First-in-human study of ONO-4578, an antagonist of prostaglandin E2 receptor 4, alone and with nivolumab in solid tumors.

EP4, a prostaglandin E2 receptor, has shown an immunosuppressive activity on cancer cells. This first-in-human study evaluated ONO-4578, a highly selective EP4 antagonist, as monotherapy and in combination with nivolumab in patients with advanced or metastatic solid tumors. A daily dose ranging from 30 mg to 100 mg of ONO-4578 monotherapy and that ranging from 2 mg to 60 mg of ONO-4578 with biweekly nivolumab 240 mg were administered. A total of 31 patients were enrolled, 10 receiving monotherapy and 21 receiving combination therapy. Overall, 26 patients experienced treatment-related adverse events. Dose-limiting toxicities were observed in three patients; one of six patients receiving 100 mg monotherapy developed grade 3 duodenal ulcer and two of six patients receiving 60 mg combination therapy developed either grade 3 erythema multiforme or grade 3 increased amylase and grade 4 increased lipase. One patient with small-cell lung cancer who received 40 mg combination therapy had a partial response, and three patients with monotherapy and six patients with combination therapy had stable disease. Pharmacodynamics analyses showed that ONO-4578 had EP4 antagonistic activity at doses as low as 2 mg. In conclusion, the maximum tolerated dose of ONO-4578 alone or in combination with nivolumab was not reached. ONO-4578 was well tolerated at the tested doses and showed signs of antitumor activity. Considering safety, efficacy, and pharmacokinetics/pharmacodynamics results, ONO-4578 40 mg daily with nivolumab 240 mg biweekly was selected as the recommended dose for future clinical trials. (Registration: JapicCTI-173,496 and NCT03155061).

Involvement of Adapter Protein Complex 4 in Hypersensitive Cell Death Induced by Avirulent Bacteria.

Plant immunity to avirulent bacterial pathogens is associated with subcellular membrane dynamics including fusion between the vacuolar and plasma membranes, resulting in hypersensitive cell death. Here, we report that ADAPTOR PROTEIN COMPLEX-4 (AP-4) subunits are involved in plant immunity associated with hypersensitive cell death. We isolated a mutant with a defect in resistance to an avirulent strain of Pseudomonas syringae pv. tomato (Pto) DC3000 avrRpm1 from a vacuolar protein sorting mutant library of Arabidopsis (Arabidopsis thaliana). The mutant was identical to gfs4-1, which has a mutation in the gene encoding the AP-4 subunit AP4B. Thus, we focused on AP4B and another subunit, AP4E. All of the mutants (ap4b-3, ap4b-4, ap4e-1, and ap4e-2) were defective in hypersensitive cell death and resistance to Pto DC3000 with the type III effector AvrRpm1 or AvrRpt2, both of which are recognized on the plasma membrane, while they showed slightly enhanced susceptibility to the type-III-secretion-deficient P. syringae strain hrcC On the other hand, both ap4b-3 and ap4b-4 showed no defect in resistance to Pto DC3000 with the type III effector AvrRps4, which is recognized in the cytosol and does not induce hypersensitive cell death. Upon infection with Pto DC3000 avrRpt2, the ap4b-3 and ap4b-4 leaf cells did not show fusion between vacuolar and plasma membranes, whereas the wild-type leaf cells did. These results suggest that AP-4 contributes to cell death-associated immunity, possibly via membrane fusion, after type III effector-recognition on the plasma membrane.

Grana-Localized Proteins, RIQ1 and RIQ2, Affect the Organization of Light-Harvesting Complex II and Grana Stacking in Arabidopsis.

Grana are stacked thylakoid membrane structures in land plants that contain PSII and light-harvesting complex II proteins (LHCIIs). We isolated two Arabidopsis thaliana mutants, reduced induction of non-photochemical quenching1 (riq1) and riq2, in which stacking of grana was enhanced. The curvature thylakoid 1a (curt1a) mutant was previously shown to lack grana structure. In riq1 curt1a, the grana were enlarged with more stacking, and in riq2 curt1a, the thylakoids were abnormally stacked and aggregated. Despite having different phenotypes in thylakoid structure, riq1, riq2, and curt1a showed a similar defect in the level of nonphotochemical quenching of chlorophyll fluorescence (NPQ). In riq curt1a double mutants, NPQ induction was more severely affected than in either single mutant. In riq mutants, state transitions were inhibited and the PSII antennae were smaller than in wild-type plants. The riq defects did not affect NPQ induction in the chlorophyll b-less mutant. RIQ1 and RIQ2 are paralogous and encode uncharacterized grana thylakoid proteins, but despite the high level of identity of the sequence, the functions of RIQ1 and RIQ2 were not redundant. RIQ1 is required for RIQ2 accumulation, and the wild-type level of RIQ2 did not complement the NPQ and thylakoid phenotypes in riq1 We propose that RIQ proteins link the grana structure and organization of LHCIIs.

PYK10 myrosinase reveals a functional coordination between endoplasmic reticulum bodies and glucosinolates in Arabidopsis thaliana.

The endoplasmic reticulum body (ER body) is an organelle derived from the ER that occurs in only three families of the order Brassicales and is suggested to be involved in plant defense. ER bodies in Arabidopsis thaliana contain large amounts of ß-glucosidases, but the physiological functions of ER bodies and these enzymes remain largely unclear. Here we show that PYK10, the most abundant ß-glucosidase in A. thaliana root ER bodies, hydrolyzes indole glucosinolates (IGs) in addition to the previously reported in vitro substrate scopolin. We found a striking co-expression between ER body-related genes (including PYK10), glucosinolate biosynthetic genes and the genes for so-called specifier proteins affecting the terminal products of myrosinase-mediated glucosinolate metabolism, indicating that these systems have been integrated into a common transcriptional network. Consistent with this, comparative metabolite profiling utilizing a number of A. thaliana relatives within Brassicaceae identified a clear phylogenetic co-occurrence between ER bodies and IGs, but not between ER bodies and scopolin. Collectively, our findings suggest a functional link between ER bodies and glucosinolate metabolism in planta. In addition, in silico three-dimensional modeling, combined with phylogenomic analysis, suggests that PYK10 represents a clade of 16 myrosinases that arose independently from the other well-documented class of six thioglucoside glucohydrolases. These findings provide deeper insights into how glucosinolates are metabolized in cruciferous plants and reveal variation of the myrosinase-glucosinolate system within individual plants.

Overexpression of the protein disulfide isomerase AtCYO1 in chloroplasts slows dark-induced senescence in Arabidopsis.

BACKGROUND: Chlorophyll breakdown is the most obvious sign of leaf senescence. The chlorophyll catabolism pathway and the associated proteins/genes have been identified in considerable detail by genetic approaches combined with stay-green phenotyping. Arabidopsis CYO1 (AtCYO1), a protein disulfide reductase/isomerase localized in the thylakoid membrane, is hypothesized to assemble the photosystem by interacting with cysteine residues of the subunits. RESULTS: In this study, we report that ectopic overexpression of AtCYO1 in leaves induces a stay-green phenotype during darkness, where oxidative conditions favor catabolism. In AtCYO1ox leaves, Fv/Fm and both chlorophyll a and chlorophyll b content remained high during dark-induced senescence. The thylakoid ultrastructure was preserved for a longer time in AtCYO1ox leaves than in wild type leaves. AtCYO1ox leaves maintained thylakoid chlorophyll-binding proteins associated with both PSII (D1, D2, CP43, CP47, LHCB2, and Cyt f) and PSI (PSA-A/B), as well as stromal proteins (Rubisco and ferredoxin-NADP+ reductase). AtCYO1ox did not affect senescence-inducible gene expression for chlorophyll catabolism or accumulation of chlorophyll catabolites. CONCLUSIONS: Our results suggest that ectopic overexpression of AtCYO1 had a negative impact on the initiation of chlorophyll degradation and proteolysis within chloroplasts. Our findings cast new light on the redox regulation of protein disulfide bonds for the maintenance of functional chloroplasts.

A novel membrane fusion-mediated plant immunity against bacterial pathogens.

Plants have developed their own defense strategies because they have no immune cells. A common plant defense strategy involves programmed cell death (PCD) at the infection site, but how the PCD-associated cell-autonomous immunity is executed in plants is not fully understood. Here we provide a novel mechanism underlying cell-autonomous immunity, which involves the fusion of membranes of a large central vacuole with the plasma membrane, resulting in the discharge of vacuolar antibacterial proteins to the outside of the cells, where bacteria proliferate. The extracellular fluid that was discharged from the vacuoles of infected leaves had both antibacterial activity and cell death-inducing activity. We found that a defect in proteasome function abolished the membrane fusion associated with both disease resistance and PCD in response to avirulent bacterial strains but not to a virulent strain. Furthermore, RNAi plants with a defective proteasome subunit PBA1 have reduced DEVDase activity, which is an activity associated with caspase-3, one of the executors of animal apoptosis. The plant counterpart of caspase-3 has not yet been identified. Our results suggest that PBA1 acts as a plant caspase-3-like enzyme. Thus, this novel defense strategy through proteasome-regulating membrane fusion of the vacuolar and plasma membranes provides plants with a mechanism for attacking intercellular bacterial pathogens.

[Evaluation of a Newly Developed Reagent "Lias Auto P-FDP" on Coapresta 2000 and Clinico-Pathological Analysis of Discrepant Samples in FDP Value].

Patients with disseminated intravascular coagulation (DIC) exhibit increased levels of fibrin/fibrinogen degradation products (FDP), and FDP levels are carefully monitored as a marker of fibrinolysis during the diagnosis of DIC and other fibrinolysis-related conditions. Although FDP levels can be measured using automatic analyzers, it is reported that measured FDP values differ depending on the reagent used. Recently, a newly developed reagent for measuring FDP levels (Lias Auto P-FDP) was launched. In this study, we used Lias Auto P-FDP in combination with the Coapresta 2000 automatic analyzer and evaluated its reactivity. We confirmed the reproducibility of the measurements obtained with the Lias Auto P-FDP reagent when the manufacturer's instructions were followed and that the Lias Auto P-FDP reagent can be used with the Coapresta 2000. In the reactivity test, the Lias Auto P-FDP reagent exhibited stronger reactivity with low molecular weight FDP than the reagent we currently use (BL2 P-FDP). Moreover, the samples that exhibited non-specific reactions to the BL2 P-FDP reagent did not display similar reactions to the Lias auto P-FDP reagent. In the clinico-pathological analysis of divergent value between Lias Auto P-FDP and BL2 P-FDP, seven cases were highly discrepant value of FDP. Interestingly, we found the description of pleural fluid and/or ascites in 85.7% cases. In conclusion, we confirmed that the Lias Auto P-FDP reagent can be used in combination with the Coapresta 2000. In addition, the reactivity of FDP varied depending on the reagent used. It is important to understand the characteristics of each FDP reagent and which automatic analyzers they can be used with. [Original].

Organ-specific quality control of plant peroxisomes is mediated by autophagy.

Peroxisomes are essential organelles that are characterized by the possession of enzymes that produce hydrogen peroxide (H2O2) as part of their normal catalytic cycle. During the metabolic process, peroxisomal proteins are inevitably damaged by H2O2 and the integrity of the peroxisomes is impaired. Here, we show that autophagy, an intracellular process for vacuolar degradation, selectively degrades dysfunctional peroxisomes. Marked accumulation of peroxisomes was observed in the leaves but not roots of autophagy-related (ATG)-knockout Arabidopsis thaliana mutants. The peroxisomes in leaf cells contained markedly increased levels of catalase in an insoluble and inactive aggregate form. The chemically inducible complementation system in ATG5-knockout Arabidopsis provided the evidence that these accumulated peroxisomes were delivered to vacuoles for degradation by autophagy. Interestingly, autophagosomal membrane structures specifically recognized the abnormal peroxisomes at the site of the aggregates. Thus, autophagy is essential for the quality control of peroxisomes in leaves and for proper plant development under natural growth conditions.

Extension of oil biosynthesis during the mid-phase of seed development enhances oil content in Arabidopsis seeds.

Regulation of oil biosynthesis in plant seeds has been extensively studied, and biotechnological approaches have been designed to increase seed oil content. Oil and protein synthesis is negatively correlated in seeds, but the mechanisms controlling interactions between these two pathways are unknown. Here, we identify the molecular mechanism controlling oil and protein content in seeds. We utilized transgenic Arabidopsis thaliana plants overexpressing WRINKLED1 (WRI1), a master transcription factor regulating seed oil biosynthesis, and knockout mutants of major seed storage proteins. Oil and protein biosynthesis in wild-type plants was sequentially activated during early and late seed development, respectively. The negative correlation between oil and protein contents in seeds arises from competition between the pathways. Extension of WRI1 expression during mid-phase of seed development significantly enhanced seed oil content. This study demonstrates that temporal activation of genes involved in oil or storage protein biosynthesis determines the oil/protein ratio in Arabidopsis seeds. These results provide novel insights into potential breeding strategies to generate crops with high oil contents in seeds.

Spatiotemporal secretion of PEROXIDASE36 is required for seed coat mucilage extrusion in Arabidopsis.

The epidermal cells of the Arabidopsis thaliana seed coat, which correspond to the second layer of the outer integument (oi2), contain large quantities of a pectic polysaccharide called mucilage within the apoplastic space beneath the outer periclinal cell wall. Immediately after seed imbibition, the mucilage is extruded and completely envelops the seed in a gel-like capsule. We found that a class III peroxidase family protein, PEROXIDASE36 (PER36), functions as a mucilage extrusion factor. Expression of PER36 occurred only in oi2 cells for a few days around the torpedo stage. A PER36-green fluorescent protein fusion was secreted into the outer cell wall in a polarized manner. per36 mutants were defective in mucilage extrusion after seed imbibition due to the failure of outer cell wall rupture, although the mutants exhibited normal monosaccharide composition of the mucilage. This abnormal phenotype of per36 was rescued by pectin solubilization, which promoted cell wall loosening. These results suggest that PER36 regulates the degradation of the outer cell wall. Taken together, this work indicates that polarized secretion of PER36 in a developmental stage-dependent manner plays a role in cell wall modification of oi2 cells.

MAIGO5 functions in protein export from Golgi-associated endoplasmic reticulum exit sites in Arabidopsis.

Plant cells face unique challenges to efficiently export cargo from the endoplasmic reticulum (ER) to mobile Golgi stacks. Coat protein complex II (COPII) components, which include two heterodimers of Secretory23/24 (Sec23/24) and Sec13/31, facilitate selective cargo export from the ER; however, little is known about the mechanisms that regulate their recruitment to the ER membrane, especially in plants. Here, we report a protein transport mutant of Arabidopsis thaliana, named maigo5 (mag5), which abnormally accumulates precursor forms of storage proteins in seeds. mag5-1 has a deletion in the putative ortholog of the Saccharomyces cerevisiae and Homo sapiens Sec16, which encodes a critical component of ER exit sites (ERESs). mag mutants developed abnormal structures (MAG bodies) within the ER and exhibited compromised ER export. A functional MAG5/SEC16A-green fluorescent protein fusion localized at Golgi-associated cup-shaped ERESs and cycled on and off these sites at a slower rate than the COPII coat. MAG5/SEC16A interacted with SEC13 and SEC31; however, in the absence of MAG5/SEC16A, recruitment of the COPII coat to ERESs was accelerated. Our results identify a key component of ER export in plants by demonstrating that MAG5/SEC16A is required for protein export at ERESs that are associated with mobile Golgi stacks, where it regulates COPII coat turnover.

Highly oxidized peroxisomes are selectively degraded via autophagy in Arabidopsis.

The positioning of peroxisomes in a cell is a regulated process that is closely associated with their functions. Using this feature of the peroxisomal positioning as a criterion, we identified three Arabidopsis thaliana mutants (peroxisome unusual positioning1 [peup1], peup2, and peup4) that contain aggregated peroxisomes. We found that the PEUP1, PEUP2, and PEUP4 were identical to Autophagy-related2 (ATG2), ATG18a, and ATG7, respectively, which are involved in the autophagic system. The number of peroxisomes was increased and the peroxisomal proteins were highly accumulated in the peup1 mutant, suggesting that peroxisome degradation by autophagy (pexophagy) is deficient in the peup1 mutant. These aggregated peroxisomes contained high levels of inactive catalase and were more oxidative than those of the wild type, indicating that peroxisome aggregates comprise damaged peroxisomes. In addition, peroxisome aggregation was induced in wild-type plants by exogenous application of hydrogen peroxide. The cat2 mutant also contained peroxisome aggregates. These findings demonstrate that hydrogen peroxide as a result of catalase inactivation is the inducer of peroxisome aggregation. Furthermore, an autophagosome marker, ATG8, frequently colocalized with peroxisome aggregates, indicating that peroxisomes damaged by hydrogen peroxide are selectively degraded by autophagy in the wild type. Our data provide evidence that autophagy is crucial for quality control mechanisms for peroxisomes in Arabidopsis.

Pattern dynamics in adaxial-abaxial specific gene expression are modulated by a plastid retrograde signal during Arabidopsis thaliana leaf development.

The maintenance and reformation of gene expression domains are the basis for the morphogenic processes of multicellular systems. In a leaf primordium of Arabidopsis thaliana, the expression of FILAMENTOUS FLOWER (FIL) and the activity of the microRNA miR165/166 are specific to the abaxial side. This miR165/166 activity restricts the target gene expression to the adaxial side. The adaxial and abaxial specific gene expressions are crucial for the wide expansion of leaf lamina. The FIL-expression and the miR165/166-free domains are almost mutually exclusive, and they have been considered to be maintained during leaf development. However, we found here that the position of the boundary between the two domains gradually shifts from the adaxial side to the abaxial side. The cell lineage analysis revealed that this boundary shifting was associated with a sequential gene expression switch from the FIL-expressing (miR165/166 active) to the miR165/166-free (non-FIL-expressing) states. Our genetic analyses using the enlarged fil expression domain2 (enf2) mutant and chemical treatment experiments revealed that impairment in the plastid (chloroplast) gene expression machinery retards this boundary shifting and inhibits the lamina expansion. Furthermore, these developmental effects caused by the abnormal plastids were not observed in the genomes uncoupled1 (gun1) mutant background. This study characterizes the dynamic nature of the adaxial-abaxial specification process in leaf primordia and reveals that the dynamic process is affected by the GUN1-dependent retrograde signal in response to the failure of plastid gene expression. These findings advance our understanding on the molecular mechanism linking the plastid function to the leaf morphogenic processes.

Role of galactolipid biosynthesis in coordinated development of photosynthetic complexes and thylakoid membranes during chloroplast biogenesis in Arabidopsis.

The galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the predominant lipids in thylakoid membranes and indispensable for photosynthesis. Among the three isoforms that catalyze MGDG synthesis in Arabidopsis thaliana, MGD1 is responsible for most galactolipid synthesis in chloroplasts, whereas MGD2 and MGD3 are required for DGDG accumulation during phosphate (Pi) starvation. A null mutant of Arabidopsis MGD1 (mgd1-2), which lacks both galactolipids and shows a severe defect in chloroplast biogenesis under nutrient-sufficient conditions, accumulated large amounts of DGDG, with a strong induction of MGD2/3 expression, during Pi starvation. In plastids of Pi-starved mgd1-2 leaves, biogenesis of thylakoid-like internal membranes, occasionally associated with invagination of the inner envelope, was observed, together with chlorophyll accumulation. Moreover, the mutant accumulated photosynthetic membrane proteins upon Pi starvation, indicating a compensation for MGD1 deficiency by Pi stress-induced galactolipid biosynthesis. However, photosynthetic activity in the mutant was still abolished, and light-harvesting/photosystem core complexes were improperly formed, suggesting a requirement for MGDG for proper assembly of these complexes. During Pi starvation, distribution of plastid nucleoids changed concomitantly with internal membrane biogenesis in the mgd1-2 mutant. Moreover, the reduced expression of nuclear- and plastid-encoded photosynthetic genes observed in the mgd1-2 mutant under Pi-sufficient conditions was restored after Pi starvation. In contrast, Pi starvation had no such positive effects in mutants lacking chlorophyll biosynthesis. These observations demonstrate that galactolipid biosynthesis and subsequent membrane biogenesis inside the plastid strongly influence nucleoid distribution and the expression of both plastid- and nuclear-encoded photosynthetic genes, independently of photosynthesis.

MAG2 and three MAG2-INTERACTING PROTEINs form an ER-localized complex to facilitate storage protein transport in Arabidopsis thaliana.

In Arabidopsis thaliana, MAIGO 2 (MAG2) is involved in protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus via its association with the ER-localized t-SNARE components SYP81/AtUfe1 and SEC20. To characterize the molecular machinery of MAG2-mediated protein transport, we explored MAG2-interacting proteins using transgenic A. thaliana plants expressing TAP-tagged MAG2. We identified three proteins, which were designated as MAG2-INTERACTING PROTEIN 1-3 [MIP1 (At2g32900), MIP2 (At5g24350) and MIP3 (At2g42700)]. Both MIP1 and MAG2 localized to the ER membrane. All of the mag2, mip1, mip2 and mip3 mutants exhibited a defect in storage protein maturation, and developed abnormal storage protein body (MAG body) structures in the ER of seed cells. These observations suggest that MIPs are closely associated with MAG2 and function in protein transport between the ER and Golgi apparatus. MIP1 and MIP2 contain a Zeste-White 10 (ZW10) domain and a Sec39 domain, respectively, but have low sequence identities (21% and 23%) with respective human orthologs. These results suggest that the plant MAG2-MIP1-MIP2 complex is a counterpart of the triple-subunit tethering complexes in yeast (Tip20p-Dsl1p-Sec39p) and humans (RINT1-ZW10-NAG). Surprisingly, the plant complex also contained a fourth member (MIP3) with a Sec1 domain. There have been no previous reports showing that a Sec1-containing protein is a subunit of ER-localized tethering complexes. Our results suggest that MAG2 and the three MIP proteins form a unique complex on the ER that is responsible for efficient transport of seed storage proteins.

Chaperone and protease functions of LON protease 2 modulate the peroxisomal transition and degradation with autophagy.

Balancing repair and degradation is essential for maintaining organellar and cellular homeostasis. Peroxisomes are ubiquitous organelles in eukaryotic cells that play pivotal roles in cell survival. However, the quality control mechanism used to maintain peroxisomes is unclear. Here, we demonstrate that LON protease 2 (LON2), which is encoded by ABERRANT PEROXISOME MORPHOLOGY 10 (APEM10), is responsible for the functional transition of peroxisomes with autophagy. The Arabidopsis apem10 mutant displayed accelerated peroxisome degradation and a dramatically reduced number of peroxisomes. LON2 deficiency caused enhanced peroxisome degradation by autophagy, and peroxisomal proteins accumulated in the cytosol due to a decrease in the number of peroxisomes. We also show the proteolytic consequence of LON2 for the degradation of peroxisomal proteins, and we demonstrated that unnecessary proteins are eliminated by LON2- and autophagy-dependent degradation pathways during the functional transition of peroxisomes. LON2 plays dual roles as an ATP-dependent protease and a chaperone. We show that the chaperone domain of LON2 is essential for the suppression of autophagy, whereas its peptidase domain interferes with this chaperone function, indicating that intramolecular modulation between the proteolysis and chaperone functions of LON2 regulates degradation of peroxisomes by autophagy.

The Plant Organelles Database 3 (PODB3) update 2014: integrating electron micrographs and new options for plant organelle research.

The Plant Organelles Database 2 (PODB2), which was first launched in 2006 as PODB, provides static image and movie data of plant organelles, protocols for plant organelle research and external links to relevant websites. PODB2 has facilitated plant organellar research and the understanding of plant organelle dynamics. To provide comprehensive information on plant organelles in more detail, PODB2 was updated to PODB3 (http://podb.nibb.ac.jp/Organellome/). PODB3 contains two additional components: the electron micrograph database and the perceptive organelles database. Through the electron micrograph database, users can examine the subcellular and/or suborganellar structures in various organs of wild-type and mutant plants. The perceptive organelles database provides information on organelle dynamics in response to external stimuli. In addition to the extra components, the user interface for access has been enhanced in PODB3. The data in PODB3 are directly submitted by plant researchers and can be freely downloaded for use in further analysis. PODB3 contains all the information included in PODB2, and the volume of data and protocols deposited in PODB3 continue to grow steadily. We welcome contributions of data from all plant researchers to enhance the utility and comprehensiveness of PODB3.

A monogalactosyldiacylglycerol synthase found in the green sulfur bacterium Chlorobaculum tepidum reveals important roles for galactolipids in photosynthesis.

Monogalactosyldiacylglycerol (MGDG), which is conserved in almost all photosynthetic organisms, is the most abundant natural polar lipid on Earth. In plants, MGDG is highly accumulated in the chloroplast membranes and is an important bulk constituent of thylakoid membranes. However, precise functions of MGDG in photosynthesis have not been well understood. Here, we report a novel MGDG synthase from the green sulfur bacterium Chlorobaculum tepidum. This enzyme, MgdA, catalyzes MGDG synthesis using UDP-Gal as a substrate. The gene encoding MgdA was essential for this bacterium; only heterozygous mgdA mutants could be isolated. An mgdA knockdown mutation affected in vivo assembly of bacteriochlorophyll c aggregates, suggesting the involvement of MGDG in the construction of the light-harvesting complex called chlorosome. These results indicate that MGDG biosynthesis has been independently established in each photosynthetic organism to perform photosynthesis under different environmental conditions. We complemented an Arabidopsis thaliana MGDG synthase mutant by heterologous expression of MgdA. The complemented plants showed almost normal levels of MGDG, although they also had abnormal morphological phenotypes, including reduced chlorophyll content, no apical dominance in shoot growth, atypical flower development, and infertility. These observations provide new insights regarding the importance of regulated MGDG synthesis in the physiology of higher plants.

The plastidic DEAD-box RNA helicase 22, HS3, is essential for plastid functions both in seed development and in seedling growth.

Plants accumulate large amounts of storage products in seeds to provide an energy reserve and to supply nutrients for germination and post-germinative growth. Arabidopsis thaliana belongs to the Brassica family, and oil is the main storage product in Arabidopsis seeds. To elucidate the regulatory mechanisms of oil biosynthesis in seeds, we screened for high density seeds (heavy seed) that have a low oil content. HS3 (heavy seed 3) encodes the DEAD-box RNA helicase 22 that is localized to plastids. The triacylglycerol (TAG) content of hs3-1 seeds was 10% lower than that of wild-type (WT) seeds, while the protein content was unchanged. The hs3-1 plants displayed a pale-green phenotype in developing seeds and seedlings, but not in adult leaves. The HS3 expression level was high in developing seeds and seedlings, but was low in stems, rosette leaves and flowers. The plastid gene expression profile of WT developing seeds and seedlings differed from that of hs3-1 developing seeds and seedlings. The expression of several genes was reduced in developing hs3-1 seeds, including accD, a gene that encodes the ß subunit of carboxyltransferase, which is one component of acetyl-CoA carboxylase in plastids. In contrast, no differences were observed between the expression profiles of WT and hs3-1 rosette leaves. These results show that HS3 is essential for proper mRNA accumulation of plastid genes during seed development and seedling growth, and suggest that HS3 ensures seed oil biosynthesis by maintaining plastid mRNA levels.

Fusing the 3'UTR of seed storage protein genes leads to massive recombinant protein accumulation in seeds.

The demand for recombinant proteins is rising dramatically, and effective production systems are currently being developed. The production of recombinant proteins in plants is a promising approach due to its low cost and low risk of contamination of the proteins with endotoxins or infectious agents from the culture serum. Plant seeds primarily accumulate seed storage proteins (SSPs), which are transcribed and translated from a few genes; therefore, the mechanism underlying SSP accumulation has been studied to help devise ways to increase recombinant protein production. We found that the 3'UTR of SSP genes are essential for SSP accumulation and can be used in the production of recombinant proteins in Arabidopsis. Fusion of the 3'UTR of SSP genes to the 3' ends of DNA sequences encoding recombinant proteins enables massive accumulation of recombinant proteins with enzymatic activity in Arabidopsis seeds. This method is also applicable to the production of human Interferon Lambda-3 (IFN-lambda 3), a candidate biopharmaceutical compound against hepatitis C infection. Considering the low cost and ease of protein production in Arabidopsis, as well as the rapid growth of this plant, our method is useful for large-scale preparation of recombinant proteins for both academic research and biopharmaceutical production.