable land. Among many microalgae considered for biofuel production, Nannochloropsis oceanica (CCMP1779) is particularly promising, because following nutrient deprivation it produces very high amounts of triacylglycerols (TAG). The committed step in TAG synthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT). Remarkably, a total of 13 putative DGAT-encoding genes have been previously identified in CCMP1779 but most have not yet been studied in detail. RESULTS: Based on their expression profile, six out of 12 type-2 DGAT-encoding genes (NoDGTT1-NoDGTT6) were chosen for their possible role in TAG biosynthesis and the respective cDNAs were expressed in a TAG synthesis-deficient mutant of yeast. Yeast expressing NoDGTT5 accumulated TAG to the highest level. Over-expression of NoDGTT5 in CCMP1779 grown in N-replete medium resulted in levels of TAG normally observed only after N deprivation. Reduced growth rates accompanied NoDGTT5 over-expression in CCMP1779. Constitutive expression of NoDGTT5 in Arabidopsis thaliana was accompanied by increased TAG content in seeds and leaves. A broad substrate specificity for NoDGTT5 was revealed, with preference for unsaturated acyl groups. Furthermore, NoDGTT5 was able to successfully rescue the Arabidopsis tag1-1 mutant by restoring the TAG content in seeds. CONCLUSIONS: Taken together, our results identified NoDGTT5 as the most promising gene for the engineering of TAG synthesis in multiple hosts among the 13 DGAT-encoding genes of N. oceanica CCMP1779. Consequently, this study demonstrates the potential of NoDGTT5 as a tool for enhancing the energy density in biomass by increasing TAG content in transgenic crops used for biofuel production.

Plant acyl-CoA-binding proteins: An emerging family involved in plant development and stress responses.

Acyl-CoA-binding protein (ACBP) was first identified in mammals as a neuropeptide, and was demonstrated to belong to an important house-keeping protein family that extends across eukaryotes and some prokaryotes. In plants, the Arabidopsis ACBP family consists of six AtACBPs (AtACBP1 to AtACBP6), and has been investigated using gene knock-out mutants and overexpression lines. Herein, recent findings on the AtACBPs are examined to provide an insight on their functions in various plant developmental processes, such as embryo and seed development, seed dormancy and germination, seedling development and cuticle formation, as well as their roles under various environmental stresses. The significance of the AtACBPs in acyl-CoA/lipid metabolism, with focus on their interaction with long to very-long-chain (VLC) acyl-CoA esters and their potential role in the formation of lipid droplets in seeds and vegetative tissues are discussed. In addition, recent findings on the rice ACBP family are presented. The similarities and differences between ACBPs from Arabidopsis and rice, that represent eudicot and monocot model plants, respectively, are analyzed and the evolution of plant ACBPs by phylogenetic analysis reviewed. Finally, we propose potential uses of plant ACBPs in phytoremediation and in agriculture related to the improvement of environmental stress tolerance and seed oil production.

Characterization of a small acyl-CoA-binding protein (ACBP) from Helianthus annuus L. and its binding affinities.

Acyl-CoA-binding proteins (ACBPs) bind to acyl-CoA esters and promote their interaction with other proteins, lipids and cell structures. Small class I ACBPs have been identified in different plants, such as Arabidopsis thaliana (AtACBP6), Brassica napus (BnACBP) and Oryza sativa (OsACBP1, OsACBP2, OsACBP3), and they are capable of binding to different acyl-CoA esters and phospholipids. Here we characterize HaACBP6, a class I ACBP expressed in sunflower (Helianthus annuus) tissues, studying the specificity of its corresponding recombinant HaACBP6 protein towards various acyl-CoA esters and phospholipids in vitro, particularly using isothermal titration calorimetry and protein phospholipid binding assays. This protein binds with high affinity to de novo synthetized derivatives palmitoly-CoA, stearoyl-CoA and oleoyl-CoA (Kd 0.29, 0.14 and 0.15 µM respectively). On the contrary, it showed lower affinity towards linoleoyl-CoA (Kd 5.6 µM). Moreover, rHaACBP6 binds to different phosphatidylcholine species (dipalmitoyl-PC, dioleoyl-PC and dilinoleoyl-PC), yet it displays no affinity towards other phospholipids like lyso-PC, phosphatidic acid and lysophosphatidic acid derivatives. In the light of these results, the possible involvement of this protein in sunflower oil synthesis is considered.

Triacylglycerol Accumulation in Photosynthetic Cells in Plants and Algae.

Plant and algal oils are some of the most energy-dense renewable compounds provided by nature. Triacylglycerols (TAGs) are the major constituent of plant oils, which can be converted into fatty acid methyl esters commonly known as biodiesel. As one of the most efficient producers of TAGs, photosynthetic microalgae have attracted substantial interest for renewable fuel production. Currently, the big challenge of microalgae based TAGs for biofuels is their high cost compared to fossil fuels. A conundrum is that microalgae accumulate large amounts of TAGs only during stress conditions such as nutrient deprivation and temperature stress, which inevitably will inhibit growth. Thus, a better understanding of why and how microalgae induce TAG biosynthesis under stress conditions would allow the development of engineered microalgae with increased TAG production during conditions optimal for growth. Land plants also synthesize TAGs during stresses and we will compare new findings on environmental stress-induced TAG accumulation in plants and microalgae especially in the well-characterized model alga Chlamydomonas reinhardtii and a biotechnologically relevant genus Nannochloropsis.

Stress-induced neutral lipid biosynthesis in microalgae - Molecular, cellular and physiological insights.

Photosynthetic microalgae have promise as biofuel feedstock. Under certain conditions, they produce substantial amounts of neutral lipids, mainly in the form of triacylglycerols (TAGs), which can be converted to fuels. Much of our current knowledge on the genetic and molecular basis of algal neutral lipid metabolism derives mainly from studies of plants, i.e. seed tissues, and to a lesser extent from direct studies of algal lipid metabolism. Thus, the knowledge of TAG synthesis and the cellular trafficking of TAG precursors in algal cells is to a large extent based on genome predictions, and most aspects of TAG metabolism have yet to be experimentally verified. The biofuel prospects of microalgae have raised the interest in mechanistic studies of algal TAG biosynthesis in recent years and resulted in an increasing number of publications on lipid metabolism in microalgae. In this review we summarize the current findings on genetic, molecular and physiological studies of TAG accumulation in microalgae. Special emphasis is on the functional analysis of key genes involved in TAG synthesis, molecular mechanisms of regulation of TAG biosynthesis, as well as on possible mechanisms of lipid droplet formation in microalgal cells. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.