Amyloplast-Localized SUBSTANDARD STARCH GRAIN4 Protein Influences the Size of Starch Grains in Rice Endosperm

Starch is a biologically and commercially important polymer of glucose and is synthesized to form starch grains (SGs) inside amyloplasts. Cereal endosperm accumulates starch to levels that are more than 90% of the total weight, and most of the intracellular space is occupied by SGs. The size of SGs differs depending on the plant species and is one of the most important factors for industrial applications of starch. However, the molecular machinery that regulates the size of SGs is unknown. In this study, we report a novel rice (Oryza sativa) mutant called substandard starch grain4 (ssg4) that develops enlarged SGs in the endosperm. Enlargement of SGs in ssg4 was also observed in other starch-accumulating tissues such as pollen grains, root caps, and young pericarps. The SSG4 gene was identified by map-based cloning. SSG4 encodes a protein that contains 2,135 amino acid residues and an amino-terminal amyloplast-targeted sequence. SSG4 contains a domain of unknown function490 that is conserved from bacteria to higher plants. Domain of unknown function490-containing proteins with lengths greater than 2,000 amino acid residues are predominant in photosynthetic organisms such as cyanobacteria and higher plants but are minor in proteobacteria. The results of this study suggest that SSG4 is a novel protein that influences the size of SGs. SSG4 will be a useful molecular tool for future starch breeding and biotechnology.Plastids originated from the endosymbiosis of cyanobacteria and can differentiate into several forms depending on their intracellular functions during the plant life cycle (Sakamoto et al., 2008). The amyloplast is a terminally differentiated plastid responsible for starch synthesis and storage. Starch forms insoluble particles in amyloplasts, referred to as starch grains (SGs). SGs are easily visualized by staining with iodine solution, and they can be observed using a light microscope. SGs are observed in storage organs such as seed endosperm, potato (Solanum tuberosum) tubers, and pollen grains. Nonstorage tissues such as endodermis and root caps also contain SGs (Morita, 2010).Cereal endosperm accumulates high levels of starch in amyloplasts. The volume of SGs is approximately the same as the volume of amyloplasts that fill most of the intracellular space. SGs in rice (Oryza sativa) endosperm are normally 10 to 20 μm in diameter (Matsushima et al., 2010). One amyloplast contains a single SG that is assembled of several dozen smaller starch granules. Each starch granule is a sharp-edged polyhedron with a typical diameter of 3 to 8 μm. This type of SG is called a compound SG (Tateoka, 1962). For compound SGs, starch granules are assembled (but not fused) to form a single SG, which is easily separated by conventional purification procedures. By contrast, simple SGs contain a single starch granule. Simple SGs are produced in several important crops, such as maize (Zea mays), sorghum (Sorghum bicolor), barley (Hordeum vulgare), and wheat (Triticum aestivum; Tateoka, 1962; Matsushima et al., 2010, 2013).The size of SGs in cereal endosperm is diverse. Maize and sorghum SGs have a uniform size distribution of approximately 10 μm in diameter (Jane et al., 1994; Matsushima et al., 2010; Ai et al., 2011). In barley and wheat, SGs of two discrete size classes (approximately 15−25 μm and less than 10 μm) coexist in the same cells (Evers, 1973; French, 1984; Jane et al., 1994; Matsushima et al., 2010). In Bromus species, intrageneric size variations of SGs are observed in which even phylogenetic neighbors develop distinctly sized SGs (Matsushima et al., 2013). The size of SGs can be controlled by manipulating the activity of starch synthetic enzymes using transgenic plants or genetic mutants (Gutiérrez et al., 2002; Bustos et al., 2004; Ji et al., 2004; Stahl et al., 2004; Matsushima et al., 2010). However, the molecular mechanism that controls the interspecific size variations of SGs has not been resolved.The SG occupies most of the amyloplast interior, because the SG is approximately the same size as the amyloplast. The size of amyloplasts may affect the size of SGs, or vice versa. Amyloplasts and chloroplasts both develop from proplastids. The size of chloroplasts is controlled by the chloroplast binary fission division machinery, especially by the ring structures that form at the division sites (Miyagishima, 2011). Proteins involved in the ring structures have been isolated, including Filamenting temperature-sensitive mutantZ (FtsZ), Minicell locusD (MinD), MinE, and ACCUMULATION AND REPLICATIONS OF CHLOROPLAST5 (ARC5). Arabidopsis (Arabidopsis thaliana) mutants that are defective in these proteins have defects in chloroplast division and contain enlarged and dumbbell-shaped chloroplasts. In contrast to the binary fission of chloroplasts, amyloplasts divide at multiple sites and generate a beads-on-a-string structure (Yun and Kawagoe, 2009). The inhibition of the chloroplast division machinery does not result in enlarged amyloplasts (Yun and Kawagoe, 2009).We recently developed a rapid method to prepare thin sections of endosperm (Matsushima et al., 2010). Using this method, SGs in mature endosperm can be easily and clearly observed. We performed genetic screening for rice mutants defective in SG morphology and size. One of the isolated mutants, substandard starch grain4 (ssg4), develops enlarged SGs in its endosperm. In this study, we characterized ssg4 phenotypes and identified the responsible gene. SSG4 encodes a protein containing 2,135 amino acid residues and an N-terminal plastid-targeted sequence. The domain of unknown function 490 (DUF490) was found at the C terminus of SSG4, where the ssg4 mutation was located. This suggests that SSG4 is a novel factor that influences the size of SGs and has potential as a molecular tool for starch breeding and biotechnology.     

Projected coral bleaching in response to future sea surface temperature rises and the uncertainties among climate models

Long-term data on surface water temperature (SWT) from 9 lakes larger than 10 km² located in different climatic regions in Austria were analysed for June–September 1965–2009. The lakes are situated north and south of the Alps, in the east bordering Hungary and in the west bordering Germany. Time series of air temperature (AT) and SWT were smoothed by the lowess function and linear trends. Water temperature for the year 2050 was estimated from (1) linear extrapolation of the time trend, (2) projection of the AT–SWT relation and (3) increase of average present day SWT (2000–2009) by 3°C in summer in the Alps as expected from models by climatologists. Results indicate a rise in SWT parallel to AT since the mid-1960s. On an annual basis, changes in water temperature were the greatest in spring and summer. A conservative estimate of the average increase of summer SWT until 2050 is 2°C (1.2–2.9°C), differentiated by region. As a consequence of warming water temperatures, the duration of thermal stratification will increase and mixing and retention time will be affected. Changes in the food web are difficult to forecast, but will strongly depend on local environmental conditions and will therefore be different for individual lakes.     

Versatile design of biohybrid light-harvesting architectures to tune location,density, and spectral coverage of attached synthetic chromophores for enhanced energy capture

Biohybrid antennas built upon chromophore–polypeptide conjugates show promise for the design of efficient light-capturing modules for specific purposes. Three new designs, each of which employs analogs of the β-polypeptide from Rhodobacter sphaeroides, have been investigated. In the first design, amino acids at seven different positions on the polypeptide were individually substituted with cysteine, to which a synthetic chromophore (bacteriochlorin or Oregon Green) was covalently attached. The polypeptide positions are at –2, –6, –10, –14, –17, –21, and –34 relative to the 0-position of the histidine that coordinates bacteriochlorophyll a (BChl a). All chromophore–polypeptides readily formed LH1-type complexes upon combination with the α-polypeptide and BChl a. Efficient energy transfer occurs from the attached chromophore to the circular array of 875 nm absorbing BChl a molecules (denoted B875). In the second design, use of two attachment sites (positions –10 and –21) on the polypeptide affords (1) double the density of chromophores per polypeptide and (2) a highly efficient energy-transfer relay from the chromophore at –21 to that at –10 and on to B875. In the third design, three spectrally distinct bacteriochlorin–polypeptides were prepared (each attached to cysteine at the –14 position) and combined in an ~1:1:1 mixture to form a heterogeneous mixture of LH1-type complexes with increased solar coverage and nearly quantitative energy transfer from each bacteriochlorin to B875. Collectively, the results illustrate the great latitude of the biohybrid approach for the design of diverse light-harvesting systems.     

Characterization of the intramolecular transfer state of marine carotenoid fucoxanthin by femtosecond pump–probe spectroscopy

Fucoxanthin, containing a carbonyl group in conjugation with its polyene backbone, is a naturally occurring pigment in marine organisms and is essential to the photosynthetic light-harvesting function in brown alga and diatom. Fucoxanthin exhibits optical characteristics attributed to an intramolecular charge transfer (ICT) state that arises in polar environments due to the presence of the carbonyl group. In this study, we report the spectroscopic properties of fucoxanthin in methanol (polar and protic solvent) observed by femtosecond pump–probe measurements in the near-infrared region, where transient absorption associated with the optically allowed S₂ (1¹B _u ⁺ ) state and stimulated emission from the strongly coupled S₁/ICT state were observed following one-photon excitation to the S₂ state. The results showed that the amplitude of the stimulated emission of the S₁/ICT state increased with decreasing excitation energy, demonstrating that the fucoxanthin form associated with the lower energy of the steady-state absorption exhibits stronger ICT character.     

Statistical considerations on the formation of circular photosynthetic light-harvesting complexes from Rhodopseudomonas palustris

Depending on growth conditions, some species of purple photosynthetic bacteria contain peripheral light-harvesting (LH2) complexes that are heterogeneous owing to the presence of different protomers (containing different αβ-apoproteins). Recent spectroscopic studies of Rhodopseudomonas palustris grown under low-light conditions suggest the presence of a C ₃-symmetric LH2 nonamer comprised of two distinct protomers. The software program Cyclaplex, which enables generation and data-mining of virtual libraries of molecular rings formed upon combinatorial reactions, has been used to delineate the possible number and type of distinct nonamers as a function of numbers of distinct protomers. The yield of the C ₃-symmetric nonamer from two protomers (A and B in varying ratios) has been studied under the following conditions: (1) statistical, (2) enriched (preclusion of the B-B sequence), and (3) seeded (pre-formation of an A-B-A block). The yield of C ₃-symmetric nonamer is at most 0.98 % under statistical conditions versus 5.6 % under enriched conditions, and can be dominant under conditions of pre-seeding with an A-B-A block. In summary, the formation of any one specific nonamer even from only two protomers is unlikely on statistical grounds but must stem from enhanced free energy of formation or a directed assembly process by as-yet unknown factors.     

Design of recycling system for poly(methyl methacrylate) (PMMA). Part 2: process hazards and material flow analysis

Introduction

In this series of papers, we present a design of poly(methyl methacrylate) (PMMA) recycling system considering environmental impacts, chemical hazards, and resource availability. We applied life cycle assessment (LCA), environment, health, and safety (EHS) assessment as well as material flow analysis to the evaluation of the recycling system.

Purpose

Recycling systems for highly functional plastics such as PMMA have not been studied sufficiently. Along with the popularization of PMMA-containing products such as liquid crystal displays (LCDs), the use of PMMA is steadily increasing, which will result in more waste of PMMA in the next decades. In this study, pyrolysis process for recycling waste PMMA into methyl methacrylate (MMA) monomer was examined, considering not only general environmental impacts quantified by life cycle assessment but also local environment, health, and safety hazards, and raw material availability.

Methods

Process EHS hazards assessment was applied to quantify the local effects of the PMMA monomer recycling process. Process hazards are strongly connected with the hazardous properties of chemical substances and stream conditions within the process. Two alternative cooling methods exist, and their difference was analyzed by LCA and EHS assessment. Besides the process hazard, the availability of waste PMMA must be an important point for the feasibility of implementing the PMMA monomer recycling process. The available amount can be quantified by analyzing the material flow of PMMA-containing products. PMMA contained in LCDs as light guide panels was selected as a feasible source of waste PMMA, and the quantity of PMMA flows in the society was evaluated.

Results and discussion

In the case of PMMA, monomer recycling has less process hazard than the production of fresh MMA from crude oil. The implementation of circulated cooling water could significantly decrease the process hazard in PMMA pyrolysis attributable to chemical hazards. Material flow analysis revealed that the availability of waste PMMA shows a fluctuating trend in the next 20 years because of the sharp peak demand for LCD television sets. The fluctuation is strongly dependent on the lifetime of LCD television sets.

Conclusions

PMMA monomer recycling has a potential to reduce environmental impacts with a less process hazards than fresh MMA production from crude oil. The availability of waste PMMA has a strong relationship with the lifetime of LCD television sets. The multiple and comprehensive assessments can reveal various aspects of a process technology.     

Sema3A chemorepellant regulates the timing and patterning of dental nerves during development of incisor tooth germ

Semaphorin 3A (Sema3A) axon repellant serves multiple developmental functions. Sema3A mRNAs are expressed in epithelial and mesenchymal components of the developing incisor in a dynamic manner. Here, we investigate the functions of Sema3A during development of incisors using Sema3A-deficient mice. We analyze histomorphogenesis and innervation of mandibular incisors using immunohistochemistry as well as computed tomography and thick tissue confocal imaging. Whereas no apparent disturbances in histomorphogenesis or hard tissue formation of Sema3A ^?/? incisors were observed, nerve fibers were prematurely seen in the presumptive dental mesenchyme of the bud stage Sema3A ^?/? tooth germ. Later, nerves were ectopically present in the Sema3A ^?/? dental papilla mesenchyme during the cap and bell stages, whereas in the Sema3A ^+/+ mice the first nerve fibers were seen in the pulp after the onset of dental hard tissue formation. However, no apparent topographic differences in innervation pattern or nerve fasciculation were seen inside the pulp between postnatal and adult Sema3A ^+/+ or Sema3A ^?/? incisors. In contrast, an abnormally large number of nerves and arborizations were observed in the Sema3A ^?/? developing dental follicle target field and periodontium and, unlike in the wild-type mice, nerve fibers were abundant in the labial periodontium. Of note, the observed defects appeared to be mostly corrected in the adult incisors. The expressions of Ngf and Gdnf neurotrophins and their receptors were not altered in the Sema3A ^?/? postnatal incisor or trigeminal ganglion, respectively. Thus, Sema3A is an essential, locally produced chemorepellant, which by creating mesenchymal exclusion areas, regulates the timing and patterning of the dental nerves during the development of incisor tooth germ.