Membrane lipid polyunsaturation mediated by FATTY ACID DESATURASE 2 (FAD2) is involved in endoplasmic reticulum stress tolerance in Arabidopsis thaliana

Unsaturation of membrane glycerolipid classes at their hydrophobic fatty acid tails critically affects the physical nature of the lipid molecule. In Arabidopsis thaliana, 7 fatty acid desaturases (FADs) differently desaturate each glycerolipid class in plastids and the endoplasmic reticulum (ER). Here, we showed that polyunsaturation of ER glycerolipids is required for the ER stress response. Through systematic screening of FAD mutants, we found that a mutant of FAD2 resulted in a hypersensitive response to tunicamycin, a chemical inducer of ER stress. FAD2 converts oleic acid to linoleic acid of the fatty acyl groups of ER‐synthesized phospholipids. Our functional in vivo reporter assay revealed the ER localization and distinct tissue‐specific expression patterns of FAD2. Moreover, glycerolipid profiling of both mutants and overexpressors of FAD2 under tunicamycin‐induced ER stress conditions, along with phenotypic screening of the mutants of the FAD family, suggested that the ratio of monounsaturated fatty acids to polyunsaturated fatty acids, particularly 18:1 to 18:2 species, may be an important factor in allowing the ER membrane to cope with ER stress. Therefore, our results suggest that membrane lipid polyunsaturation mediated by FAD2 is involved in ER stress tolerance in Arabidopsis.     

Histone acetyltransferase general control non‐repressed protein 5 (GCN5) affects the fatty acid composition of Arabidopsis thaliana seeds by acetylating fatty acid desaturase3 (FAD3)

Seed oils are important natural resources used in the processing and preparation of food. Histone modifications represent key epigenetic mechanisms that regulate gene expression, plant growth and development. However, histone modification events during fatty acid (FA) biosynthesis are not well understood. Here, we demonstrate that a mutation of the histone acetyltransferase GCN5 can decrease the ratio of α‐linolenic acid (ALA) to linoleic acid (LA) in seed oil. Using RNA‐Seq and ChIP assays, we identified FAD3, LACS2, LPP3 and PLAIIIβ as the targets of GCN5. Notably, the GCN5‐dependent H3K9/14 acetylation of FAD3 determined the expression levels of FAD3 in Arabidopsis thaliana seeds, and the ratio of ALA/LA in the gcn5 mutant was rescued to the wild‐type levels through the overexpression of FAD3. The results of this study indicated that GCN5 modulated FA biosynthesis by affecting the acetylation levels of FAD3. We provide evidence that histone acetylation is involved in FA biosynthesis in Arabidopsis seeds and might contribute to the optimization of the nutritional structure of edible oils through epigenetic engineering.     

Ultra‐high α‐linolenic acid accumulating developmental defective embryo was rescued by lysophosphatidic acid acyltransferase 2

For decades, genetic engineering approaches to produce unusual fatty acids (UFAs) in crops has reached a bottleneck, including reduced seed oil production and seed vigor. Currently, plant models in the field of research are primarily used to investigate defects in oil production and seedling development, while the role of UFAs in embryonic developmental defects remains unknown. In this study, we developed a transgenic Arabidopsis plant model, in which the embryo exhibits severely wrinkled appearance owing to α‐linolenic acid (ALA) accumulation. RNA‐sequencing analysis in the defective embryo suggested that brassinosteroid synthesis, FA synthesis and photosynthesis were inhibited, while FA degradation, endoplasmic reticulum stress and oxidative stress were activated. Lipidomics analysis showed that ultra‐accumulated ALA is released from phosphatidylcholine as a free FA in cells, inducing severe endoplasmic reticulum and oxidative stress. Furthermore, we identified that overexpression of lysophosphatidic acid acyltransferase 2 rescued the defective phenotype. In the rescue line, the pool capacity of the Kennedy pathway was increased, and the esterification of ALA indirectly to triacylglycerol was enhanced to avoid stress. This study provides a plant model that aids in understanding the molecular mechanism of embryonic developmental defects and generates strategies to produce higher levels of UFAs.     

A dominant‐negative form of Arabidopsis AP‐3 β‐adaptin improves intracellular pH homeostasis

Intracellular pH (pH_i) is a crucial parameter in cellular physiology but its mechanisms of homeostasis are only partially understood. To uncover novel roles and participants of the pH_i regulatory system, we have screened an Arabidopsis mutant collection for resistance of seed germination to intracellular acidification induced by weak organic acids (acetic, propionic, sorbic). The phenotypes of one identified mutant, weak acid‐tolerant 1‐1D (wat1‐1D) are due to the expression of a truncated form of AP‐3 β‐adaptin (encoded by the PAT2 gene) that behaves as a as dominant‐negative. During acetic acid treatment the root epidermal cells of the mutant maintain a higher pH_i and a more depolarized plasma membrane electrical potential than wild‐type cells. Additional phenotypes of wat1‐1D roots include increased rates of acetate efflux, K⁺ uptake and H⁺ efflux, the latter reflecting the in vivo activity of the plasma membrane H⁺‐ATPase. The in vitro activity of the enzyme was not increased but, as the H⁺‐ATPase is electrogenic, the increased ion permeability would allow a higher rate of H⁺ efflux. The AP‐3 adaptor complex is involved in traffic from Golgi to vacuoles but its function in plants is not much known. The phenotypes of the wat1‐1D mutant can be explained if loss of function of the AP‐3 β‐adaptin causes activation of channels or transporters for organic anions (acetate) and for K⁺ at the plasma membrane, perhaps through miss‐localization of tonoplast proteins. This suggests a role of this adaptin in trafficking of ion channels or transporters to the tonoplast.     

Multi‐level regulation of the chloroplast ATP synthase: the chloroplast NADPH thioredoxin reductase C (NTRC) is required for redox modulation specifically under low irradiance

The chloroplast ATP synthase is known to be regulated by redox modulation of a disulfide bridge on the γ‐subunit through the ferredoxin–thioredoxin regulatory system. We show that a second enzyme, the recently identified chloroplast NADPH thioredoxin reductase C (NTRC), plays a role specifically at low irradiance. Arabidopsis mutants lacking NTRC (ntrc) displayed a striking photosynthetic phenotype in which feedback regulation of the light reactions was strongly activated at low light, but returned to wild‐type levels as irradiance was increased. This effect was caused by an altered redox state of the γ‐subunit under low, but not high, light. The low light‐specific decrease in ATP synthase activity in ntrc resulted in a buildup of the thylakoid proton motive force with subsequent activation of non‐photochemical quenching and downregulation of linear electron flow. We conclude that NTRC provides redox modulation at low light using the relatively oxidizing substrate NADPH, whereas the canonical ferredoxin–thioredoxin system can take over at higher light, when reduced ferredoxin can accumulate. Based on these results, we reassess previous models for ATP synthase regulation and propose that NTRC is most likely regulated by light. We also find that ntrc is highly sensitive to rapidly changing light intensities that probably do not involve the chloroplast ATP synthase, implicating this system in multiple photosynthetic processes, particularly under fluctuating environmental conditions.     

Arabidopsis plants expressing only the redox‐regulated Rca‐α isoform have constrained photosynthesis and plant growth

Rubisco activase (Rca) facilitates the release of sugar‐phosphate inhibitors from the active sites of Rubisco and thereby plays a central role in initiating and sustaining Rubisco activation. In Arabidopsis, alternative splicing of a single Rca gene results in two Rca isoforms, Rca‐α and Rca‐β. Redox modulation of Rca‐α regulates the function of Rca‐α and Rca‐β acting together to control Rubisco activation. Although Arabidopsis Rca‐α alone less effectively activates Rubisco in vitro, it is not known how CO₂ assimilation and plant growth are impacted. Here, we show that two independent transgenic Arabidopsis lines expressing Rca‐α in the absence of Rca‐β (‘Rca‐α only’ lines) grew more slowly in various light conditions, especially under low light or fluctuating light intensity, and in a short day photoperiod compared to wildtype. Photosynthetic induction was slower in the Rca‐α only lines, and they maintained a lower rate of CO₂ assimilation during both photoperiod types. Our findings suggest Rca oligomers composed of Rca‐α only are less effective in initiating and sustaining the activation of Rubisco than when Rca‐β is also present. Currently there are no examples of any plant species that naturally express Rca‐α only but numerous examples of species expressing Rca‐β only. That Rca‐α exists in most plant species, including many C3 and C4 food and bioenergy crops, implies its presence is adaptive under some circumstances.     

The hybrid Four‐CBS‐Domain KINβγ subunit functions as the canonical γ subunit of the plant energy sensor SnRK1

The AMPK/SNF1/SnRK1 protein kinases are a family of ancient and highly conserved eukaryotic energy sensors that function as heterotrimeric complexes. These typically comprise catalytic α subunits and regulatory β and γ subunits, the latter function as the energy‐sensing modules of animal AMPK through adenosine nucleotide binding. The ability to monitor accurately and adapt to changing environmental conditions and energy supply is essential for optimal plant growth and survival, but mechanistic insight in the plant SnRK1 function is still limited. In addition to a family of γ‐like proteins, plants also encode a hybrid βγ protein that combines the Four‐Cystathionine β‐synthase (CBS)‐domain (FCD) structure in γ subunits with a glycogen‐binding domain (GBD), typically found in β subunits. We used integrated functional analyses by ectopic SnRK1 complex reconstitution, yeast mutant complementation, in‐depth phylogenetic reconstruction, and a seedling starvation assay to show that only the hybrid KINβγ protein that recruited the GBD around the emergence of the green chloroplast‐containing plants, acts as the canonical γ subunit required for heterotrimeric complex formation. Mutagenesis and truncation analysis further show that complex interaction in plant cells and γ subunit function in yeast depend on both a highly conserved FCD and a pre‐CBS domain, but not the GBD. In addition to novel insight into canonical AMPK/SNF/SnRK1 γ subunit function, regulation and evolution, we provide a new classification of plant FCD genes as a convenient and reliable tool to predict regulatory partners for the SnRK1 energy sensor and novel FCD gene functions.     

Light modulated activity of root alkaline/neutral invertase involves the interaction with 14‐3‐3 proteins

Alkaline/neutral invertases (A/N‐Invs) are now recognized as essential proteins in plant life. They catalyze the irreversible breakdown of sucrose into glucose and fructose and thus supply the cells with energy as well as signaling molecules. In this study we report on a mechanism that affects the activity of the cytosolic invertase AtCINV1 (At‐A/N‐InvG or AT1G35580). We demonstrate that Ser547 at the extreme C‐terminus of the AtCINV1 protein is a substrate of calcium‐dependent kinases (CPK3 and 21) and that phosphorylation creates a high‐affinity binding site for 14‐3‐3 proteins. The invertase as such has basal activity, but we provide evidence that interaction with 14‐3‐3 proteins enhances its activity. The analysis of three quadruple 14‐3‐3 mutants generated from six T‐DNA insertion mutants of the non‐epsilon family shows both specificity as well as redundancy for this function of 14‐3‐3 proteins. The strong reduction in hexose levels in the roots of one 14‐3‐3 quadruple mutant plant is in line with the activating function of 14‐3‐3 proteins. The physiological relevance of this mechanism that affects A/N‐invertase activity is underscored by the light‐induced activation and is another example of the central role of 14‐3‐3 proteins in mediating dark/light signaling. The nature of the light‐induced signal that travels from the shoot to root and the question whether this signal is transmitted via cytosolic Ca⁺⁺ changes that activate calcium‐dependent kinases, await further study.     

A TIR–NBS protein encoded by Arabidopsis Chilling Sensitive 1 (CHS1) limits chloroplast damage and cell death at low temperature

Survival of plants at low temperature depends on mechanisms for limiting physiological damage and maintaining growth. We mapped the chs1‐1 (chilling sensitive1‐1) mutation in Arabidopsis accession Columbia to the TIR‐NBS gene At1g17610. In chs1‐1, a single amino acid exchange at the CHS1 N‐terminus close to the conserved TIR domain creates a stable mutant protein that fails to protect leaves against chilling stress. The sequence of another TIR‐NBS gene (At5g40090) named CHL1 (CHS1‐like 1) is related to that of CHS1. Over‐expression of CHS1 or CHL1 alleviates chilling damage and enhances plant growth at moderate (24°C) and chilling (13°C) temperatures, suggesting a role for both proteins in growth homeostasis. chs1‐1 mutants show induced salicylic acid production and defense gene expression at 13°C, indicative of autoimmunity. Genetic analysis of chs1‐1 in combination with defense pathway mutants shows that chs1‐1 chilling sensitivity requires the TIR‐NBS‐LRR and basal resistance regulators encoded by EDS1 and PAD4 but not salicylic acid. By following the timing of metabolic, physiological and chloroplast ultrastructural changes in chs1‐1 leaves during chilling, we have established that alterations in photosynthetic complexes and thylakoid membrane integrity precede leaf cell death measured by ion leakage. At 24°C, the chs1‐1 mutant appears normal but produces a massive necrotic response to virulent Pseudomonas syringae pv. tomato infection, although this does not affect bacterial proliferation. Our results suggest that CHS1 acts at an intersection between temperature sensing and biotic stress pathway activation to maintain plant performance over a range of conditions.     

The diversion of 2‐C‐methyl‐d‐erythritol‐2,4‐cyclodiphosphate from the 2‐C‐methyl‐d‐erythritol 4‐phosphate pathway to hemiterpene glycosides mediates stress responses in Arabidopsis thaliana

2‐C‐Methyl‐d ‐erythritol‐2,4‐cyclodiphosphate (MEcDP) is an intermediate of the plastid‐localized 2‐C‐methyl‐d ‐erythritol‐4‐phosphate (MEP) pathway which supplies isoprenoid precursors for photosynthetic pigments, redox co‐factor side chains, plant volatiles, and phytohormones. The Arabidopsis hds‐3 mutant, defective in the 1‐hydroxy‐2‐methyl‐2‐(E)‐butenyl‐4‐diphosphate synthase step of the MEP pathway, accumulates its substrate MEcDP as well as the free tetraol 2‐C‐methyl‐d ‐erythritol (ME) and glucosylated ME metabolites, a metabolic diversion also occurring in wild type plants. MEcDP dephosphorylation to the free tetraol precedes glucosylation, a process which likely takes place in the cytosol. Other MEP pathway intermediates were not affected in hds‐3. Isotopic labeling, dark treatment, and inhibitor studies indicate that a second pool of MEcDP metabolically isolated from the main pathway is the source of a signal which activates salicylic acid induced defense responses before its conversion to hemiterpene glycosides. The hds‐3 mutant also showed enhanced resistance to the phloem‐feeding aphid Brevicoryne brassicae due to its constitutively activated defense response. However, this MEcDP‐mediated defense response is developmentally dependent and is repressed in emerging seedlings. MEcDP and ME exogenously applied to adult leaves mimics many of the gene induction effects seen in the hds‐3 mutant. In conclusion, we have identified a metabolic shunt from the central MEP pathway that diverts MEcDP to hemiterpene glycosides via ME, a process linked to balancing plant responses to biotic stress.     

ABC1K1/PGR6 kinase: a regulatory link between photosynthetic activity and chloroplast metabolism

Arabidopsis proton gradient regulation (pgr) mutants have high chlorophyll fluorescence and reduced non‐photochemical quenching (NPQ) caused by defects in photosynthetic electron transport. Here, we identify PGR6 as the chloroplast lipid droplet (plastoglobule, PG) kinase ABC1K1 (activity of bc1 complex kinase 1). The members of the ABC1/ADCK/UbiB family of atypical kinases regulate ubiquinone synthesis in bacteria and mitochondria, and impact various metabolic pathways in plant chloroplasts. Here, we demonstrate that abc1k1 has a unique photosynthetic and metabolic phenotype that is distinct from that of the abc1k3 homolog. The abc1k1/pgr6 single mutant is specifically deficient in the electron carrier plastoquinone, as well as in β–carotene and the xanthophyll lutein, and is defective in membrane antioxidant tocopherol metabolism. After 2 days of continuous high light stress, abc1k1/pgr6 plants suffer extensive photosynthetic and metabolic perturbations, strongly affecting carbohydrate metabolism. Remarkably, however, the mutant acclimates to high light after 7 days together with a recovery of carotenoid levels and a drastic alteration in the starch‐to‐sucrose ratio. Moreover, ABC1K1 behaves as an active kinase and phosphorylates VTE1, a key enzyme of tocopherol (vitamin E) metabolism in vitro. Our results indicate that the ABC1K1 kinase constitutes a new type of regulatory link between photosynthetic activity and chloroplast metabolism.     

The E3 ubiquitin ligase SP1‐like 1 plays a positive role in peroxisome biogenesis in Arabidopsis

Peroxisomes are dynamic organelles crucial for a variety of metabolic processes during the development of eukaryotic organisms, and are functionally linked to other subcellular organelles, such as mitochondria and chloroplasts. Peroxisomal matrix proteins are imported by peroxins (PEX proteins), yet the modulation of peroxin functions is poorly understood. We previously reported that, besides its known function in chloroplast protein import, the Arabidopsis E3 ubiquitin ligase SP1 (suppressor of ppi1 locus1) also targets to peroxisomes and mitochondria, and promotes the destabilization of the peroxisomal receptor–cargo docking complex components PEX13 and PEX14. Here we present evidence that in Arabidopsis, SP1's closest homolog SP1‐like 1 (SPL1) plays an opposite role to SP1 in peroxisomes. In contrast to sp1, loss‐of‐function of SPL1 led to reduced peroxisomal β‐oxidation activity, and enhanced the physiological and growth defects of pex14 and pex13 mutants. Transient co‐expression of SPL1 and SP1 promoted each other's destabilization. SPL1 reduced the ability of SP1 to induce PEX13 turnover, and it is the N‐terminus of SP1 and SPL1 that determines whether the protein is able to promote PEX13 turnover. Finally, SPL1 showed prevalent targeting to mitochondria, but rather weak and partial localization to peroxisomes. Our data suggest that these two members of the same E3 protein family utilize distinct mechanisms to modulate peroxisome biogenesis, where SPL1 reduces the function of SP1. Plants and possibly other higher eukaryotes may employ this small family of E3 enzymes to differentially modulate the dynamics of several organelles essential to energy metabolism via the ubiquitin‐proteasome system.     

Redox signaling mediates the expression of a sulfate‐deprivation‐inducible microRNA395 in Arabidopsis

MicroRNA395 (miR395) is a conserved miRNA that targets a low‐affinity sulfate transporter (AST68) and three ATP sulfurylases (APS1, APS3 and APS4) in higher plants. In this study, At2g28780 was confirmed as another target of miR395 in Arabidopsis. Interestingly, several dicots contained genes homologous to At2g28780 and a cognate miR395 complementary site but possess a gradient of mismatches at the target site. It is well established that miR395 is induced during S deprivation in Arabidopsis; however, the signaling pathways that mediate this regulation are unknown. Several findings in the present study demonstrate that redox signaling plays an important role in induction of miR395 during S deprivation. These include the following results: (i) glutathione (GSH) supplementation suppressed miR395 induction in S‐deprived plants (ii) miR395 is induced in Arabidopsis seedlings exposed to Arsenate or Cu²⁺, which induces oxidative stress (iii), S deprivation‐induced oxidative stress, and (iv) compromised induction of miR395 during S deprivation in cad2 mutant (deficient in GSH biosynthesis) that is defective in glutaredoxin‐dependent redox signaling and ntra/ntrb (defective in thioredoxin reductases a and b) double mutants that are defective in thioredoxin‐dependent redox signaling. Collectively, these findings strongly support the involvement of redox signaling in inducing the expression of miR395 during S deprivation in Arabidopsis.