Ubiquitin-specific protease 8 deubiquitinates Sec31A and decreases large COPII carriers and collagen IV secretion.

Nascent cargo proteins in the endoplasmic reticulum are transported to the Golgi by COPII carriers. Typical COPII vesicles are 60-70 nm in diameter, and much larger macromolecules, such as procollagen, are transported by atypical large COPII carriers in mammalian cells. The formation of large COPII carriers is enhanced by Cul3 ubiquitin ligase, which mono-ubiquitinates Sec31A, a COPII coat protein. However, the deubiquitinating enzyme for Sec31A was unclear. Here, we show that the deubiquitinating enzyme USP8 interacts with and deubiquitinates Sec31A. The interaction was mediated by the adaptor protein STAM1. USP8 overexpression inhibited the formation of large COPII carriers. By contrast, USP8 knockdown caused the accumulation of COPII coat proteins around the cis-Golgi, promoted the intracellular trafficking of procollagen IV from the endoplasmic reticulum to the Golgi, and increased collagen IV secretion. We concluded that USP8 deubiquitinates Sec31A and inhibits the formation of large COPII carriers, thereby suppressing collagen IV secretion.

Amputation of a C-terminal helix of the γ subunit increases ATP-hydrolysis activity of cyanobacterial F1 ATP synthase.

F1 is a soluble part of FoF1-ATP synthase and performs a catalytic process of ATP hydrolysis and synthesis. The γ subunit, which is the rotary shaft of F1 motor, is composed of N-terminal and C-terminal helices domains, and a protruding Rossman-fold domain located between the two major helices parts. The N-terminal and C-terminal helices domains of γ assemble into an antiparallel coiled-coil structure, and are almost embedded into the stator ring composed of α3ß3 hexamer of the F1 molecule. Cyanobacterial and chloroplast γ subunits harbor an inserted sequence of 30 or 39 amino acids length within the Rossman-fold domain in comparison with bacterial or mitochondrial γ. To understand the structure-function relationship of the γ subunit, we prepared a mutant F1-ATP synthase of a thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1, in which the γ subunit is split into N-terminal α-helix along with the inserted sequence and the remaining C-terminal part. The obtained mutant showed higher ATP-hydrolysis activities than those containing the wild-type γ. Contrary to our expectation, the complexes containing the split γ subunits were mostly devoid of the C-terminal helix. We further investigated the effect of post-assembly cleavage of the γ subunit. We demonstrate that insertion of the nick between two helices of the γ subunit imparts resistance to ADP inhibition, and the C-terminal α-helix is dispensable for ATP-hydrolysis activity and plays a crucial role in the assembly of F1-ATP synthase.

Arabidopsis Phosphatidic Acid Phosphohydrolases Are Essential for Growth under Nitrogen-Depleted Conditions.

The Arabidopsis homologs of mammalian lipin, PAH1 and PAH2, are cytosolic phosphatidic acid phosphohydrolases that are involved in phospholipid biosynthesis and are essential for growth under phosphate starvation. Here, pah1 pah2 double-knockout mutants were found to be hypersensitive to nitrogen (N) starvation, whereas transgenic plants overexpressing PAH1 or PAH2 in the pah1 pah2 mutant background showed a similar growth phenotype as compared with wild type (WT) under N starvation. The chlorophyll content of pah1 pah2 was significantly lower than that of WT, whereas the chlorophyll content and photosynthetic activity of the transgenic plants were significantly higher than those of WT under N-depleted conditions. Membrane glycerolipid composition of the pah1 pah2 mutants showed a significant decrease in the mole percent of chloroplast lipids to other phospholipids, whereas membrane lipid composition did not differ between transgenic plants and WT plants. Pulse-chase labeling experiments using plants grown under N-depleted conditions showed that, in pah1 pah2 plants, the labeling percent of chloroplast lipids such as phosphatidylglycerol and monogalactosyldiacylglycerol in the total glycerolipids was significantly lower than in WT. Moreover, N starvation-induced degradation of chloroplast structure was enhanced in pah1 pah2 mutants, and the membrane structure was recovered by complementation with PAH1. Thus, PAH is involved in maintaining chloroplast membrane structure and is required for growth under N-depleted conditions.

Tangled evolutionary processes with commonality and diversity in plastidial glycolipid synthesis in photosynthetic organisms.

In photosynthetic organisms, the photosynthetic membrane constitutes a scaffold for light-harvesting complexes and photosynthetic reaction centers. Three kinds of glycolipids, namely monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and sulfoquinovosyldiacylglycerol, constitute approximately 80-90% of photosynthetic membrane lipids and are well conserved from tiny cyanobacteria to the leaves of huge trees. These glycolipids perform a wide variety of functions beyond biological membrane formation. In particular, the capability of adaptation to harsh environments through regulation of membrane glycolipid composition is essential for healthy growth and development of photosynthetic organisms. The genome analysis and functional genetics of the model seed plant Arabidopsis thaliana have yielded many new findings concerning the biosynthesis, regulation, and functions of glycolipids. Nevertheless, it remains to be clarified how the complex biosynthetic pathways and well-organized functions of glycolipids evolved in early and primitive photosynthetic organisms, such as cyanobacteria, to yield modern photosynthetic organisms like land plants. Recently, genome data for many photosynthetic organisms have been made available as the fruit of the rapid development of sequencing technology. We also have reported the draft genome sequence of the charophyte alga Klebsormidium flaccidum, which is an intermediate organism between green algae and land plants. Here, we performed a comprehensive phylogenic analysis of glycolipid biosynthesis genes in oxygenic photosynthetic organisms including K. flaccidum. Based on the results together with membrane lipid analysis of this alga, we discuss the evolution of glycolipid synthesis in photosynthetic organisms. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

An engineered lipid remodeling system using a galactolipid synthase promoter during phosphate starvation enhances oil accumulation in plants.

Inorganic phosphate (Pi) depletion is a serious problem for plant growth. Membrane lipid remodeling is a defense mechanism that plants use to survive Pi-depleted conditions. During Pi starvation, phospholipids are degraded to supply Pi for other essential biological processes, whereas galactolipid synthesis in plastids is up-regulated via the transcriptional activation of monogalactosyldiacylglycerol synthase 3 (MGD3). Thus, the produced galactolipids are transferred to extraplastidial membranes to substitute for phospholipids. We found that, Pi starvation induced oil accumulation in the vegetative tissues of various seed plants without activating the transcription of enzymes involved in the later steps of triacylglycerol (TAG) biosynthesis. Moreover, the Arabidopsis starchless phosphoglucomutase mutant, pgm-1, accumulated higher TAG levels than did wild-type plants under Pi-depleted conditions. We generated transgenic plants that expressed a key gene involved in TAG synthesis using the Pi deficiency-responsive MGD3 promoter in wild-type and pgm-1 backgrounds. During Pi starvation, the transgenic plants accumulated higher TAG amounts compared with the non-transgenic plants, suggesting that the Pi deficiency-responsive promoter of galactolipid synthase in plastids may be useful for producing transgenic plants that accumulate more oil under Pi-depleted conditions.

Differential regulation of two types of monogalactosyldiacylglycerol synthase in membrane lipid remodeling under phosphate-limited conditions in sesame plants.

Phosphate (Pi) limitation causes drastic lipid remodeling in plant membranes. Glycolipids substitute for the phospholipids that are degraded, thereby supplying Pi needed for essential biological processes. Two major types of remodeling of membrane lipids occur in higher plants: whereas one involves an increase in the concentration of sulfoquinovosyldiacylglycerol in plastids to compensate for a decreased concentration of phosphatidylglycerol, the other involves digalactosyldiacylglycerol (DGDG) synthesis in plastids and the export of DGDG to extraplastidial membranes to compensate for reduced abundances of phospholipids. Lipid remodeling depends on an adequate supply of monogalactosyldiacylglycerol (MGDG), which is a substrate that supports the elevated rate of DGDG synthesis that is induced by low Pi availability. Regulation of MGDG synthesis has been analyzed most extensively using the model plant Arabidopsis thaliana, although orthologous genes that encode putative MGDG synthases exist in photosynthetic organisms from bacteria to higher plants. We recently hypothesized that two types of MGDG synthase diverged after the appearance of seed plants. This divergence might have both enabled plants to adapt to a wide range of Pi availability in soils and contributed to the diversity of seed plants. In the work presented here, we found that membrane lipid remodeling also takes place in sesame, which is one of the most common traditional crops grown in Asia. We identified two types of MGDG synthase from sesame (encoded by SeMGD1 and SeMGD2) and analyzed their enzymatic properties. Our results show that both genes correspond to the Arabidopsis type-A and -B isoforms of MGDG synthase. Notably, whereas Pi limitation up-regulates only the gene encoding the type-B isoform of Arabidopsis, low Pi availability up-regulates the expression of both SeMGD1 and SeMGD2. We discuss the significance of the different responses to low Pi availability in sesame and Arabidopsis.

Loss of function of the IAA-glucose hydrolase gene TGW6 enhances rice grain weight and increases yield.

Increases in the yield of rice, a staple crop for more than half of the global population, are imperative to support rapid population growth. Grain weight is a major determining factor of yield. Here, we report the cloning and functional analysis of THOUSAND-GRAIN WEIGHT 6 (TGW6), a gene from the Indian landrace rice Kasalath. TGW6 encodes a novel protein with indole-3-acetic acid (IAA)-glucose hydrolase activity. In sink organs, the Nipponbare tgw6 allele affects the timing of the transition from the syncytial to the cellular phase by controlling IAA supply and limiting cell number and grain length. Most notably, loss of function of the Kasalath allele enhances grain weight through pleiotropic effects on source organs and leads to significant yield increases. Our findings suggest that TGW6 may be useful for further improvements in yield characteristics in most cultivars.

Time-related identification of chromosome regions affecting plant elongation in rice (Oryza sativa L.).

We investigated the time-related changes of Chromosome Regions that Affect Traits (CRATs) for elongation rate in rice (Oryza sativa L.) using chromosome segment substitution lines (CSSLs) carrying a single chromosome segment of the cultivar Kasalath (indica) in a Koshihikari (japonica) genetic background. The growth period of rice was partitioned into eight stages (each lasting 5-7days) from 18days after transplanting, and the elongation rate was determined as the increase of total plant height per time at each growth stage. CRATs for plant elongation rate were determined based on graphical genotype data of CSSLs that showed a significantly higher or lower elongation rate than Koshihikari. In total, 23 CRATs for plant elongation rate were detected, and different CRATs acted at different growth stages. Fifteen CRATs increased the elongation rate through Kasalath alleles, and eight increased it through Koshihikari alleles. These results suggest that plant height in rice is regulated in a stage-specific manner by a variety of genetic mechanisms that control plant elongation rate. Kasalath alleles of PE1-9 increased the elongation rate at an early growth stage (18-25days after transplanting), while Koshihikari alleles of PE8-3 decreased the elongation rate at a late growth stage (68-74days after transplanting). In a line that contained both of these CRATs, the elongation rate at the early growth stage was increased without affecting plant height at harvesting. We conclude that stage-specific optimization of plant height in rice may be achieved by combining CRATs that control plant elongation at specific stages.

New target for rice lodging resistance and its effect in a typhoon.

We demonstrated the new target for lodging resistance in rice (Oryza sativa L.) by the analysis of physiological function of a locus for lodging resistance in a typhoon (lrt5) with the near isogenic line under rice "Koshihikari" genetic background (tentatively named S1). The higher lodging resistance of S1 was observed during a typhoon in September 2004 (28 days after heading), when most other plants in "Koshihikari" became lodged. Visual observations showed that bending of the upper stems triggered lodging during the typhoon; the upper stem of "Koshihikari" buckled completely, whereas that of S1 remained straight. In addition to the strong rain and winds during the typhoon, the weight of the buckled upper plant parts increased the pressure on adjacent plants and caused a domino effect in "Koshihikari". Young's modulus, an indicator of the rigidity of the culm, was significantly higher in S1 than in "Koshihikari". In the upper culm, the starch content in S1 was 4.8 times the value in "Koshihikari", and senescence was delayed in the upper leaves of S1. These results suggest that the rigidity of the upper culm by the higher starch content (as a result of delayed senescence in the upper leaves) may be responsible for the higher lodging resistance during a typhoon in rice.

Quantitative trait loci for sucrose, starch, and hexose accumulation before heading in rice.

We studied the storage of sucrose, starch, and hexose before heading in rice (Oryza sativa L.) plants by quantitative trait locus (QTL) analysis with a population of backcross inbred lines (BILs) of japonica cv. Nipponbare x indica cv. Kasalath. Carbohydrates are accumulated in the rice plant before heading and are translated to the panicle after heading. A higher capacity for accumulation is thus a main target for improvement in yield. The form of carbohydrate (sucrose, starch, or hexose) differs depending on the organ in which it is stored. There was no correlation between starch and sucrose or hexose contents in BILs, and the positions of QTLs controlling starch differed from those for sucrose and hexose accumulation. These results suggest that the genetic control of accumulation differs between starch and sugars. QTLs that control the ratio of sucrose to starch content were detected, suggesting the existence of a mechanism(s) that determines this ratio. On chromosome 1, sucrose-phosphate synthase 1, the key enzyme in sucrose synthesis was close to the peaks of the likelihood odds ratios in QTLs for sucrose or hexose content. These results suggest that SPS1 is related to conversion of carbohydrate to sucrose as accumulated form in a plant before heading.

Locus prl5 improves lodging resistance of rice by delaying senescence and increasing carbohydrate reaccumulation.

We investigated the reason for carbohydrate retention in the stem of rice (Oryza sativa L.) at full-ripe stage in a near-isogenic line (NIL63) carrying prl5, which confers lodging resistance without yield loss. NIL63 showed higher lodging resistance than Nipponbare (control) without reduced yield. At heading, the carbohydrate content in the NIL63 stem (culm and leaf sheathes) was the same as in Nipponbare. At 2 weeks after heading, the carbohydrate content in NIL63 was significantly higher than in Nipponbare. At 4 weeks after heading, the carbohydrate content in NIL63 had decreased to near the level in Nipponbare. At 6 weeks after heading, NIL63 showed higher carbohydrate reaccumulation. Chlorophyll degradation in the leaf blades of NIL63 was slower, and the chlorophyll content at 6 weeks after heading was higher than in Nipponbare. These results suggest that the delay in leaf senescence by prl5 results in carbohydrate reaccumulation in the stem after grain filling, increasing lodging resistance.

Identification and physiological analyses of a locus for rice yield potential across the genetic background.

A new locus responsible for increased yield potential across the genetic background in rice (Oryza sativa L.) was identified and evaluated. Quantitative trait loci (QTLs) were analysed for the ratio of filled grains, a yield component, in backcrossed inbred lines of a japonica 'Nipponbare'xindica 'Kasalath' cross for 3 years. Only one QTL (rg5), with a positive Kasalath allele, was detected across environments (years). The physiological functions of rg5 were clarified in a near-isogenic line (NILrg5) with a Kasalath chromosome segment containing rg5 in a Nipponbare genetic background. In NILrg5, carbohydrate storage capacity before heading or sink activity in the first or last stages of the reproductive phase was significantly higher than in Nipponbare (control). The ratio of filled grains and yield per plant were significantly higher in NILrg5 than in Nipponbare, by 5% (P <0.01) and 15% (P <0.05), respectively. These results suggest that rg5 improves carbohydrate storage capacity and keeps sink activity higher in the reproductive stage, and consequently increases yield potential. Greater capacity to accumulate carbohydrate is the main target for increasing rice yield potential; therefore, rg5 might function under other genetic backgrounds. Substitution of the rice cv. Kasalath chromosome segment containing rg5 gave higher yield potential in the top premium rice cv. Koshihikari. These results suggest that rg5 might be able to affect yield under different genetic backgrounds, and physiological analyses of the targeted locus might reveal these effects.

Chloroplast transformation with modified accD operon increases acetyl-CoA carboxylase and causes extension of leaf longevity and increase in seed yield in tobacco.

Acetyl-CoA carboxylase (ACCase) in plastids is a key enzyme regulating the rate of de novo fatty acid biosynthesis in plants. Plastidic ACCase is composed of three nuclear-encoded subunits and one plastid-encoded accD subunit. To boost ACCase levels, we examined whether overexpression of accD elevates ACCase production. Using homologous recombination, we replaced the promoter of the accD operon in the tobacco plastid genome with a plastid rRNA-operon (rrn) promoter that directs enhanced expression in photosynthetic and non-photosynthetic organs, and successfully raised the total ACCase levels in plastids. This result suggests that the level of the accD subunit is a determinant of ACCase levels, and that enzyme levels are in part controlled post-transcriptionally at the level of subunit assembly. The resultant transformants grew normally and the fatty acid content was significantly increased in leaves, but not significantly in seeds. However, the transformants displayed extended leaf longevity and a twofold increase of seed yield over the control value, which eventually almost doubled the fatty acid production per plant of the transformants relative to control and wild-type plants. These findings offer a potential method for raising plant productivity and oil production.