Deciphering proteomic mechanisms explaining the role of glutathione as an aid in improving plant fitness and tolerance against cadmium-toxicity in Brassica napus L.

Cadmium (Cd) hazard is a serious limitation to plants, soils and environments. Cd-toxicity causes stunted growth, chlorosis, necrosis, and plant yield loss. Thus, ecofriendly strategies with understanding of molecular mechanisms of Cd-tolerance in plants is highly demandable. The Cd-toxicity caused plant growth retardation, leaf chlorosis and cellular damages, where the glutathione (GSH) enhanced plant fitness and Cd-toxicity in Brassica through Cd accumulation and antioxidant defense. A high-throughput proteome approach screened 4947 proteins, wherein 370 were differently abundant, 164 were upregulated and 206 were downregulated. These proteins involved in energy and carbohydrate metabolism, CO2 assimilation and photosynthesis, signal transduction and protein metabolism, antioxidant defense response, heavy metal detoxification, cytoskeleton and cell wall structure, and plant development in Brassica. Interestingly, several key proteins including glutathione S-transferase F9 (A0A078GBY1), ATP sulfurylase 2 (A0A078GW82), cystine lyase CORI3 (A0A078FC13), ferredoxin-dependent glutamate synthase 1 (A0A078HXC0), glutaredoxin-C5 (A0A078ILU9), glutaredoxin-C2 (A0A078HHH4) actively involved in antioxidant defense and sulfur assimilation-mediated Cd detoxification process confirmed by their interactome analyses. These candidate proteins shared common gene networks associated with plant fitness, Cd-detoxification and tolerance in Brassica. The proteome insights may encourage breeders for enhancing multi-omics assisted Cd-tolerance in Brassica, and GSH-mediated hazard free oil seed crop production for global food security.

Proteome insights of citric acid-mediated cadmium toxicity tolerance in Brassica napus L.

Cadmium (Cd) is a toxic substance that is uptake by plants from soils, Cd easily transfers into the food chain. Considering global food security, eco-friendly, cost-effective, and metal detoxification strategies are highly demandable for sustainable food crop production. The purpose of this study was to investigate how citric acid (CA) alleviates or tolerates Cd toxicity in Brassica using a proteome approach. In this study, the global proteome level was significantly altered under Cd toxicity with or without CA supplementation in Brassica. A total of 4947 proteins were identified using the gel-free proteome approach. Out of these, 476 proteins showed differential abundance between the treatment groups, wherein 316 were upregulated and 160 were downregulated. The gene ontology analysis reveals that differentially abundant proteins were involved in different biological processes including energy and carbohydrate metabolism, CO2 assimilation and photosynthesis, signal transduction and protein metabolism, antioxidant defense, heavy metal detoxification, plant development, and cytoskeleton and cell wall structure in Brassica leaves. Interestingly, several candidate proteins such as superoxide dismutase (A0A078GZ68) L-ascorbate peroxidase 3 (A0A078HSG4), glutamine synthetase (A0A078HLB2), glutathione S-transferase DHAR1 (A0A078HPN8), glutamine synthetase (A0A078HLB2), cysteine synthase (A0A078GAD3), S-adenosylmethionine synthase 2 (A0A078JDL6), and thiosulfate/3-mercaptopyruvate sulfur transferase 2 (A0A078H905) were involved in antioxidant defense system and sulfur assimilation-involving Cd-detoxification process in Brassica. These findings provide new proteome insights into CA-mediated Cd-toxicity alleviation in Brassica, which might be useful to oilseed crop breeders for enhancing heavy metal tolerance in Brassica using the breeding program, with sustainable and smart Brassica production in a metal-toxic environment.

Characterization of fluorescence properties of wounds on soybean seedlings during healing process using excitation emission matrix and fluorescence imaging.

To establish a simple and nondestructive method for measuring plant wound-healing ability, we characterized the fluorescence characteristics of wounds on hypocotyl of soybean seedlings during healing process. Wounds were manually created on the stem of soybean seedlings 7 days after sowing. The fluorescence time-series characteristics of the wounds were measured until 96 h after wounding using excitation emission matrix (EEM) and fluorescence images excited by wavelength of 365 nm. In the EEM of wounds, three main fluorescence peaks were observed, and the intensity decreased with time after wounding. The reddish color due to chlorophyll in fluorescence images also decreased with healing process. In addition, microscopic observation of the wounded tissue using a confocal laser microscope showed that the intensity of lignin or suberin like fluorescence increased with healing time, which might have blocked the excitation light. These results suggest that UV-excited fluorescence can be a new indicator of the healing ability of plant tissues.

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.

Analysis of Physiological Variations and Genetic Architecture for Photosynthetic Capacity of Japanese Soybean Germplasm.

The culmination of conventional yield improving parameters has widened the margin between food demand and crop yield, leaving the potential yield productivity to be bridged by the manipulation of photosynthetic processes in plants. Efficient strategies to assess photosynthetic capacity in crops need to be developed to identify suitable targets that have the potential to improve photosynthetic efficiencies. Here, we assessed the photosynthetic capacity of the Japanese soybean mini core collection (GmJMC) using a newly developed high-throughput photosynthesis measurement system "MIC-100" to analyze physiological mechanisms and genetic architecture underpinning photosynthesis. K-means clustering of light-saturated photosynthesis (Asat ) classified GmJMC accessions into four distinct clusters with Cluster2 comprised of highly photosynthesizing accessions. Genome-wide association analysis based on the variation of Asat revealed a significant association with a single nucleotide polymorphism (SNP) on chromosome 17. Among the candidate genes related to photosynthesis in the genomic region, variation in expression of a gene encoding G protein alpha subunit 1 (GPA1) showed a strong correlation (r = 0.72, p < 0.01) with that of Asat . Among GmJMC accessions, GmJMC47 was characterized by the highest Asat , stomatal conductance (gs ), stomatal density (SDensity ), electron transfer rate (ETR), and light use efficiency of photosystem II (Fv'/Fm') and the lowest non-photochemical quenching [NPQ(t)], indicating that GmJMC47 has greater CO2 supply and efficient light-harvesting systems. These results provide strong evidence that exploration of plant germplasm is a useful strategy to unlock the potential of resource use efficiencies for photosynthesis.

High source-sink ratio at and after sink capacity formation promotes green stem disorder in soybean.

Green stem disorder (GSD) of soybean is characterized by delayed leaf and stem maturation despite normal pod maturation. Previous studies have suggested that GSD occurrence is promoted by a high source-sink ratio, which is produced by thinning or shade removal at the R5 growth stage (the beginning of seed filling). Here the effects of different times and durations of shade removal after the R5 stage on GSD severity were analyzed. First, shade removal for more than 28 days after R5 increased GSD severity by more than 0.4 point in GSD score. Thinning treatment at R5 increased specific leaf weight by 23%, suppressed stem dry weight reduction, and upregulated 19 genes including those encoding vegetative storage proteins at R5 + 28d, indicating excess source ability relative to sink size. On the contrary, shade removal for 14 days after R5 decreased GSD severity by 0.5 point in GSD score. In this treatment, seed size was smaller, while seed number was significantly larger than control, suggesting that shortage of source ability relative to sink size. These results implied that soybean plants regulate GSD occurrences either positively or negatively according to a source-sink ratio during the R5 to R5 + 28d growth stages.

Proteome Changes Reveal the Protective Roles of Exogenous Citric Acid in Alleviating Cu Toxicity in Brassica napus L.

Citric acid (CA), as an organic chelator, plays a vital role in alleviating copper (Cu) stress-mediated oxidative damage, wherein a number of molecular mechanisms alter in plants. However, it remains largely unknown how CA regulates differentially abundant proteins (DAPs) in response to Cu stress in Brassica napus L. In the present study, we aimed to investigate the proteome changes in the leaves of B. L. seedlings in response to CA-mediated alleviation of Cu stress. Exposure of 21-day-old seedlings to Cu (25 and 50 µM) and CA (1.0 mM) for 7 days exhibited a dramatic inhibition of overall growth and considerable increase in the enzymatic activities (POD, SOD, CAT). Using a label-free proteome approach, a total of 6345 proteins were identified in differentially treated leaves, from which 426 proteins were differentially expressed among the treatment groups. Gene ontology (GO) and KEGG pathways analysis revealed that most of the differential abundance proteins were found to be involved in energy and carbohydrate metabolism, photosynthesis, protein metabolism, stress and defense, metal detoxification, and cell wall reorganization. Our results suggest that the downregulation of chlorophyll biosynthetic proteins involved in photosynthesis were consistent with reduced chlorophyll content. The increased abundance of proteins involved in stress and defense indicates that these DAPs might provide significant insights into the adaptation of Brassica seedlings to Cu stress. The abundances of key proteins were further verified by monitoring the mRNA expression level of the respective transcripts. Taken together, these findings provide a potential molecular mechanism towards Cu stress tolerance and open a new route in accelerating the phytoextraction of Cu through exogenous application of CA in B. napus.

Impacts of the continuous maize cultivation on soil properties in Sainyabuli province, Laos.

In tropical mountainous areas, soil degradation and yield decrease have been anticipated due to conversion from shifting to continuous cultivation and the introduction of cash crops. In our previous report, we quantified the decrease in maize yield under continuous cultivation in farmers' fields in Laos. In this report, we focused on soil nutritional conditions under continuous cultivation in the farmers' fields. For the purpose, twelve soil properties were investigated over two years from three sample sites in each of the 40 farmers' fields with the duration of continuous cultivation varying from 1 to 30 years. Total carbon (TC), total nitrogen (TN), available phosphorus, exchangeable potassium, and exchangeable calcium in the soil decreased with increasing duration of continuous cultivation in the sloped fields. These soil nutrients decreased to around half of the initial content in these 30 years. However, the decreasing rates of TC and TN were negligible in the flat fields. Other soil properties such as clay and exchangeable magnesium were not related to the duration of continuous cultivation in both sloped and flat fields. The reduction in maize yield was mainly explained by TC, but the determination coefficient was only 0.24. Although further analysis is required to quantify the effect of soil nutrients on maize production, the development of integrated soil management would be necessary in the sloped fields for sustainable crop production in the study site.

Two novel quantitative trait loci affecting the variation in leaf photosynthetic capacity among soybeans.

There is a large variation in CO2 assimilation rate per unit of leaf area (A) within or among crop species, which can be exploited to improve A by elucidating the mechanisms underlying such variation. The objective of the present study is to elucidate the genetic factors affecting the variation in leaf photosynthetic capacity among soybeans. Here, we conducted field experiments over three years, using Enrei, a leading variety in Japan, Peking, a landrace from China and the chromosome segment substitution lines derived from their progenies. The gas exchange measurements were conducted to evaluate A among soybean. Peking showed higher A than Enrei after the flowering in all the years. The genetic analysis identified two novel quantitative trait loci (QTLs) related to variation in A, which were located on chromosome 13 (qLPC13) and 20 (qLPC20). The Peking allele at qLPC13 increased A by 8.3 % in the Enrei genetic background, while the Peking allele at qLPC20 decreased A by 15.3 %. The present study is the first report on QTLs affecting a genotypic variation in leaf photosynthetic capacity among field-grown soybeans. The identification of the causal genes in these QTLs can provide a novel strategy to enhance leaf photosynthetic capacity with soybean breeding.

Genetic Diversity in Stomatal Density among Soybeans Elucidated Using High-throughput Technique Based on an Algorithm for Object Detection.

The stomatal density (SD) can be a promising target to improve the leaf photosynthesis in soybeans (Glycine max (L.) Merr). In a conventional SD evaluation, the counting process of the stomata during a manual operation can be time-consuming. We aimed to develop a high-throughput technique for evaluating the SD and elucidating the variation in the SD among various soybean accessions. The central leaflet of the first trifoliolate was sampled, and microscopic images of the leaflet replica were obtained among 90 soybean accessions. The Single Shot MultiBox Detector, an algorithm for an object detection based on deep learning, was introduced to develop an automatic detector of the stomata in the image. The developed detector successfully recognized the stomata in the microscopic image with high-throughput. Using this technique, the value of R2 reached 0.90 when the manually and automatically measured SDs were compared in the 150 images. This technique discovered a variation in SD from 93 ± 3 to 166 ± 4 mm-2 among the 90 accessions. Our detector can be a powerful tool for a SD evaluation with a large-scale population in crop species, accelerating the identification of useful alleles related to the SD in future breeding programs.

Parameterization of the vertical distribution of leaf area index (LAI) in rice (Oryza sativa L.) using a plant canopy analyzer.

Monitoring the vertical distribution of leaf area index (LAI) is an effective method for evaluating canopy photosynthesis and biomass productivity. In this study, we proposed a novel method to characterize LAI vertical distribution non-destructively by utilizing LAI-2200 plant canopy analyzer, followed by the application of statistical moment equations. Field experiments were conducted with 5 rice cultivars under 2 fertilizer treatments in 2013 and with 3 rice cultivars under 3 plant density treatments in 2014. LAI readings obtained by a plant canopy analyzer for non-destructive stratified measurements were relatively consistent with LAI estimations using the stratified clipping method for every cultivar and treatment. The parameters calculated using the statistical moment equations numerically showed the changes in LAI vertical distribution with plant growth up to the heading stage. The differences in the parameters also quantified the effect of cultivar, fertilizer, and plant density treatments. These results suggest that the non-destructive stratified measurements and the statistical moments evaluated in this study provide quantitative, reliable information on the dynamics of LAI vertical distribution. The method is expected to be utilized by researchers in various research fields sharing common interests.

Factors underlying genotypic differences in the induction of photosynthesis in soybean [Glycine max (L.) Merr].

Crop leaves are subject to continually changing light levels in the field. Photosynthetic efficiency of a crop canopy and productivity will depend significantly on how quickly a leaf can acclimate to a change. One measure of speed of response is the rate of photosynthesis increase toward its steady state on transition from low to high light. This rate was measured for seven genotypes of soybean [Glycine max (L.) Merr.]. After 10 min of illumination, cultivar 'UA4805' (UA) had achieved a leaf photosynthetic rate (Pn ) of 23.2 µmol · m(-2) · s(-1) , close to its steady-state rate, while the slowest cultivar 'Tachinagaha' (Tc) had only reached 13.0 µmol · m(-2) · s(-1) and was still many minutes from obtaining steady state. This difference was further investigated by examining induction at a range of carbon dioxide concentrations. Applying a biochemical model of limitations to photosynthesis to the responses of Pn to intercellular CO2 concentration (Ci ), it was found that the speed of apparent in vivo activation of ribulose-1:5-bisphosphate carboxylase/oxygenase (Rubisco) was responsible for this difference. Sequence analysis of the Rubisco activase gene revealed single nucleotide polymorphisms that could relate to this difference. The results show a potential route for selection of cultivars with increased photosynthetic efficiency in fluctuating light.

Seed yield and its components of indeterminate and determinate lines in recombinant inbred lines of soybean.

The present study was conducted to evaluate the benefits of indeterminate growth habit in breeding to improve yield potential of Japanese soybean varieties, which exclusively have determinate growth habit. Two populations of recombinant inbred lines (RILs) derived from crosses between determinate Japanese cultivars and indeterminate US cultivars were grown in Akita and Kyoto, and seed weight per plant (SW) and its components were compared between indeterminate and determinate RILs. The difference of SW between the two growth habits in RILs varied depending on maturation time. The SW of early indeterminate lines was significantly higher than that of early determinate ones in Akita, but not in Kyoto. Among yield components, the number of seeds per pod was constantly larger in indeterminate lines than that in determinate ones irrespective of maturation time. The number of seeds per plant and the number of pods per plant of the indeterminate lines were greater than those of the determinate lines in early maturation in Akita. These results suggest that the indeterminate growth habit is an advantageous characteristic in breeding for high yield of early maturing soybean varieties in the Tohoku region.

A major and stable QTL associated with seed weight in soybean across multiple environments and genetic backgrounds.

KEY MESSAGE: We detected a QTL for single seed weight in soybean that was stable across multiple environments and genetic backgrounds with the use of two recombinant inbred line populations. Single seed weight (SSW) in soybean is a key determinant of both seed yield and the quality of soy food products, and it exhibits wide variation. SSW is under genetic control, but the molecular mechanisms of such control remain unclear. We have now investigated quantitative trait loci (QTLs) for SSW in soybean and have identified such a QTL that is stable across multiple environments and genetic backgrounds. Two populations of 225 and 250 recombinant inbred lines were developed from crosses between Japanese and US cultivars of soybean that differ in SSW by a factor of ~2, and these populations were grown in at least three different environments. A whole-genome panel comprising 304 simple sequence repeat (SSR) loci was applied to mapping in each population. We identified 15 significant QTLs for SSW dispersed among 11 chromosomes in the two populations. One QTL located between Sat_284 and Sat_292 on chromosome 17 was detected (3.6 < LOD < 14.1) in both populations grown in all environments. This QTL, tentatively designated qSw17-1, accounted for 9.4-20.9 % of phenotypic variation in SSW, with a dominant allele being associated with increased SSW. Given its substantial effect on SSW, qSw17-1 is an attractive target for positional cloning, and SSR markers closely associated with this locus may prove useful for marker-assisted selection for SSW control in soybean.

Stem growth habit affects leaf morphology and gas exchange traits in soybean.

BACKGROUNDS AND AIMS: The stem growth habit, determinate or indeterminate, of soybean, Glycine max, varieties affects various plant morphological and developmental traits. The objective of this study is to identify the effect of stem growth habit in soybean on the stomatal conductance of single leaves in relation to their leaf morphology in order to better understand the ecological and agronomic significance of this plant trait. METHODS: The stomatal conductance of leaves on the main stem was measured periodically under favourable field conditions to evaluate g(max), defined as the maximum stomatal conductance at full leaf expansion, for four varieties of soybean and their respective determinate or indeterminate near isogenic lines (NILs). Leaf morphological traits including stomatal density, guard cell length and vein density were also measured. KEY RESULTS: The value of g(max) ranged from 0.383 to 0.754 mol H(2)O m(-2) s(-1) across all the genotypes for both years. For the four pairs of varieties, the indeterminate lines exhibited significantly greater g(max), stomatal density, numbers of epidermal cells per unit area and total vein length per unit area than their respective determinate NILs in both years. The guard cell length, leaf mass per area and single leaf size all tended to be greater in the determinate types. The variation of g(max) across genotypes and years was well explained by the product of stomatal density and guard cell length (r = 0.86, P < 0.01). CONCLUSIONS: The indeterminate stem growth habit resulted in a greater maximum stomatal conductance for soybean than the determinate habit, and this was attributed to the differences in leaf structure. This raises the further hypothesis that the difference in stem growth habit results in different water use characteristics of soybean plants in the field. Stomatal conductance under favourable conditions can be modified by leaf morphological traits.

Evaluation of genotypic variation in leaf photosynthetic rate and its associated factors by using rice diversity research set of germplasm.

In order to evaluate genotypic variation, we measured leaf photosynthetic rate (Pn) and its associated factors for the rice diversity research set of germplasm (RDRS) selected from the Genebank in National Institute of Agrobiological Sciences (NIAS). Pn showed large genotypic variation from 11.9 to 32.1 micromol m(-2 )s(-1). The variation in stomatal conductance to CO2 (Gs) explained about 50% of that in Pn, while that in nitrogen concentration (N) in leaves explained about 35%. The genotype group which mainly consists of aus type indica tended to have higher Gs, and the genotype group which corresponds to japonica had a higher nitrogen concentration (N) in leaves. The relationships of Pn with Gs and N were not significantly different among genotype groups, suggesting photosynthetic efficiencies are similar among genotype groups.

A model explaining genotypic and ontogenetic variation of leaf photosynthetic rate in rice (Oryza sativa) based on leaf nitrogen content and stomatal conductance.

BACKGROUNDS AND AIMS: Identification of physiological traits associated with leaf photosynthetic rate (Pn) is important for improving potential productivity of rice (Oryza sativa). The objectives of this study were to develop a model which can explain genotypic variation and ontogenetic change of Pn in rice under optimal conditions as a function of leaf nitrogen content per unit area (N) and stomatal conductance (g(s)), and to quantify the effects of interaction between N and g(s) on the variation of Pn. METHODS: Pn, N and g(s) were measured at different developmental stages for the topmost fully expanded leaves in ten rice genotypes with diverse backgrounds grown in pots (2002) and in the field (2001 and 2002). A model of Pn that accounts for carboxylation and CO diffusion processes, and assumes that the ratio of internal conductance to g(s) is constant, was constructed, and its goodness of fit was examined. KEY RESULTS: Considerable genotypic differences in Pn were evident for rice throughout development in both the pot and field experiments. The genotypic variation of Pn was correlated with that of g(s) at a given stage, and the change of Pn with plant development was closely related to the change of N. The variation of g(s) among genotypes was independent of that of N. The model explained well the variation in Pn of the ten genotypes grown under different conditions at different developmental stages. Conclusions The response of Pn to increased N differs with g(s), and the increase in Pn of genotypes with low g(s) is smaller than that of genotypes with high g(s). Therefore, simultaneous improvements of these two traits are essential for an effective breeding of rice genotypes with increased Pn.

Genotypic difference in canopy diffusive conductance measured by a new remote-sensing method and its association with the difference in rice yield potential.

There have been few practical ways of measuring physiological determinants of rice yield. Rapid evaluation of yield determination traits may expedite breeding of high-yielding rice. Here, we report a new remote-sensing technique for the evaluation of canopy ecophysiological status under field conditions developed based on simultaneous measurements of sunlit and suddenly shaded canopy temperatures. This technique has the advantage of instantaneous estimation of aerodynamic resistance (r(a)) and canopy diffusive resistance (r(c) without measuring wind velocity. Canopy diffusive conductance (1 / r(c)) estimated by the remote sensing method was closely related to leaf stomatal conductance (g(s)) measured with a portable gas exchange system. This result supported the validity of this new method for quantitative estimation of canopy physiological characteristics. Significant genotypic differences were obtained in canopy-air temperature difference (Tc-Ta), r(c) and 1 / r(c) during the 2-week period preceding full heading for two years, and 1 / r(c) was highly correlated with crop growth rate (CGR), which was closely related to the final yield. These results suggest that 1 / r(c) can be an effective criterion for the selection of high-yielding rice genotypes, and the remote sensing technique proposed here can be a powerful tool for the rapid evaluation of 1 / r(c) under field conditions.

Time-related mapping of quantitative trait loci controlling grain-filling in rice (Oryza sativa L.).

Grain-filling is a crucial process that determines final grain yield in rice (Oryza sativa L.). To understand the genetic basis of dynamics of grain-filling, quantitative trait locus (QTL) analysis was conducted using time-related phenotypic data on grain-filling collected from a population of 155 recombinant inbred lines (F12), derived from a cross between Milyang 23 and Akihikari. Two QTLs detected on chromosomes 8 and 12 were strongly associated with increased filling percentage per panicle. These QTLs were not linked with those controlling spikelet numbers per panicle. This result confers the possibility of improving grain-filling together with an enlargement of sink size. The QTL for filling percentage per panicle on chromosome 8 exactly overlapped that for non-structural carbohydrate (NSC) content in the culm and leaf sheaths during grain-filling, and the Milyang 23 allele associated with increased grain-filling percentage per panicle was associated with decreased NSC content. Therefore, this QTL may be directly involved in NSC translocation from the culm and leaf sheaths to panicle. In addition, the Milyang 23 alleles of QTLs associated with greater spikelet number per panicle on chromosomes 1 and 6 were also related with a reduction in NSC content in the culm and leaf sheaths during grain-filling. These results indicate that NSC dynamics during grain-filling is partly dependent on sink size. NSC accumulation in the culm and leaf sheaths at the heading stage was mainly controlled by different genetic regulations from NSC dynamics during grain-filling. Nitrogen dynamics during grain-filling may also be involved in carbohydrate dynamics.