Silicon (Si) plays an important role in reducing plant susceptibility against a variety of different biotic and abiotic stresses; and also has an important regulatory role in soil to avoid heavy metal toxicity and providing suitable growing conditions for plants. A full-length cDNAs of 696bp of serine-rich protein was cloned from mangrove plant (Rhizophora apiculata) by amplification of cDNA ends from an expressed sequence tag homologous to groundnut (Arachis hypogaea), submitted to NCBI (KF211374). This serine-rich protein gene encodes a deduced protein of 223 amino acids. The transcript titre of the serine-rich protein was found to be strongly enriched in roots compared with the leaves of two month old mangrove plants and expression level of this serine-rich protein was found to be strongly induced when the mangrove seedlings were exposed to SiO2. Expression of the serine-rich protein transgenic was detected in transgenic Arabidopsis thaliana, where the amount of serine increased from 1.02 to 37.8mg/g. The same trend was also seen in Si content in the roots (14.3%) and leaves (7.4%) of the transgenic A. thaliana compared to the wild-type plants under Si treatment. The biological results demonstrated that the accumulation of the serine amino acid in the vegetative tissues of the transgenic plants enhanced their ability to absorb and accumulate more Si in the roots and leaves and suggests that the serine-rich protein gene has potential for use in genetic engineering of different stress tolerance characteristics.
Bacterial toxin-antitoxin (TA) systems have various cellular functions, including as part of the general stress response. The genome of the Gram-positive human pathogen Streptococcus pneumoniae harbors several putative TA systems, including yefM-yoeBSpn, which is one of four systems that had been demonstrated to be biologically functional. Overexpression of the yoeBSpn toxin gene resulted in cell stasis and eventually cell death in its native host, as well as in Escherichia coli. Our previous work showed that induced expression of a yoeBSpn toxin-Green Fluorescent Protein (GFP) fusion gene apparently triggered apoptosis and was lethal in the model plant, Arabidopsis thaliana. In this study, we investigated the effects of co-expression of the yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic A. thaliana. When co-expressed in Arabidopsis, the YefMSpn antitoxin was found to neutralize the toxicity of YoeBSpn-GFP. Interestingly, the inducible expression of both yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic hybrid Arabidopsis resulted in larger rosette leaves and taller plants with a higher number of inflorescence stems and increased silique production. To our knowledge, this is the first demonstration of a prokaryotic antitoxin neutralizing its cognate toxin in plant cells.
Matched MeSH terms: Plants, Genetically Modified/growth & development
Wild relatives of domesticated crop species harbor multiple, diverse, disease resistance (R) genes that could be used to engineer sustainable disease control. However, breeding R genes into crop lines often requires long breeding timelines of 5-15 years to break linkage between R genes and deleterious alleles (linkage drag). Further, when R genes are bred one at a time into crop lines, the protection that they confer is often overcome within a few seasons by pathogen evolution. If several cloned R genes were available, it would be possible to pyramid R genes in a crop, which might provide more durable resistance. We describe a three-step method (MutRenSeq)-that combines chemical mutagenesis with exome capture and sequencing for rapid R gene cloning. We applied MutRenSeq to clone stem rust resistance genes Sr22 and Sr45 from hexaploid bread wheat. MutRenSeq can be applied to other commercially relevant crops and their relatives, including, for example, pea, bean, barley, oat, rye, rice and maize.
Suppression subtractive hybridization (SSH) is an effective method to identify different genes with different expression levels involved in a variety of biological processes. This method has often been used to study molecular mechanisms of plants in complex relationships with different pathogens and a variety of biotic stresses. Compared to other techniques used in gene expression profiling, SSH needs relatively smaller amounts of the initial materials, with lower costs, and fewer false positives present within the results. Extraction of total RNA from plant species rich in phenolic compounds, carbohydrates, and polysaccharides that easily bind to nucleic acids through cellular mechanisms is difficult and needs to be considered. Remarkable advancement has been achieved in the next-generation sequencing (NGS) field. As a result of progress within fields related to molecular chemistry and biology as well as specialized engineering, parallelization in the sequencing reaction has exceptionally enhanced the overall read number of generated sequences per run. Currently available sequencing platforms support an earlier unparalleled view directly into complex mixes associated with RNA in addition to DNA samples. NGS technology has demonstrated the ability to sequence DNA with remarkable swiftness, therefore allowing previously unthinkable scientific accomplishments along with novel biological purposes. However, the massive amounts of data generated by NGS impose a substantial challenge with regard to data safe-keeping and analysis. This review examines some simple but vital points involved in preparing the initial material for SSH and introduces this method as well as its associated applications to detect different novel genes from different plant species. This review evaluates general concepts, basic applications, plus the probable results of NGS technology in genomics, with unique mention of feasible potential tools as well as bioinformatics.
In the past decade, interest in the production of recombinant pharmaceutical proteins in plants has tremendously progressed because plants do not harbor mammalian viruses, are economically competitive, easily scalable, and capable of carrying out complex post-translational modifications required for recombinant pharmaceutical proteins. Mucuna bracteata is an essential perennial cover crop species widely planted as an underground cover in oil palm and rubber plantations. As a legume, they have high biomass, thrive in its habitat, and can fix nitrogen. Thus, M. bracteata is a cost-efficient crop that shows ideal characteristics as a platform for mass production of recombinant protein. In this study, we established a new platform for the transient production of a recombinant protein in M. bracteata via vacuum-assisted agro-infiltration. Five-week-old M. bracteata plants were vacuum infiltrated with Agrobacterium tumefaciens harboring a plasmid that encodes for an anti-toxoplasma immunoglobulin (IgG) under different parameters, including trifoliate leaf positional effects, days to harvest post-infiltration, and the Agrobacterium strain used. Our results showed that vacuum infiltration of M. bracteata plant with A. tumefaciens strain GV3101 produced the highest concentration of heterologous protein in its bottom trifoliate leaf at 2 days post-infiltration. The purified anti-toxoplasma IgG was then analyzed using Western blot and ELISA. It was demonstrated that, while structural heterogeneity existed in the purified anti-toxoplasma IgG from M. bracteata, its transient expression level was two-fold higher than the model platform, Nicotiana benthamiana. This study has laid the foundation towards establishing M. bracteata as a potential platform for the production of recombinant pharmaceutical protein.
Monodehydroascorbate reductase (MDHAR), an important enzyme of the ascorbate-glutathione cycle, is involved in salt tolerance of plants through scavenging of reactive oxygen species (ROS). In this study, a cDNA encoding MDHAR from the mangrove plant Acanthus ebracteatus was introduced into rice to examine its role in salt tolerance. Three stable transgenic lines (MT22, MT24 and MT25) overexpressing AeMDHAR were selected in vitro using hygromycin and confirmed by PCR, quantitative reverse-transcription (qRT) PCR and enzyme assay. The transgenic line MT24 was predicted to possess a single copy of the transgene while the other two transgenic lines were predicted to have multiple transgene integrations. The AeMDHAR transcripts were detected only in transgenic rice lines but not in untransformed rice. The abundance of AeMDHAR transcripts in transgenic lines MT22 and MT25 was approximately 2.75 times the amount found in MT24. The transgenic rice lines overexpressing AeMDHAR showed a significant increase in MDHAR enzyme activity compared to untransformed plants under both NaCl and control conditions. All transgenic lines showed better yield attributes such as a higher tiller number and increased 1000-grain weight compared to non-transgenics. They also showed tolerance to salt at germination and seedling stages. The transgenic line MT24, which harbors a single copy of AeMDHAR, displayed a lower rate of sterility, a higher number of tillers and longer panicle compared to untransformed plants when subjected to salt stress.
The stearoyl-acyl-carrier-protein (ACP) desaturase is a plastid-localized enzyme that catalyzes the conversion of stearoyl-ACP to oleoyl-ACP and plays an important role in the determination of the properties of the majority of cellular glycerolipids. Functional characterization of the fatty acid desaturase genes and their specific promoters is a prerequisite for altering the composition of unsaturated fatty acids of palm oil by genetic engineering. In this paper, the specificity and strength of the oil palm stearoyl-ACP desaturase gene promoter (Des) was evaluated in transgenic tomato plants. Transcriptional fusions between 5' deletions of the Des promoter (Des1-4) and the β-glucuronidase (GUS) reporter gene were generated and their expression analyzed in different tissues of stably transformed tomato plants. Histochemical analysis of the Des promoter deletion series revealed that GUS gene expression was confined to the tomato fruits. No expression was detected in vegetative tissues of the transgenic plants. The highest levels of GUS activity was observed in different tissues of ripe red fruits (vascular tissue, septa, endocarp, mesocarp and columella) and in seeds, which harbored the promoter region located between -590 and +10. A comparison of the promoter-deletion constructs showed that the Des4 promoter deletion (314bp) produced a markedly low level of GUS expression in fruits and seeds. Fluorometric analysis of the GUS activity revealed a 4-fold increase in the activity of the full-length Des promoter compared to the CaMV35S promoter. RNA-hybridization analyses provided additional evidence of increased GUS expression in fruits driven by a Des fragment. Taken together, these results demonstrate the potential of the Des promoter as a tool for the genetic engineering of oil palms and other species, including dicots, in improving the quality and nutritional value of the fruits.
Metallothioneins (MTs) are cysteine-rich metal-binding proteins that are involved in cell growth regulation, transportation of metal ions and detoxification of heavy metals. A mesocarp-specific metallothionein-like gene (MT3-A) promoter was isolated from the oil palm (Elaeis guineensis Jacq). A vector construct containing the MT3-A promoter fused to the β-glucuronidase (GUS) gene in the pCAMBIA 1304 vector was produced and used in Agrobacterium-mediated transformation of tomato. Histochemical GUS assay of different tissues of transgenic tomato showed that the MT3-A promoter only drove GUS expression in the reproductive tissues and organs, including the anther, fruit and seed coat. Competitive RT-PCR and GUS fluorometric assay showed changes in the level of GUS mRNA and enzyme activity in the transgenic tomato (T(0)). No GUS mRNA was found in roots and leaves of transgenic tomato. In contrast, the leaves of transgenic tomato seedlings (T(1)) produced the highest GUS activity when treated with 150 μM Cu(2+) compared to the control (without Cu(2+)). However, Zn(2+) and Fe(2+) treatments did not show GUS expression in the leaves of the transgenic tomato seedlings. Interestingly, the results showed a breaking-off tissue-specific activity of the oil palm MT3-A promoter in T(1) seedlings of tomato when subjected to Cu(2+) ions.
Brassinosteroid Insensitive 1 (BRI1)-Associated Kinase I (BAK1) has been reported to interact with BRI1 for brassinosteroid (BR) perception and signal transduction that regulate plant growth and development. The aim of this study is to investigate the functions of a rice OsBAK1 homologue, designated as OsI-BAK1, which is highly expressed after heading. Silencing of OsI-BAK1 in rice plants produced a high number of undeveloped green and unfilled grains compared to the untransformed plants. Histological analyses demonstrated that embryos were either absent or retarded in their development in these unfilled rice grains of OsI-BAK1 RNAi plants. Down regulation of OsI-BAK1 caused a reduction in cell number and enlargement in leaf bulliform cells. Furthermore, transgenic rice plants overexpressing OsI-BAK1 were demonstrated to have corrugated and twisted leaves probably due to increased cell number that caused abnormal bulliform cell structure which were enlarged and plugged deep into leaf epidermis. The current findings suggest that OsI-BAK1 may play an important role in the developmental processes of rice grain filling and leaf cell including the bulliform cells.
Matched MeSH terms: Plants, Genetically Modified/growth & development
Consumption of white rice predisposes some Asian populations to increased risk of type 2 diabetes. We compared the postprandial glucometabolic responses to three newly-developed crossbred red rice variants (UKMRC9, UKMRC10, UKMRC11) against three selected commercial rice types (Thai red, Basmati white, Jasmine white) using 50-g carbohydrate equivalents provided to 12 normoglycaemic adults in a crossover design. Venous blood was drawn fasted and postprandially for three hours. Glycaemic (GI) and insulin (II) indices, incremental areas-under-the-curves for glucose and insulin (IAUCins), indices of insulin sensitivity and secretion, lactate and peptide hormones (motilin, neuropeptide-Y, orexin-A) were analyzed. The lowest to highest trends for GI and II were similar i.e., UKMRC9 < Basmati < Thai red < UKMRC10 < UKMRC11 < Jasmine. Postprandial insulinaemia and IAUCins of only UKMRC9 were significantly the lowest compared to Jasmine. Crude protein and fiber content correlated negatively with the GI values of the test rice. Although peptide hormones were not associated with GI and II characteristics of test rice, early and late phases of prandial neuropeptide-Y changes were negatively correlated with postprandial insulinaemia. This study indicated that only UKMRC9 among the new rice crossbreeds could serve as an alternative cereal option to improve diet quality of Asians with its lowest glycaemic and insulinaemic burden.
Three different morphological callus types, identified as type A, B and C, and tips of in vitro inflorescences were used as target tissues for genetic transformation. Five different DNA plasmids carrying a synthetic green fluorescent protein (gfp) gene driven by different promoters, CaMV 35S, HBT, and Ubi1 were tested for the genetic transformation of Dendrobium Sonia 17. 35S-sgfp-TYG-nos (p35S) with the CaMV 35S promoter showed the highest GFP transient expression rate, while the HBT and Ubi1 promoters showed a relatively lower expression rate in all of the target tissues tested. The highest number of GFP-expressing cells was observed on day 2 post-bombardment, and the number declined gradually over the course of the next 2 weeks. Type A and B callus were found to be the best potential target tissues for genetic transformation.
Magnaporthe oryzae is a rice blast fungus and plant pathogen that causes a serious rice disease and, therefore, poses a threat to the world's second most important food security crop. Plant transformation technology has become an adaptable system for cultivar improvement and to functionally analyze genes in plants. The objective of this study was to determine the effects (through over-expressing and using the CaMV 35S promoter) of Pikh on MR219 resistance because it is a rice variety that is susceptible to the blast fungus pathotype P7.2. Thus, a full DNA and coding DNA sequence (CDS) of the Pikh gene, 3172 bp, and 1206 bp in length, were obtained through amplifying the gDNA and cDNA template from a PH9-resistant rice variety using a specific primer. Agrobacterium-mediated transformation technology was also used to introduce the Pikh gene into the MR219 callus. Subsequently, transgenic plants were evaluated from the DNA to protein stages using polymerase chain reaction (PCR), semi-quantitative RT-PCR, real-time quantitative PCR and high performance liquid chromatography (HPLC). Transgenic plants were also compared with a control using a real-time quantification technique (to quantify the pathogen population), and transgenic and control plants were challenged with the local most virulent M. oryzae pathotype, P7.2. Based on the results, the Pikh gene encodes a hydrophilic protein with 18 sheets, 4 helixes, and 21 coils. This protein contains 401 amino acids, among which the amino acid sequence from 1 to 376 is a non-cytoplasmic region, that from 377 to 397 is a transmembrane region, and that from 398 to 401 is a cytoplasmic region with no identified disordered regions. The Pikh gene was up-regulated in the transgenic plants compared with the control plants. The quantity of the amino acid leucine in the transgenic rice plants increased significantly from 17.131 in the wild-type to 47.865 mg g(-1) in transgenic plants. The M. oryzae population was constant at 31, 48, and 72 h after inoculation in transgenic plants, while it was increased in the inoculated control plants. This study successfully clarified that over-expression of the Pikh gene in transgenic plants can improve their blast resistance against the M. oryzae pathotype P7.2.
Physical and biological parameters affecting DNA delivery into oil palm embryogenic calli using the biolistic device are optimized. Five different promoters are also evaluated to identify the most suitable promoter for use in oil palm transformation. Finally, the effectiveness of kanamycin, geneticin (G418), neomycin, hygromycin, and herbicide Basta as selection agents to inhibit growth of oil palm embryogenic calli is evaluated. Combination of optimized parameters, best promoter and selection agent is later used to transform oil palm embryogenic calli for producing transgenic oil palm plants. Bombarded embryogenic calli are exposed to 50 mg/l of Basta after 3 weeks. Basta-resistant embryogenic calli started to emerge five to six months in medium containing Basta. The Basta-resistant embryogenic calli are proliferated until they reach a specific size, and the Basta-resistant calli are later individually isolated and regenerated to produce complete plantlets. The complete regenerated plantlets are evaluated for the presence of transgenes by PCR, Southern and thin layer chromatography analyses.
Oil palm is a major economic crop for Malaysia. The major challenges faced by the industry are labor shortage, availability of arable land and unstable commodity price. This has caused the industry to diversify its applications into higher value products besides increasing its yield. While conventional breeding has its limitations, biotechnology was identified as one of the tools for overcoming the above challenges. Research on biotechnology of oil palm began more than two decades ago leveraging a multidisciplinary approach involving biochemical studies, gene and promoter isolation, transformation vector construction and finally genetic transformation to produce the targeted products. The main target of oil palm biotechnology research is to increase oleic acid in the mesocarp. Other targets are stearic acid, palmitoleic acid, ricinoleic acid, lycopene (carotenoid) and biodegradable plastics. Significant achievements were reported for the biochemical studies, isolation of useful oil palm genes and characterization of important promoters. A large number of transformation constructs for various targeted products were successfully produced using the isolated oil palm genes and promoters. Finally transformation of these constructs into oil palm embryogenic calli was carried out while the regeneration of transgenic oil palm harboring the useful genes is in progress.
Pharmaceutically active compounds from medical plants are attractive as a major source for new drug development. Prenylated stilbenoids with increased lipophilicity are valuable secondary metabolites which possess a wide range of biological activities. So far, many prenylated stilbenoids have been isolated from Morus alba but the enzyme responsible for the crucial prenyl modification remains unknown. In the present study, a stilbenoid-specific prenyltransferase (PT), termed Morus alba oxyresveratrol geranyltransferase (MaOGT), was identified and functionally characterized in vitro. MaOGT recognized oxyresveratrol and geranyl diphosphate (GPP) as natural substrates, and catalyzed oxyresveratrol prenylation. Our results indicated that MaOGT shared common features with other aromatic PTs, e.g. multiple transmembrane regions, conserved functional domains and targeting to plant plastids. This distinct PT represents the first stilbenoid-specific PT accepting GPP as a natural prenyl donor, and could help identify additional functionally varied PTs in moraceous plants. Furthermore, MaOGT might be applied for high-efficiency and large-scale prenylation of oxyresveratrol to produce bioactive compounds for potential therapeutic applications.
Genetic improvement is an important approach for crop improvement towards yield stability in stress-prone areas. Functional analysis of candidate stress response genes can provide key information to allow the selection and modification of improved crop varieties. In this study, the constitutive expression of a banana cDNA, MaRHD3 in Arabidopsis improved the ability of transgenic lines to adapt to drought conditions. Transgenic Arabidopsis plants expressing MaRHD3 had roots with enhanced branching and more root hairs when challenged with drought stress. The MaRHD3 plants had higher biomass accumulation, higher relative water content, higher chlorophyll content and an increase in activity of reactive oxygen species (ROS) scavenging enzymes; SOD, CAT, GR, POD and APX with reduced water loss rates compared to control plants. The analysis of oxidative damage indicated lower cell membrane damage in transgenic lines compared to control plants. These findings, together with data from higher expression of ABF-3 and higher ABA content of drought-stressed transgenic MaRHD3 expressing plants, support the involvement of the ABA signal pathway and ROS scavenging enzyme systems in MaRHD3 mediated drought tolerance.
Abiotic stress reduces plant growth and crop productivity. However, the mechanism underlying posttranscriptional regulations of stress response remains elusive. Herein, we report the posttranscriptional mechanism of nucleocytoplasmic RNA transport of stress-responsive transcripts mediated by EgRBP42, a heterogeneous nuclear ribonucleoprotein-like RNA-binding protein from oil palm, which could be necessary for rapid protein translation to confer abiotic stress tolerance in plants. Transgenic Arabidopsis overexpressing EgRBP42 showed early flowering through alteration of gene expression of flowering regulators and exhibited tolerance towards heat, cold, drought, flood, and salinity stresses with enhanced poststress recovery response by increasing the expression of its target stress-responsive genes. EgRBP42 harbours nucleocytoplasmic shuttling activity mediated by the nuclear localization signal and the M9-like domain of EgRBP42 and interacts directly with regulators in the nucleus, membrane, and the cytoplasm. EgRBP42 regulates the nucleocytoplasmic RNA transport of target stress-responsive transcripts through direct binding to their AG-rich motifs. Additionally, EgRBP42 transcript and protein induction by environmental stimuli are regulated at the transcriptional and posttranscriptional levels. Taken together, the posttranscriptional regulation of RNA transport mediated by EgRBP42 may change the stress-responsive protein profiles under abiotic stress conditions leading to a better adaptation of plants to environmental changes.
Cassava brown streak disease (CBSD) is a leading cause of cassava losses in East and Central Africa, and is currently having a severe impact on food security. The disease is caused by two viruses within the Potyviridae family: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), which both encode atypical Ham1 proteins with highly conserved inosine triphosphate (ITP) pyrophosphohydrolase (ITPase) domains. ITPase proteins are widely encoded by plant, animal, and archaea. They selectively hydrolyse mutagenic nucleotide triphosphates to prevent their incorporation into nucleic acid and thereby function to reduce mutation rates. It has previously been hypothesized that U/CBSVs encode Ham1 proteins with ITPase activity to reduce viral mutation rates during infection. In this study, we investigate the potential roles of U/CBSV Ham1 proteins. We show that both CBSV and UCBSV Ham1 proteins have ITPase activities through in vitro enzyme assays. Deep-sequencing experiments found no evidence of the U/CBSV Ham1 proteins providing mutagenic protection during infections of Nicotiana hosts. Manipulations of the CBSV_Tanza infectious clone were performed, including a Ham1 deletion, ITPase point mutations, and UCBSV Ham1 chimera. Unlike severely necrotic wild-type CBSV_Tanza infections, infections of Nicotiana benthamiana with the manipulated CBSV infectious clones do not develop necrosis, indicating that that the CBSV Ham1 is a necrosis determinant. We propose that the presence of U/CBSV Ham1 proteins with highly conserved ITPase motifs indicates that they serve highly selectable functions during infections of cassava and may represent a euphorbia host adaptation that could be targeted in antiviral strategies.
MAIN CONCLUSION: Rice sheath blight research should prioritise optimising biological control approaches, identification of resistance gene mechanisms and application in genetic improvement and smart farming for early disease detection. Rice sheath blight, caused by Rhizoctonia solani AG1-1A, is one of the most devasting diseases of the crop. To move forward with effective crop protection against sheath blight, it is important to review the published information related to pathogenicity and disease management and to determine areas of research that require deeper study. While progress has been made in the identification of pathogenesis-related genes both in rice and in the pathogen, the mechanisms remain unclear. Research related to disease management practices has addressed the use of agronomic practices, chemical control, biological control and genetic improvement: Optimising nitrogen fertiliser use in conjunction with plant spacing can reduce spread of infection while smart agriculture technologies such as crop monitoring with Unmanned Aerial Systems assist in early detection and management of sheath blight disease. Replacing older fungicides with natural fungicides and use of biological agents can provide effective sheath blight control, also minimising environmental impact. Genetic approaches that show promise for the control of sheath blight include treatment with exogenous dsRNA to silence pathogen gene expression, genome editing to develop rice lines with lower susceptibility to sheath blight and development of transgenic rice lines overexpressing or silencing pathogenesis related genes. The main challenges that were identified for effective crop protection against sheath blight are the adaptive flexibility of the pathogen, lack of resistant rice varieties, abscence of single resistance genes for use in breeding and low access of farmers to awareness programmes for optimal management practices.
KEY MESSAGE: This study highlights the changes in umami-related nucleotide and glutamate levels when the AMP deaminase gene was elevated in transgenic tomato. Taste is perceived as one of a combination of five sensations, sweet, sour, bitter, salty, and umami. The umami taste is best known as a savoury sensation and plays a central role in food flavour, palatability, and eating satisfaction. Umami flavour can be imparted by the presence of glutamate and is greatly enhanced by the addition of ribonucleotides, such as inosine monophosphate (IMP) and guanosine monophosphate (GMP). The production of IMP is regulated by the enzyme adenosine monophosphate (AMP) deaminase which functions to convert AMP into IMP. We have generated transgenic tomato (Solanum lycopersicum) lines over expressing AMP deaminase under the control of a fruit-specific promoter. The transgenic lines showed substantially enhanced levels of AMP deaminase expression in comparison to the wild-type control. Elevated AMP deaminase levels resulted in the reduced accumulation of glutamate and increased levels of the umami nucleotide GMP. AMP concentrations were unchanged. The effects on the levels of glutamate and GMP were unexpected and are discussed in relation to the metabolite flux within this pathway.