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Sulfur assimilation in soybean ( Glycine max [L.] Merr.): molecular cloning and characterization of a cytosolic isoform of serine acetyltransferase.

A full-length cDNA clone encoding a cytosolic isoform of serine acetyltransferase (SATase; EC 2.3.1.30) was isolated by screening a soybean seedling cDNA library with a (32)P-labeled expressed sequence tag. Nucleotide sequence analysis of the isolated cDNA revealed a single open-reading frame of 858 base pairs encoding a 30-kDa polypeptide. The deduced amino acid sequence of soybean SATase revealed significant homology with other plant SATases. Analysis of genomic DNA by Southern blotting indicated that SATase is encoded by a small gene family. The authenticity of the isolated SATase cDNA was confirmed by the expression of the cDNA in an Escherichia coli cysteine-auxotrophic mutant resulting in the growth of the mutant in minimal medium without cysteine. Expression of soybean SATase in E. coli resulted in the production of a 34-kDa protein that was subsequently purified by nickel-affinity column chromatography. The purified protein exhibited SATase activity, indicating that the E. coli-expressed protein is a functionally active SATase. The recombinant soybean SATase was inhibited by l-cysteine, the end product of the cysteine biosynthetic pathway. Antibodies raised against the recombinant soybean SATase cross-reacted with a 34-kDa protein from Arabidopsis leaves, but failed to detect any proteins from soybean leaves and seeds. Reverse transcriptase-polymerase chain reaction analysis indicated that SATase mRNA was expressed at low levels during soybean seed development. In comparison to Arabidopsis leaves, the SATase activity was several-fold lower in soybean leaves and seeds, suggesting that SATase is a low-abundance enzyme.

Acetyltransferases↗

Identification of differentially expressed genes in seeds of two near-isogenic Brassica napus lines with different oil content.

The regulation of seed oil synthesis in rapeseed is largely unknown. In this study, we compared the gene expression during seed development between two lines of Brassica napus with a 10% difference in oil content. We isolated the immature seeds 15 and 25 days after flowering at periods preceding and including the major accumulation of storage oils and proteins. The differentially expressed gene clones between the two rape lines were isolated by subtractive suppression hybridization (SSH). All SSH clones were arrayed and screened by dot blot hybridization, followed by RT-PCR analysis for selected clones. A total of 217 cDNA clones corresponding to 30 genes were found to have a high expression in seeds with high oil content. Six genes were highly expressed in seeds with low oil content. Northern blot and enzyme activity analysis demonstrated a change in expression pattern of several genes. The results provide information on gene-encoding factors responsible for the regulation of oil synthesis. The possible role of these genes in seeds is discussed. The genes in this study may be suitable as novel targets for genetic improvement of seed oil content and may also provide molecular markers for studies of rape breeding.

Brassica napus↗

The introduction of a phytase gene from Bacillus subtilis improved the growth performance of transgenic tobacco.

Phytate, the main form of phosphorus storage in plant seeds, is well known to be an anti-nutrient and a major source of phosphorus pollution in animal manure. To improve phosphorus bio-availability, we introduced a recently characterized phytase from Bacillus subtilis into the cytoplasm of tobacco cells. Although the introduction of acid fungal phytase from Aspergillus niger in previous studies did not result in any phenotypic changes in tobacco, here we show that a tobacco line transformed with a neutral phytase exhibited phenotypic changes in flowering, seed development, and response to phosphate deficiency. The transgenic line showed an increase in flower and fruit numbers, small seed syndrome, lower seed IP6/IP5 ratio, and enhanced growth under phosphate-starvation conditions compared with the wildtype. The results suggest that the over-expression of Bacillus phytase in the cytoplasm of tobacco cells shifts the equilibrium of the inositol phosphate biosynthesis pathway, thereby making more phosphate available for primary metabolism. The approach presented here can be applied as a strategy for boosting productivity in agriculture and horticulture.

6-Phytase↗

Molecular cloning of two classes of Em-like proteins from the seeds of the leguminous tree Robinia pseudoacacia.

To check for the presence of Em-like proteins in seeds of the leguminous tree Robinia pseudoacacia L. (black locust), a cDNA library constructed from mRNA isolated from developing seeds was screened using radiolabeled cDNA encoding the Em protein from Vigna radiata as a probe. Sequence analysis of the identified cDNA clones revealed two classes of Em proteins in Robinia. The nucleotide sequence data for each class have been submitted to Genbank/EMBL Data library and are available under the accession numbers U40820 and U40821. Northern blot analysis demonstrated that the Robinia Em proteins are translated from mRNAs of approximately 800 nucleotides, which are abundant in the seed but are not expressed in the bark. Southern blot analysis indicated that the Em proteins in Robinia are encoded by at least two genes. The Robinia Em proteins have a high degree of sequence homology with previously characterized Em-like proteins from both monocots and dicots.

Journal Article↗

Microgametophyte population sizes and plant reproductive output in the insect-pollinated Prunella grandiflora (Lamiaceae).

Intraspecific variation in pollen deposition and number of pollen tubes per style is rarely quantified, but is essential for assessing the occurrence of pollen limitation and pollen competition and their evolutionary implications. Moreover, pollen deposition, pollen tube growth, and the fate of fertilized ovules are rarely distinguished in field studies. Here we present such a study in eight natural populations of Prunella grandiflora. We quantified microgametophyte population sizes and inferred pollen limitation when the number of fertilizable ovules exceeded pollen tubes, and assessed seed set and fate after open pollination. Two and three populations had on average significantly fewer pollen grains and pollen tubes per flower, respectively, than the fixed number of fertilizable ovules per fruit, while one population experienced significant pollen competition. Style length was positively correlated with the number of pollen tubes. While pollen availability was very variable, seed abortion was significantly less frequent in denser populations, and in one population the proportion of well-developed seeds was significantly, positively correlated with the number of pollen tubes in the style. Less pollen deposition, lower numbers of pollen tubes reaching the base of the style, lower pollen quality and therefore increased abortion of fertilized ovules can all reduce seed set in natural P. grandiflora stands. Substantial intraspecific variability implies that microgametophyte competition also occurs in this species. Finally, style morphology may affect pollen receipt.

Animals↗

Evaluation and host specificity of two seed flies Mesoclanis polana and M. magnipalpis (Diptera: Tephritidae): biological control agents for Chrysanthemoides monilifera (Asteraceae) in Australia.

Larvae of the South African tephritid flies Mesoclanis polana Munro and M. magnipalpis Bezzi feed in the developing seeds of Chrysanthemoides monilifera. Host specificity evaluation using 109 plant species from 25 families indicated that complete development was restricted to their natural host C. monilifera. Minor feeding and limited development was detected on 18 species, but was of no ecological or economic significance. Mesoclanis polana and M. magnipalpis have been released in Australia and M. polana has established and dispersed widely. Mesoclanis magnipalpis has not yet become naturalized. Parasitism of M. polana in Australia by several species of Hymenoptera has been detected, but is not expected to limit the establishment and impact of these flies.

Animals↗

Developmental anatomy and morphology of the ovule and seed of heliconia (heliconiaceae, zingiberales).

The developmental anatomy and morphology of the ovule and seed in several species of Heliconia were investigated as part of an embryological study of the Heliconiaceae and to provide a better understanding of their relationships with the other families of the Zingiberales. Heliconia species have an ovule primordium with an outer integument of both dermal and subdermal origin. The archesporial cell is divided into a megasporocyte and a single parietal cell, which in turn are divided only anticlinally to form a single parietal layer, disintegrating later during gametogenesis. The embryo sac was fully developed prior to anthesis. In the developing seed, the endosperm was nuclear, with wall formation in the globular stage; a nucellar pad was observed during embryo development, but later became compressed. The ripe fruit contained seeds enveloped by a lignified endocarp that formed the pyrenes, with each pyrene having an operculum at the basal end; the embryo was considered to be differentiated. Most of these characteristics are shared with other Zingiberales, although the derivation of the operculum from the funicle and the formation of the main mechanical layer by the endocarp are unique to the Heliconiaceae.

Flowering Tops↗

Characterisation of a developmentally related polypeptide with glutelin solubility characteristics from Lupinus albus L.

Proteins from Lupinus albus L. cv. Rio Maior seeds were fractionated according to solubility criteria. Patterns of concanavalin A (ConA)-binding polypeptides from the different classes, albumins, globulins, glutelins and prolamins, were established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two bands of apparent molecular masses of 29 and 23.5 kDa with glutelin solubility characteristics bound the lectin. The 23.5-kDa band was separated by two-dimensional electrophoresis into two components: one glycosylated and heterogeneous with an isoelectric point of approx. 10 (designated as G23) and another, not detected with ConA, precipitating in the first dimension. The amino acid and hexosamine analysis of G23 showed that it is particularly rich in Gly (11.2%), Glx (10.0%), Ser (9.0%), Leu (8.2%), Asx (7.5%), and Pro (6.7%) and that it has a considerable content of the sulphur-containing amino acids Met (2.0%) and Cys (5.8%) and contains glucosamine. The determined N-terminal amino acid sequence of G23 was: 1KG(R)V5KGTGD10(T)PXXV15XLY(N)R20T, and this had no significant similarity to any of the amino acid sequences contained in the data bank SWISS-PROT 26. The glycoprotein G23 was completely deglycosylated with peptide-N-glycosidase F, yielding a homogeneous 21-kDa polypeptide composed of approximately 191 amino acids. The structures of the major N-linked neutral oligosaccharides of G23, determined by exoglycosidase sequencing, were as follows: Man alpha 2Man alpha 6(Man alpha 3) Man alpha 6(Man alpha 2Man alpha 2Man alpha 3)Man beta 4GlcNAc beta 4 GlcNAc (13%); +/- Man alpha 2Man alpha 6(Man alpha 3)Man alpha 6(+/- Man alpha 2 Man alpha 2 Man alpha 3)Man beta 4GlcNAc beta 4GlcNAc (29%); Man alpha 6(Man alpha 3) Man alpha 6(Man alpha 2Man alpha 3)Man beta 4GlcNAc beta 4GlcNAc (13%); Man alpha 6(Man alpha 3)Man alpha 6(Man alpha 3)Man beta 4GlcNAc beta 4GlcNAc (16%); Man alpha 6(Man alpha 3)(Xyl beta 2)Man beta 4GlcNAc beta 4GlcNAc (28%). Changes in G23 abundance during seed development, germination and seedling growth were monitored with a specific antibody. The glycoprotein G23 started to accumulate appreciably during seed formation between the 40th and the 50th days after anthesis and was detected following seed imbibition, until the 9th day in cotyledons, the 2nd day in roots and the 4th day in hypocotyls and leaves.

Amino Acid Sequence↗

Sequences responsible for the tissue specific promoter activity of a pea legumin gene in tobacco.

Maturing pea cotyledons accumulate large quantities of storage proteins at a specific time in seed development. To examine the sequences responsible for this regulated expression, a series of deletion mutants of the legA major seed storage protein gene were made and transferred to tobacco using the Bin19 disarmed Agrobacterium vector system. A promoter sequence of 97 bp including the CAAT and TATA boxes was insufficient for expression. Expression was first detected in a construct with 549 bp of upstream flanking sequence which contained the the leg box element, a 28 bp conserved sequence found in the legumin-type genes of several legume species. Constructs containing -833 and -1203 bp of promoter sequence significantly increased levels of expression. All expressing constructs preserved seed specificity and temporal regulation. The results indicate that promoter sequences between positions -97 and -549 bp are responsible for promoter activity, seed specificity, and temporal regulation of the pea legA gene. Sequences between positions -549 and -1203 bp appear to function as enhancer-like elements, to increase expression.

Base Sequence↗

Functional characterization of beta-ketoacyl-CoA synthase genes from Brassica napus L.

Seed-specifically expressed beta-ketoacyl-CoA synthase genes of Brassica napus (Bn-FAE1.1 genes) were cloned from two cultivars, namely Askari, a high-erucic-acid type, and Drakkar, a low-erucic-acid type. The genes from the two cultivars were found to be nearly identical. They encode proteins of 507 amino acids, the sequences of which differ only at position 282. The Bn-FAE1.1 gene of Askari, unlike that of Drakkar, was functionally expressed in yeast cells suggesting that the single amino acid exchange effects the low erucic acid phenotype at the E1 gene locus. In yeast cells the beta-ketoacyl-CoA synthase of Askari elongated not only oleoyl but also palmitoleoyl groups as well as saturated acyl groups in such a way that monounsaturated acyl groups of 22 carbons and saturated ones of 26 carbons were formed as main products. A reporter gene fused to the promoter region of the Bn-FAE1.1 gene from Askari showed seed-specific expression in transgenic rapeseed plants. Over-expression of the coding region of the Askari gene in developing seeds of transgenic Drakkar plants resulted in a significant increase in the levels of eicosenoic acid and erucic acid esterified in the seed oil. On the other hand, in transgenic high-erucic-acid rapeseed plants the increase in erucic acid level was at most 60% although the chimeric Bn-FAE1.1 gene was co-expressed with an erucoyl-CoA-specific lysophosphatidate acyltransferase gene enabling trierucoyl glycerol to accumulate in the seed oil.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗

Differential expression of two related amino acid transporters with differing substrate specificity in Arabidopsis thaliana.

A general amino acid permease cDNA (AAP2) was isolated from Arabidopsis by complementation of a yeast mutant defective in citrulline uptake. Direct transport measurements in yeast show that the protein mediates uptake of L-[14C]-citrulline and L-[14C]-proline. Detailed analyses of the substrate specificity by competition studies demonstrate that all proteogenic amino acids are recognized by the carrier, including those that represent the major transport forms of reduced nitrogen in many species, i.e. glutamine, glutamate and asparagine. Thus, AAP2 is less selective as compared with AAP1 and transports basic amino acids such as histidine as shown by expression in a histidine transport-deficient yeast strain. The predicted polypeptide of 53 kDa is highly hydrophobic with 12 putative membrane-spanning regions and shows significant homologies to the Arabidopsis broad specificity permease AAP1, and a limited homology to bacterial branched chain amino acid transporters, but not to any other known proteins. Alterations in the charged residues as compared with AAP1 in four regions might be involved in the difference in selectivity towards basic amino acids. Both genes are highly expressed in developing pods indicating a role in supplying the developing seeds with reduced nitrogen. AAP2 is selectively expressed in the stem and might therefore play a role in xylem-to-phloem transfer of amino acids during seed filling. Furthermore in situ hybridization shows that both genes are expressed in the vascular system of cotyledons in developing seedlings.

Amino Acid Sequence↗

A deletion mutation at the ep locus causes low seed coat peroxidase activity in soybean.

The Ep locus severely affects the amount of peroxidase enzyme in soybean seed coats. Plants containing the dominant Ep allele accumulate large amounts of peroxidase in the hourglass cells of the sub-epidermis. Homozygous recessive epep genotypes do not accumulate peroxidase in the hourglass cells and are much reduced in total seed coat peroxidase activity. To isolate the gene encoding the seed coat peroxidase and to determine whether it corresponds to the Ep locus, a cDNA library was constructed from developing seed coats and an abundant 1.3 kb peroxidase transcript was cloned. The corresponding structural gene was also isolated from a genomic library. Sequence analysis shows that the seed coat peroxidase is translated as a 352 amino acid precursor protein of 38 kDa. Processing of a putative 26 amino acid signal sequence results in a mature protein of 326 residues with a calculated mass of 35 kDa and a pl of 4.4. Using probes derived from the cDNA, genomic DNA blot hybridization and polymerase chain reaction analysis detected polymorphisms that distinguished EpEp and epep genotypes. Co-segregation of the polymorphisms in an F2 population from a cross of EpEp and epep plants shows that the Ep locus encodes the seed coat peroxidase protein. Comparison of Ep and ep alleles indicates that the recessive gene lacks 87 bp of sequence encompassing the translation start codon. Analysis by RNA blot hybridization shows that epep plants have drastically reduced amounts of peroxidase transcript compared with EpEp plants. The peroxidase mRNA is abundant in seed coat tissues of EpEp plants during the late stages of seed maturation, and could also be detected in root tissues, but not in the flower, embryo, pod or leaf. The results indicate that the lack of peroxidase accumulation in seed coats of homozygous recessive epep plants is due to a mutation of the structural gene that reduces transcript abundance.

Amino Acid Sequence↗

EMF genes maintain vegetative development by repressing the flower program in Arabidopsis.

The EMBRYONIC FLOWER (EMF) genes EMF1 and EMF2 are required to maintain vegetative development and repress flower development. EMF1 encodes a putative transcriptional regulator, and EMF2 encodes a Polycomb group protein homolog. We examined expression profiles of emf mutants using GeneChip technology. The high degree of overlap in expression changes from the wild type among the emf1 and emf2 mutants was consistent with the functional similarity between the two genes. Expression profiles of emf seedlings before flower development were similar to that of Arabidopsis flowers, indicating the commitment of germinating emf seedlings to the reproductive fate. The germinating emf seedlings ectopically expressed flower organ genes, suggesting that vegetative development in wild-type plants results from EMF repression of the flower program, directly or indirectly. In addition, the seed development program is derepressed in the emf1 mutants. Gene expression analysis showed no clear regulation of CONSTANS (CO), FLOWERING LOCUS T (FT), LEAFY (LFY), and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 by EMF1. Consistent with epistasis results that co, lfy, or ft cannot rescue rosette development in emf mutants, these data show that the mechanism of EMF-mediated repression of flower organ genes is independent of these flowering genes. Based on these findings, a new mechanism of EMF-mediated floral repression is proposed.

Arabidopsis↗

Dark chilling inhibition of photosynthesis and symbiotic nitrogen fixation in soybean during pod filling.

The growth stage of a soybean [Glycine max (L.) Merrill] plant may influence its physiological response to dark chilling. Opposed to vegetative development, the intense nutrient and energy requirements of the developing seeds during pod filling could cause additional chilling damage and decreased recovery capacity. Previously, we investigated dark chilling tolerance during vegetative development in two soybean genotypes, 'Maple Arrow' and 'Fiskeby V' and consistently found that photosynthesis and symbiotic nitrogen fixation (SNF) was less affected by dark chilling in 'Maple Arrow'. In this study we describe the dark chilling response of the same genotypes during pod filling. Our aim was to establish whether the potential selection criteria for dark chilling tolerance, identified during vegetative development, was equally sensitive during pod filling. The results indicate that photosynthesis is less affected by dark chilling in 'Maple Arrow' than in 'Fiskeby V', not only during vegetative development, but also during the critical reproductive stage of pod filling. 'Fiskeby V' also lacks the ability to restore normal photosynthetic capacity during an extended recovery treatment. The decrease of nodule ureide content indicates that SNF was inhibited to a similar extent in both genotypes. Nodule ureide content was reduced more than stem ureide content, suggesting that the former is a more sensitive indicator of chilling stress effects on SNF. The results indicate that certain photosynthetic and fluorescence parameters are sensitive indicators of dark chilling tolerance throughout plant development and should prove valuable in future breeding programmes aimed at increasing the chilling tolerance of soybean.

Carbon Dioxide↗

Differential manifestation of seed mortality induced by seed-specific expression of the gene for diphtheria toxin A chain in Arabidopsis and tobacco.

A pea vicilin promoter-diphtheria toxin A (DTx-A) chain gene fusion was introduced into Arabidopsis and tobacco. The chimeric Dtx-A gene behaves as a dominant, seed-lethal, Mendelian factor, and the segregation ratios are consistent with the numbers of integrated copies as revealed by Southern blotting. Germination deficiency results from distinct developmental abnormalities, thus allowing genetic dissection of seed development. The endosperm is affected first in both species. In Arabidopsis, full cellularization of the initially syncytial endosperm does not take place, which results in shrinkage and a shriveled appearance of the mature dry seed. The embryo, which appears structurally normal and lacks visible lesions, ceases to develop at the partially recurved cotyledon stage and does not use the remaining endosperm. In tobacco, peripheral degeneration and premature termination of cellular endosperm development occurs at the cotyledon initiation stage. Lesions appear in the cotyledons at the advanced cotyledon stage, but the embryo continues to grow and attains nearly the same size and level of differentiation as mature wild-type embryos before degeneration and intracellular disintegration take place throughout. Accumulation of protein bodies and other cytoplasmic inclusions is very limited and occurs only in few cells. The timing and distribution of lesions follow a pattern typical for accumulation of protein bodies in wild-type seeds. These observations are consistent with expression of the vicilin promoter in the enlargement phase of cell differentiation. A novel tissue interaction arises, when the embryo uses up all the arrested endosperm: the embryo proves to be capable of absorbing the parenchyma layers of the integument, which are normally obliterated by, and incorporated into, the endosperm.(ABSTRACT TRUNCATED AT 250 WORDS)

Arabidopsis↗

The resistance of cowpea seeds to bruchid beetles is not related to levels of cysteine proteinase inhibitors.

A cDNA encoding a cysteine proteinase inhibitor was isolated from a cDNA library prepared from developing seeds of an insect-resistant line of cowpea. The sequence of the encoded protein was homologous with those of other plant cysteine endoproteinase inhibitors, and with Type 2 cystatins from animals. Southern blot analyses indicated that small gene families were present in both resistant and susceptible lines of cowpea, while northern blot analyses showed similar levels of expression. It is concluded that the levels of expression of the inhibitor do not account for the differences in insect resistance of the two lines.

Amino Acid Sequence↗

Plant reproduction during spaceflight: importance of the gaseous environment.

Plant reproduction is a complex developmental process likely to be disrupted by the unusual environmental conditions in orbital spacecraft. Previous results, reviewed herein, indicated difficulties in obtaining successful seen production in orbit, often relating to delayed plant development during the long-term growth necessary for a complete plant life cycle. Using short-duration exposure to spaceflight, we studied plant reproduction in Arabidopsis thaliana (L.) Heynh, during three flight experiments: CHROMEX-03 on STS-54 (6 d), CHROMEX-04 on STS-51 (10 d), and CHROMEX-05 on STS-68 (11 d). Plants were 13 - 14 d old (rosettes) at time of launch and initiated flowering shoots while in orbit. Plants were retrieved from the orbiters 2 - 3 h after landing and reproductive material was immediately processed for in-vivo observations of pollen viability, pollen tube growth, and esterase activity in the stigma, or fixed for later microscopy. Plants produced equal numbers of flowers to those controls growing on the ground but required special environmental conditions to permit fertilization and early seed development during spaceflight. In CHROMEX-03, plants were grown in closed plant growth chambers (PGCs), and male and female gametophyte development aborted at an early stage in the flight material. In CHROMEX-04, carbon dioxide enrichment was provided to the closed PGCs and reproductive development proceeded normally until the pollination stage, when there was an obstacle to pollen transfer in the spaceflight material. In CHROMEX-05, an air-exchange system was used to provide a slow purging of the PGCs with filtered cabin air. Under these conditions, the spaceflight plants apparently had reproductive development comparable to the ground controls, and immature seeds were produced. In every aspect examined, these seeds are similar to those produced by the ground control plants. The results suggest that if the physical environment around the plant under spaceflight conditions meets the physiological demands of the plant, then reproductive development can proceed normally on orbit.

Arabidopsis↗

Expression of a yeast-derived invertase in developing cotyledons of Vicia narbonensis alters the carbohydrate state and affects storage functions.

In plants the carbohydrate state provides signals to adjust metabolism to specific physiological conditions. Storage-active sink organs like seeds often contain high levels of sucrose. In order to change the sugar status during seed development a yeast-derived invertase gene was expressed in Vicia narbonensis under control of the LeguminB4 promoter. A signal sequence targeted the invertase to the apoplast in maturing embryos. In the cotyledons, sucrose was decreased whereas hexoses strongly accumulated. There was a major reduction of starch whereas proteins were less affected. Vacuoles of cotyledon cells were enlarged and dry seeds wrinkled. Transcripts and enzyme activity of sucrose synthase, the small and large subunit of ADP-glucose pyrophosphorylase as well as vicilin were downregulated. Sucrose phosphate synthase and legumin-mRNAs were not affected. Analysing single seeds with different sucrose levels revealed a positive correlation of sucrose concentration to mRNA levels of sucrose synthase and most pronounced to ADP-glucose pyrophosphorylase-mRNA levels as well as to starch content. Glucose on the other hand did not show any correlation. After feeding 14C-sucrose in vitro, the invertase-expressing cotyledons partitioned less carbon into starch compared to the wild-type. In the transgenic cotyledons, a relatively higher amount was directed into proteins compared to starch. We conclude that starch accumulation in developing cotyledons could be a function of sucrose concentration. Our results are consistent with a possible sucrose-mediated induction of storage-associated differentiation indicated by upregulation of specific genes of the starch synthesis pathway.

Carbohydrate Metabolism↗