The Evolution of C4 Photosynthesis in Flaveria (Asteraceae): Insights from the Flaveria linearis Complex.

Flaveria is a leading model for C4 plant evolution due to the presence of a dozen C3-C4 intermediate species, many of which are associated with a phylogenetic complex centered around Flaveria linearis. To investigate C4 evolution in Flaveria, we updated the Flaveria phylogeny and evaluated gas exchange, starch δ13C, and activity of C4 cycle enzymes in 19 Flaveria species and 28 populations within the F. linearis complex. A principal component analysis identified six functional clusters: (1) C3, (2) sub-C2, (3) full C2, (4) enriched C2, (5) sub-C4, and (6) fully C4 species. The sub-C2 species lacked a functional C4 cycle, while a gradient was present in the C2 clusters from little to modest C4 cycle activity as indicated by δ13C and enzyme activities. Three Yucatan populations of F. linearis had photosynthetic CO2 compensation points equivalent to C4 plants but showed little evidence for an enhanced C4 cycle, indicating they have an optimized C2 pathway that recaptures all photorespired CO2 in the bundle sheath (BS) tissue. All C2 species had enhanced aspartate aminotransferase activity relative to C3 species and most had enhanced alanine aminotransferase activity. These aminotransferases form aspartate and alanine from glutamate and in doing so could help return photorespiratory nitrogen (N) from BS to mesophyll cells, preventing glutamate feedback onto photorespiratory N assimilation. Their use requires upregulation of parts of the C4 metabolic cycle to generate carbon skeletons to sustain N return to the mesophyll, and thus could facilitate the evolution of the full C4 photosynthetic pathway.

Genome-wide association study of leaf photosynthesis using a high-throughput gas exchange system in rice.

Enhancing leaf photosynthetic capacity is essential for improving the yield of rice (Oryza sativa L.). Although the exploitation of natural genetic resources is considered a promising approach to enhance photosynthetic capacity, genomic factors related to the genetic diversity of leaf photosynthetic capacity have yet to be fully elucidated due to the limitation of measurement efficiency. In this study, we aimed to identify novel genomic regions for the net CO2 assimilation rate (A) by combining genome-wide association study (GWAS) and the newly developed rapid closed gas exchange system MIC-100. Using three MIC-100 systems in the field at the vegetative stage, we measured A of 168 temperate japonica rice varieties with six replicates for three years. We found that the modern varieties exhibited higher A than the landraces, while there was no significant relationship between the release year and A among the modern varieties. Our GWAS scan revealed two major peaks located on chromosomes 4 and 8, which were repeatedly detected in the different experiments and in the generalized linear modelling approach. We suggest that high-throughput gas exchange measurements combined with GWAS is a reliable approach for understanding the genetic mechanisms underlying photosynthetic diversities in crop species.

Development of 12 sets of chromosome segment substitution lines that enhance allele mining in Asian cultivated rice.

Many agronomic traits that are important in rice breeding are controlled by multiple genes. The extensive time and effort devoted so far to identifying and selecting such genes are still not enough to target multiple agronomic traits in practical breeding in Japan because of a lack of suitable plant materials in which to efficiently detect and validate beneficial alleles from diverse genetic resources. To facilitate the comprehensive analysis of genetic variation in agronomic traits among Asian cultivated rice, we developed 12 sets of chromosome segment substitution lines (CSSLs) with the japonica background, 11 of them in the same genetic background, using donors representing the genetic diversity of Asian cultivated rice. Using these materials, we overviewed the chromosomal locations of 1079 putative QTLs for seven agronomic traits and their allelic distribution in Asian cultivated rice through multiple linear regression analysis. The CSSLs will allow the effects of putative QTLs in the highly homogeneous japonica background to be validated.

Towards improved dynamic photosynthesis in C3 crops by utilizing natural genetic variation.

Under field environments, fluctuating light conditions induce dynamic photosynthesis, which affects carbon gain by crop plants. Elucidating the natural genetic variations among untapped germplasm resources and their underlying mechanisms can provide an effective strategy to improve dynamic photosynthesis and, ultimately, improve crop yields through molecular breeding approaches. In this review, we first overview two processes affecting dynamic photosynthesis, namely (i) biochemical processes associated with CO2 fixation and photoprotection and (ii) gas diffusion processes from the atmosphere to the chloroplast stroma. Next, we review the intra- and interspecific variations in dynamic photosynthesis in relation to each of these two processes. It is suggested that plant adaptations to different hydrological environments underlie natural genetic variation explained by gas diffusion through stomata. This emphasizes the importance of the coordination of photosynthetic and stomatal dynamics to optimize the balance between carbon gain and water use efficiency under field environments. Finally, we discuss future challenges in improving dynamic photosynthesis by utilizing natural genetic variation. The forward genetic approach supported by high-throughput phenotyping should be introduced to evaluate the effects of genetic and environmental factors and their interactions on the natural variation in dynamic photosynthesis.

Metabolomic analysis of rice brittle culm mutants reveals each mutant- specific metabolic pattern in each organ.

INTRODUCTION: Plant cell walls play an important role in providing physical strength and defence against abiotic stress. Rice brittle culm (bc) mutants are a strength-decreased mutant because of abnormal cell walls, and it has been reported that the causative genes of bc mutants affect cell wall composition. However, the metabolic alterations in each organ of bc mutants have remained unknown. OBJECTIVES: To evaluate the metabolic changes in rice bc mutants, comparative analysis of the primary metabolites was conducted. METHODS: The primary metabolites in leaves, internodes, and nodes of rice bc mutants and wild-type control were measured using CE- and LC-MS/MS. Multivariate analyses using metabolomic data was performed. RESULTS: We found that mutations in each bc mutant had different effects on metabolism. For example, higher oxalate content was observed in bc3 and bc1 bc3 mutants, suggesting that surplus carbon that was not used for cell wall components might be used for oxalate synthesis. In addition, common metabolic alterations such as a decrease of sugar nucleotides in nodes were found in bc1 and Bc6, in which the causative genes are involved in cellulose accumulation. CONCLUSION: These results suggest that metabolic analysis of the bc mutants could elucidate the functions of causative gene and improve the cell wall components for livestock feed or bioethanol production.

Anisohydric characteristics of a rice genotype 'ARC 11094' contribute to increased photosynthetic carbon fixation in response to high light.

Photosynthetic induction, which is the response of the CO2 assimilation rate to a stepwise increase in light intensity, potentially affects plant carbon gain and crop productivity in field environments. Although natural variations in photosynthetic induction are determined by CO2 supply and its fixation, detailed factors, especially CO2 supply, are unclear. This study investigated photosynthesis at steady and non-steady states in three rice (Oryza sativa L.) genotypes: ARC 11094, Takanari and Koshihikari. Stomatal traits and water relations in the plants were evaluated to characterise CO2 supply. Photosynthetic induction in ARC 11094 and Takanari was superior to that in Koshihikari owing to an efficient CO2 supply. The CO2 supply in Takanari is attributed to its high stomatal density, small guard cell length and extensive root mass, whereas that in ARC 11094 is attributed to its high stomatal conductance per stoma and stomatal opening in leaves with insufficient water (i.e., anisohydric stomatal behaviour). Our results suggest that there are various mechanisms for realising an efficient CO2 supply during the induction response. These characteristics can be useful for improving photosynthetic induction and, thus, crop productivity in field environments in future breeding programmes.

Genomic Basis of Transcriptome Dynamics in Rice under Field Conditions.

How genetic variations affect gene expression dynamics of field-grown plants remains unclear. Expression quantitative trait loci (eQTL) analysis is frequently used to find genomic regions underlying gene expression polymorphisms. This approach requires transcriptome data for the complete set of the QTL mapping population under the given conditions. Therefore, only a limited range of environmental conditions is covered by a conventional eQTL analysis. We sampled sparse time series of field-grown rice from chromosome segment substitution lines (CSSLs) and conducted RNA sequencing (RNA-Seq). Then, by using statistical analysis integrating meteorological data and the RNA-Seq data, we identified 1,675 eQTLs leading to polymorphisms in expression dynamics under field conditions. A genomic region on chromosome 11 influences the expression of several defense-related genes in a time-of-day- and scaled-age-dependent manner. This includes the eQTLs that possibly influence the time-of-day- and scaled-age-dependent differences in the innate immunity between Koshihikari and Takanari. Based on the eQTL and meteorological data, we successfully predicted gene expression under environments different from training environments and in rice cultivars with more complex genotypes than the CSSLs. Our novel approach of eQTL identification facilitated the understanding of the genetic architecture of expression dynamics under field conditions, which is difficult to assess by conventional eQTL studies. The prediction of expression based on eQTLs and environmental information could contribute to the understanding of plant traits under diverse field conditions.

Analysis of carbon flow at the metabolite level reveals that starch synthesis from hexose is a limiting factor in a high-yielding rice cultivar.

Understanding the limiting factors of grain filling is essential for the further improvement of grain yields in rice (Oryza sativa). The relatively slow grain growth of the high-yielding cultivar 'Momiroman' is not improved by increasing carbon supply, and hence low sink activity (i.e. the metabolic activity of assimilate consumption/storage in sink organs) may be a limiting factor for grain filling. However, there is no metabolic evidence to corroborate this hypothesis, partly because there is no consensus on how to define and quantify sink activity. In this study, we investigated the carbon flow at a metabolite level from photosynthesis in leaves to starch synthesis in grains of three high-yielding cultivars using the stable isotope 13C. We found that a large amount of newly fixed carbon assimilates in Momiroman was stored as hexose instead of being converted to starch. In addition, the activity of ADP-glucose pyrophosphorylase and the expression of AGPS2b, which encodes a subunit of the ADP-glucose pyrophosphorylase enzyme, were both lower in Momiroman than in the other two cultivars in grains in superior positions on panicle branches. Hence, slower starch synthesis from hexose, which is partly explained by the low expression level of AGPS2b, may be the primary metabolic reason for the lower sink activity observed in Momiroman.

The Simons Observatory: metamaterial microwave absorber and its cryogenic applications.

Controlling stray light at millimeter wavelengths requires special optical design and selection of absorptive materials that should be compatible with cryogenic operating environments. While a wide selection of absorptive materials exists, these typically exhibit high indices of refraction and reflect/scatter a significant fraction of light before absorption. For many lower index materials such as commercial microwave absorbers, their applications in cryogenic environments are challenging. In this paper, we present a new tool to control stray light: metamaterial microwave absorber tiles. These tiles comprise an outer metamaterial layer that approximates a lossy gradient index anti-reflection coating. They are fabricated via injection molding commercially available carbon-loaded polyurethane (25% by mass). The injection molding technology enables mass production at low cost. The design of these tiles is presented, along with thermal tests to 1 K. Room temperature optical measurements verify their control of reflectance to less than 1% up to 65∘ angles of incidence, and control of wide angle scattering below 0.01%. The dielectric properties of the bulk carbon-loaded material used in the tiles is also measured at different temperatures, confirming that the material maintains similar dielectric properties down to 3 K.

Methyl substitution effects on the non-adiabatic dynamics of benzene: lifting three-state quasi-degeneracy at conical intersections.

Previously, theoretical calculations on the non-adiabatic dynamics of benzene from the S2 state have indicated that the S2/S1 and S1/S0 conical intersections (CIs) facilitate ballistic nuclear wavepacket motion from S2 to S0 (fast channel) and branching to S1 (slow channel). In this paper, we present time-resolved photoelectron spectra of benzene and its methyl-derivatives (toluene and o-xylene) measured with a vacuum-UV laser, which clearly reveal both the fast and slow channels. The extremely short propagation time of the wavepacket between the two CIs of benzene indicates that the two are in close proximity to each other, while methyl substitution extends the propagation time and decreases the branching ratio into the fast channel. The results suggest that the quasi-degeneracy of the three states in benzene is lifted by the geometrical shifts of the CIs by methyl substitution.

High-yielding rice Takanari has superior photosynthetic response to a commercial rice Koshihikari under fluctuating light.

Leaves within crop canopies experience variable light over the course of a day, which greatly affects photosynthesis and crop productivity. Little is known about the mechanisms of the photosynthetic response to fluctuating light and their genetic control. Here, we examined gas exchange, metabolite levels, and chlorophyll fluorescence during the photosynthetic induction response in an Oryza sativa indica cultivar with high yield (Takanari) and a japonica cultivar with lower yield (Koshihikari). Takanari had a faster induction response to sudden increases in light intensity than Koshihikari, as demonstrated by faster increases in net CO2 assimilation rate, stomatal conductance, and electron transport rate. In a simulated light regime that mimicked a typical summer day, the faster induction response in Takanari increased daily CO2 assimilation by 10%. The faster response of Takanari was explained in part by its maintenance of a larger pool of Calvin-Benson cycle metabolites. Together, the rapid responses of electron transport rate, metabolic flux, and stomatal conductance in Takanari contributed to the greater daily carbon gain under fluctuating light typical of natural environments.

Genetic architecture of leaf photosynthesis in rice revealed by different types of reciprocal mapping populations.

The improvement of leaf net photosynthetic rate (An) is a major challenge in enhancing crop productivity. However, the genetic control of An among natural genetic accessions is still poorly understood. The high-yielding indica cultivar Takanari has the highest An of all rice cultivars, 20-30% higher than that of the high-quality japonica cultivar Koshihikari. By using reciprocal backcross inbred lines and chromosome segment substitution lines derived from a cross between Takanari and Koshihikari, we identified three quantitative trait loci (QTLs) where the Takanari alleles enhanced An in plants with a Koshihikari genetic background and five QTLs where the Koshihikari alleles enhanced An in plants with a Takanari genetic background. Two QTLs were expressed in plants with both backgrounds (type I QTL). The expression of other QTLs depended strongly on genetic background (type II QTL). These beneficial alleles increased stomatal conductance, the initial slope of An versus intercellular CO2 concentration, or An at CO2 saturation. Pyramiding of these alleles consistently increased An. Some alleles positively affected biomass production and grain yield. These alleles associated with photosynthesis and yield can be a valuable tool in rice breeding programs via DNA marker-assisted selection.

Probing ultrafast dynamics during and after passing through conical intersections.

Time-resolved photoelectron spectroscopy using vacuum-UV probe pulses enables observing ultrafast dynamics during and after passing through conical intersections (CIs). The ring-puckering CI plays a prominent role following the ππ* photoexcitation of furan. More than 90% of the excited molecules safely return to the original ground state, while the remaining 10% transforms into isomers after passing through the puckering CI.

A high-performance system of multiple gas-exchange chambers with a laser spectrometer to estimate leaf photosynthesis, stomatal conductance, and mesophyll conductance.

Direct measurements of ecophysiological processes such as leaf photosynthesis are often hampered due to the excessive time required for gas-exchange measurements and the limited availability of multiple gas analyzers. Although recent advancements in commercially available instruments have improved the ability to take measurements more conveniently, the amount of time required for each plant sample to acclimate to chamber conditions has not been sufficiently reduced. Here we describe a system of multiple gas-exchange chambers coupled with a laser spectrometer that employs tunable diode laser absorption spectroscopy (TDLAS) to measure leaf photosynthesis, stomatal conductance, and mesophyll conductance. Using four gas-exchange chambers minimizes the time loss associated with acclimation for each leaf sample. System operation is semiautomatic, and leaf temperature, humidity, and CO2 concentration can be regulated and monitored remotely by a computer system. The preliminary results with rice leaf samples demonstrated that the system is capable of high-throughput measurements, which is necessary to obtain better representativeness of the ecophysiological characteristics of plant samples.

Some like it hot: the physiological ecology of C4 plant evolution.

The evolution of C4 photosynthesis requires an intermediate phase where photorespiratory glycine produced in the mesophyll cells must flow to the vascular sheath cells for metabolism by glycine decarboxylase. This glycine flux concentrates photorespired CO2 within the sheath cells, allowing it to be efficiently refixed by sheath Rubisco. A modest C4 biochemical cycle is then upregulated, possibly to support the refixation of photorespired ammonia in sheath cells, with subsequent increases in C4 metabolism providing incremental benefits until an optimized C4 pathway is established. 'Why' C4 photosynthesis evolved is largely explained by ancestral C3 species exploiting photorespiratory CO2 to improve carbon gain and thus enhance fitness. While photorespiration depresses C3 performance, it produces a resource (photorespired CO2) that can be exploited to build an evolutionary bridge to C4 photosynthesis. 'Where' C4 evolved is indicated by the habitat of species branching near C3-to-C4 transitions on phylogenetic trees. Consistent with the photorespiratory bridge hypothesis, transitional species show that the large majority of > 60 C4 lineages arose in hot, dry, and/or saline regions where photorespiratory potential is high. 'When' C4 evolved has been clarified by molecular clock analyses using phylogenetic data, coupled with isotopic signatures from fossils. Nearly all C4 lineages arose after 25 Ma when atmospheric CO2 levels had fallen to near current values. This reduction in CO2, coupled with persistent high temperature at low-to-mid-latitudes, met a precondition where photorespiration was elevated, thus facilitating the evolutionary selection pressure that led to C4 photosynthesis.

QTLs maintaining grain fertility under salt stress detected by exome QTL-seq and interval mapping in barley.

Enhancing salt stress tolerance is a key strategy for increasing global food production. We previously found that long-term salinity stress significantly reduced grain fertility in the salt-sensitive barley (Hordeum vulgare) accession, 'OUC613', but not in the salt-tolerant accession, 'OUE812', resulting in large differences in grain yield. Here, we examined the underlying causes of the difference in grain fertility between these accessions under long-term treatment with 150 or 200 mM NaCl from the seedling stage to harvest and identified quantitative trait loci (QTLs) for maintaining grain fertility. In an artificial pollination experiment of the two accessions, grain fertility was significantly reduced only in OUC613 plants produced using pollen from plants grown under NaCl stress, suggesting that the low grain fertility of OUC613 was mainly due to reduced pollen fertility. Using QTL-seq combined with exome-capture sequencing and composite interval mapping of recombinant inbred lines derived from a cross between OUE812 and OUC613, we identified a QTL (qRP-2Hb) for grain fertility on chromosome 2H. The QTL region includes two genes encoding an F-box protein and a TIFY protein that are associated with male sterility, highlighting the importance of this region for maintaining grain fertility under salt stress.

Leaf photosynthetic rate and mesophyll cell anatomy changes during ontogenesis in backcrossed indica × japonica rice inbred lines.

The high-yielding indica rice variety, 'Takanari', has the high rate of leaf photosynthesis compared with the commercial japonica varieties. Among backcrossed inbred lines from a cross between 'Takanari' and a japonica variety, 'Koshihikari', two lines, BTK-a and BTK-b, showed approximately 20% higher photosynthetic rate than that of 'Takanari' for a flag leaf at full heading. This is a highest recorded rate of rice leaf photosynthesis. Here, the timing and cause of the increased leaf photosynthesis in the BTK lines were investigated by examining the photosynthesis and related parameters, as well as mesophyll cell anatomy during ontogenesis. Their photosynthetic rate was greater than that of 'Takanari' in the 13th leaf, as well as the flag leaf, but there were no differences in the 7th and 10th leaves. There were no consistent differences in the stomatal conductance, or the leaf nitrogen and Rubisco contents in the 13th and flag leaves. The total surface area of mesophyll cells per leaf area (TAmes) in the 13th and flag leaves increased significantly in the BTK lines due to the increased number and developed lobes of mesophyll cells compared with in 'Takanari'. The mesophyll conductance (g m) became greater in the BTK lines compared with 'Takanari' in the flag leaves but not in the 10th leaves. A close correlation was observed between TAmes and g m. We concluded that the increased mesophyll conductance through the development of mesophyll cells during the reproductive period is a probable cause of the greater photosynthetic rate in the BTK lines.

Self-compression of femtosecond deep-ultraviolet pulses by filamentation in krypton.

We demonstrate self-compression of deep-ultraviolet (DUV) pulses by filamentation in krypton. In contrast to self-compression in the near-infrared, that in the DUV is associated with a red-shifted sub-pulse appearing in the pulse temporal profile. The achieved pulse width of 15 fs is the shortest among demonstrated sub-mJ deep-ultraviolet pulses.

Photoisomerization of Vibrationally Hot Tetramethylethylene Produced by Ultrafast Internal Conversion from the Excited State.

Isomerization of tetramethylethylene (TME) following ultrafast internal conversion was investigated using time-resolved photoelectron spectroscopy with vacuum-ultraviolet probe pulses. The difference photoelectron spectrum at τ = 15 ps was reasonably well reproduced using a linear combination of static photoelectron spectra of TME and its isomers. The isomers were produced as a consequence of unimolecular reaction of vibrationally hot TME, created by internal conversion from the excited state.

Wavelength Dependence of UV Photoemission from Solvated Electrons in Bulk Water, Methanol, and Ethanol.

We have measured the wavelength dependence (340-215 nm) of one-photon photoemission from the ground electronic state of solvated electrons in bulk water, methanol, and ethanol. In every case, the vertical electron binding energy (VBE) gradually increased with photon energy, indicating that the photoelectron kinetic energy diminishes as a result of electron-vibration inelastic scattering prior to emission from the liquid surface. In contrast, the VBE of the Rydberg electron in DABCO (1,4-diazabicyclo[2,2,2]octane), which has a surface-excess density, revealed no clear wavelength dependence. These results suggest that the solvated electrons are created predominantly in the bulk and that VBEs measured using UV photoemission spectroscopy of liquids generally require energy corrections to account for inelastic scattering effects. From the wavelength dependence, we have re-estimated the VBEs of solvated electrons in bulk water, methanol, and ethanol to be 3.3, 3.1, and 3.1 eV, respectively. Hydrated electrons were also identified by photoemission spectroscopy using 90 nm radiation.