ALA Synthesis Research Articles

Study on the Mechanism of Exogenous 5-Aminolevulinic Acid (ALA) in Regulating the Photosynthetic Efficiency of Pear Leaves

To provide a theoretical basis for the application of ALA in pear production, the effects of exogenous 5-aminolevulinic acid (ALA) treatment on leaf photosynthetic gas exchange parameters, chlorophyll fast fluorescence properties, and relative expression of the related genes were investigated using pear (Pyrus pyrifolia Nakai cv. ‘Whasan’) as a material in the study. The results show that exogenous ALA treatment improved the photosynthetic gas exchange parameters of pear leaves, upregulated the expression of multiple key genes which are related to ALA biosynthesis, metabolism, and transformation into chlorophylls. GUS staining in tobacco leaves showed that exogenous ALA activated the promoter activity of PypHEMA and PypCHLH genes, implying that the synthesis of endogenous ALA and chlorophylls was promoted by exogenous ALA. Furthermore, ALA promoted the expression of the genes encoding photosystem II (PSII) reaction center proteins, such as core protein D1, inner light-harvesting pigment proteins CP43 and CP47, and cytochrome b559. This led to increased PSII reaction center activity. In addition, ALA alleviated the donor side oxygen-evolving complex inhibition and reduced the closure rate on the receptor side, allowing for increased photochemical electron transfer and reduced heat dissipation while improving the photosynthetic performance index PIabs and PItotal. The findings of this study contribute to a better understanding of ALA’s promotion of plant photosynthetic efficiency, providing valuable insights for further research and potential applications in pear production.

Comparative Transcriptomics Uncovers Upstream Factors Regulating BnFAD3 Expression and Affecting Linolenic Acid Biosynthesis in Yellow-Seeded Rapeseed (Brassica napus L.).

α-Linolenic acid (ALA) is an important nutrient component in rapeseed oil, and rapeseed breeders want to either restrain or enhance the function of fatty acid desaturases (FADs) in the ALA biosynthesis pathway. To determine the reason for the upregulation of rapeseed BnFAD genes in two high-ALA accessions, R8Q10 and YH25005, we compared their transcriptome profiles in the seed at 24 days after pollination (DAP) with those of two low-ALA lines, A28 and SW. The expression levels of twenty-eight important genes in the seed samples at 20, 27, and 34 DAP were also investigated using an RT-qPCR. The expression levels of genes involved in flavonoid and proanthocyanidin synthesis, including BnCHS, BnCHI, BnDFR, BnFLS1, BnLDOX, BnBAN, BnTT10, and BnTT12 and genes encoding the transcription factors BnTT1, BnTT2, BnTT8, and BnTT16 were lower in R8Q10 and YH25005 than in A28 and SW. The expression levels of genes encoding master transcription factors in embryo development, such as BnLEC1, BnABI3, BnFUS3, BnL1L, BnAREB3, and BnbZIP67, were elevated significantly in the two high-ALA accessions. Combined with previous results in the Arabidopsis and rapeseed literature, we speculated that the yellow-seededness genes could elevate the activity of BnLEC1, BnABI3, BnFUS3, and BnbZIP67, etc., by reducing the expression levels of several transparent testa homologs, resulting in BnFAD3 and BnFAD7 upregulation and the acceleration of ALA synthesis. Yellow-seededness is a favorable factor to promote ALA synthesis in the two high-ALA accessions with the yellow-seeded trait. These findings provide initial insights into the transcriptomic differences between high-/low-ALA germplasms and a theoretic basis for seed quality breeding.

FC2 stabilizes POR and suppresses ALA formation in the tetrapyrrole biosynthesis pathway.

During photoperiodic growth, the light-dependent nature of chlorophyll synthesis in angiosperms necessitates robust control of the production of 5-aminolevulinic acid (ALA), the rate-limiting step in the initial stage of tetrapyrrole biosynthesis (TBS). We are interested in dissecting the post-translational control of this process, which suppresses ALA synthesis for chlorophyll synthesis in dark-grown plants. Using biochemical approaches for analysis of Arabidopsis wild-type (WT) and mutant lines as well as complementation lines, we show that the heme-synthesizing ferrochelatase 2 (FC2) interacts with protochlorophyllide oxidoreductase and the regulator FLU which both promote the feedback-controlled suppression of ALA synthesis by inactivation of glutamyl-tRNA reductase, thus preventing excessive accumulation of potentially deleterious tetrapyrrole intermediates. Thereby, FC2 stabilizes POR by physical interaction. When the interaction between FC2 and POR is perturbed, suppression of ALA synthesis is attenuated and photoreactive protochlorophyllide accumulates. FC2 is anchored in the thylakoid membrane via its membrane-spanning CAB (chlorophyll-a-binding) domain. FC2 is one of the two isoforms of ferrochelatase catalyzing the last step of heme synthesis. Although FC2 belongs to the heme-synthesizing branch of TBS, its interaction with POR potentiates the effects of the GluTR-inactivation complex on the chlorophyll-synthesizing branch and ensures reciprocal control of chlorophyll and heme synthesis.

How aminomethylphosphonic acid (AMPA), the main glyphosate metabolite, interferes with chlorophyll biosynthesis?

Aminomethylphosphonic acid (AMPA) has been proposed to interfere with chlorophyll biosynthesis in photoautotrophic organisms – which may explain its greater toxicity to plants in relation to animals. By using the aquatic macrophyte Lemna minor , we investigated the AMPA toxicity and its effects on chlorophyll biosynthesis. Environmentally relevant concentrations (35–50 µg l -1 ) of AMPA reduced L. minor chlorophyll concentration with an EC 10 and EC 50 of 1.3 and 902.9 µg AMPA l -1 after seven days of exposure respectively but did not affect plant fresh weight/growth rate (EC 10 and EC 50 of 178.6/244.4 and 11,922.2/12,513.2 µg AMPA l -1 , respectively). AMPA decreased the activity of glycine decarboxylase (GDC), leading to glycine accumulation and glutamate (Glu) deprivation in plants. The lack of Glu prevents the synthesis of δ-aminolevulinic acid (ALA) in chloroplasts, interfering on chlorophyll synthesis. The negative effect of AMPA on chlorophyll concentration was reversible by supplementing growth media with Glu or ALA. The presence of AMPA in the aquatic ecosystems may contribute to their alteration by its negative effects on photosynthetic organisms through chlorophyll biosynthesis inhibition. Furthermore, we should stress out that AMPA could be toxic for animals and humans due to known toxicity of glycine induced by GDC inhibition. • Environmental relevant concentrations of AMPA reduce chlorophyll concentrations. • AMPA decreased the activity of the enzyme glycine decarboxylase (GDC). • AMPA induces the lack of glutamate and prevents the synthesis of ALA in chloroplasts. • Due to its negative effect on GDC, AMPA could be toxic for animals and humans.

Glutamate 1-semialdehyde aminotransferase is connected to GluTR by GluTR-binding protein and contributes to the rate-limiting step of 5-aminolevulinic acid synthesis

Tetrapyrroles play fundamental roles in crucial processes including photosynthesis, respiration, and catalysis. In plants, 5-aminolevulinic acid (ALA) is the common precursor of tetrapyrroles. ALA is synthesized from activated glutamate by the enzymes glutamyl-tRNA reductase (GluTR) and glutamate-1-semialdehyde aminotransferase (GSAAT). ALA synthesis is recognized as the rate-limiting step in this pathway. We aimed to explore the contribution of GSAAT to the control of ALA synthesis and the formation of a protein complex with GluTR. In Arabidopsis thaliana, two genes encode GSAAT isoforms: GSA1 and GSA2. A comparison of two GSA knockout mutants with the wild-type revealed the correlation of reduced GSAAT activity and ALA-synthesizing capacity in leaves with lower chlorophyll content. Growth and green pigmentation were more severely impaired in gsa2 than in gsa1, indicating the predominant role of GSAAT2 in ALA synthesis. Interestingly, GluTR accumulated to higher levels in gsa2 than in the wild-type and was mainly associated with the plastid membrane. We propose that the GSAAT content modulates the amount of soluble GluTR available for ALA synthesis. Several different biochemical approaches revealed the GSAAT-GluTR interaction through the assistance of GluTR-binding protein (GBP). A modeled structure of the tripartite protein complex indicated that GBP mediates the stable association of GluTR and GSAAT for adequate ALA synthesis.

A Secondary Metabolic Enzyme Functioned as an Evolutionary Seed of a Primary Metabolic Enzyme.

The antibiotic alaremycin has a structure that resembles that of 5-aminolevulinic acid (ALA), a universal precursor of porphyrins, and inhibits porphyrin biosynthesis. Genome sequencing of the alaremycin-producing bacterial strain and enzymatic analysis revealed that the first step of alaremcyin biosynthesis is catalysed by the enzyme, AlmA, which exhibits a high degree of similarity to 5-aminolevulinate synthase (ALAS) expressed by animals, protozoa, fungi, and α-proteobacteria. Site-directed mutagenesis of AlmA revealed that the substitution of two amino acids residues around the substrate binding pocket transformed its substrate specificity from that of alaremycin precursor synthesis to ALA synthesis. To estimate the evolutionary trajectory of AlmA and ALAS, we performed an ancestral sequence reconstitution analysis based on a phylogenetic tree of AlmA and ALAS. The reconstructed common ancestral enzyme of AlmA and ALAS exhibited alaremycin precursor synthetic activity, rather than ALA synthetic activity. These results suggest that ALAS evolved from an AlmA-like enzyme. We propose a new evolutionary hypothesis in which a non-essential secondary metabolic enzyme acts as an ‘evolutionary seed’ to generate an essential primary metabolic enzyme.

An alanine to valine mutation of glutamyl-tRNA reductase enhances 5-aminolevulinic acid synthesis in rice.

An alanine to valine mutation of glutamyl-tRNA reductase's 510th amino acid improves 5-aminolevulinic acid synthesis in rice. 5-aminolevulinic acid (ALA) is the common precursor of all tetrapyrroles and plays animportant role in plant growth regulation. ALA is synthesized from glutamate, catalyzed by glutamyl-tRNA synthetase (GluRS), glutamyl-tRNA reductase (GluTR), and glutamate-1-semialdehyde aminotransferase (GSAT). In Arabidopsis, ALA synthesis is the rate-limiting step in tetrapyrrole production via GluTR post-translational regulations. In rice, mutations of GluTR and GSAT homologs are known to confer chlorophyll deficiency phenotypes; however, the enzymatic activity of rice GluRS, GluTR, and GSAT and the post-translational regulation of rice GluTR have not been investigated experimentally. We have demonstrated that a suppressor mutation in rice partially reverts the xantha trait. In the present study, we first determine that the suppressor mutation results from a G → A nucleotide substitution of OsGluTR (and an A → V change of its 510th amino acid). Protein homology modeling and molecular docking show that the OsGluTRA510V mutation increases its substrate binding. We then demonstrate that the OsGluTRA510V mutation increases ALA synthesis in Escherichia coli without affecting its interaction with OsFLU. We further explore homologous genes encoding GluTR across 193 plant species and find that the amino acid (A) is 100% conserved at the position, suggesting its critical role in GluTR. Thus, we demonstrate that the gain-of-function OsGluTRA510V mutation underlies suppression of the xantha trait, experimentally proves the enzymatic activity of rice GluRS, GluTR, and GSAT in ALA synthesis, and uncovers conservation of the alanine corresponding to the 510th amino acid of OsGluTR across plant species.

Manganese toxicity-induced chlorosis in sugarcane seedlings involves inhibition of chlorophyll biosynthesis

Manganese (Mn) toxicity-induced leaf chlorosis limits crop production in acidic soils, but its underlying mechanisms remain unknown. The effects of excessive Mn on chlorophyll (Chl) biosynthesis in sugarcane (Saccharum officinarum L.) leaves were investigated. Under Mn treatment, Chl concentration decreased with Mn accumulation and chlorosis appeared in expanding leaves. Before that, levels of the initial Chl precursor 5-aminolevulinic acid (ALA) and its downstream intermediates decreased, whereas magnesium-protoporphyrin IX monomethyl ester (MgPME) accumulated. Overaccumulation of Mn in leaves downregulated the ALA biosynthetic gene GluTR (encoding glutamyl-tRNA reductase) and MgPME conversion gene MgPMEC (encoding MgPME cyclase), upregulated the ALA biosynthesis inhibitor FLU (encoding FLUORESCENT), but had no significant effect on the expression of other Chl biosynthetic genes. The above Mn-induced changes of Chl precursors and expression of corresponding genes commenced before the Chl decline and leaf chlorosis, and were reversed by ALA supplementation. Thus, excessive Mn-induced chlorosis in sugarcane is mediated by a Chl-biosynthesis disorder resulting from the inhibition of ALA synthesis and MgPME conversion.

Mechanistic modelling of enzyme-restoration effects of new recombinant liver-targeted proteins in acute intermittent porphyria.

Background and PurposeAcute intermittent porphyria (AIP) is a rare disease caused by a genetic mutation in the hepatic activity of the porphobilinogen‐deaminase. We aimed to develop a mechanistic model of the enzymatic restoration effects of a novel therapy based on the administration of different formulations of recombinant human‐PBGD (rhPBGD) linked to the ApoAI lipoprotein. This fusion protein circulates in blood, incorporating into HDL and penetrating hepatocytes.Experimental ApproachSingle i.v. dose of different formulations of rhPBGD linked to ApoAI were administered to AIP mice in which a porphyric attack was triggered by i.p. phenobarbital. Data consist on 24 h urine excreted amounts of heme precursors, 5‐aminolevulinic acid (ALA), PBG and total porphyrins that were analysed using non‐linear mixed‐effects analysis.Key ResultsThe mechanistic model successfully characterized over time the amounts excreted in urine of the three heme precursors for different formulations of rhPBGD and unravelled several mechanisms in the heme pathway, such as the regulation in ALA synthesis by heme. Treatment with rhPBGD formulations restored PBGD activity, increasing up to 51 times the value of the rate of tPOR formation estimated from baseline. Model‐based simulations showed that several formulation prototypes provided efficient protective effects when administered up to 1 week prior to the occurrence of the AIP attack.Conclusion and ImplicationsThe model developed had excellent performance over a range of doses and formulation type. This mechanistic model warrants use beyond ApoAI‐conjugates and represents a useful tool towards more efficient drug treatments of other enzymopenias as well as for acute intermittent porphyria.

Impact of Porphyrin Binding to GENOMES UNCOUPLED 4 on Tetrapyrrole Biosynthesis in planta.

Plant tetrapyrrole biosynthesis (TPS) provides the indispensable chlorophyll (Chl) and heme molecules in photosynthetic organisms. Post-translational mechanisms control the enzymes to ensure a balanced flow of intermediates in the pathway and synthesis of appropriate amounts of both endproducts. One of the critical regulators of TPS is GENOMES UNCOUPLED 4 (GUN4). GUN4 interacts with magnesium chelatase (MgCh), and its binding of the catalytic substrate and product of the MgCh reaction stimulates the insertion of Mg2+ into protoporphyrin IX. Despite numerous in vitro studies, knowledge about the in vivo function of the GUN4:porphyrin interaction for the whole TPS pathway, particularly in plants, is still limited. To address this, we focused on two highly conserved amino acids crucial for porphyrin-binding to GUN4 and analyzed GUN4-F191A, R211A, and R211E substitution mutants in vitro and in vivo. Our analysis confirmed the importance of these amino acids for porphyrin-binding and the stimulation of plant MgCh by GUN4 in vitro. Expression of porphyrin-binding deficient F191A, R211A, and R211E in the Arabidopsis gun4-2 knockout mutant background revealed that, unlike in cyanobacteria and green algae, GUN4:porphyrin interactions did not affect the stability of GUN4 or other Arabidopsis TPS pathway enzymes in vivo. In addition, although they shared diminished porphyrin-binding and MgCh activation in vitro, expression of the different GUN4 mutants in gun4-2 had divergent effects on the TPS and the accumulation of Chl and Chl-binding proteins. For instance, expression of R211E, but not R211A, induced a substantial decrease of ALA synthesis rate, lower TPS intermediate and Chl level, and strongly impaired accumulation of photosynthetic complexes compared to wild-type plants. Furthermore, the presence of R211E led to significant growth retardation and paler leaves compared to GUN4 knockdown mutants, indicating that the exchange of R211 to glutamate compromised TPS and Chl accumulation more substantially than the almost complete lack of GUN4. Extensive in vivo analysis of GUN4 point mutants suggested that F191 and R211 might also play a role beyond porphyrin-binding.

Efficient conversion of lactic acid to alanine over noble metal supported on Ni@C catalysts.

Alanine (Ala), regarded as the building block for protein synthesis, has been widely used in the field of food processing, pharmaceutical, and bio-based plastic industries. Containing plenty of oxygenic functional groups, biomass-derived chemicals are proper for Ala synthesis in an economic and green way via amination. In this work, lactic acid (LA) derived from renewable biomass and waste glycerol (the major by-product of biodiesel industry) was used to produce Ala. Here, a series of magnetic catalysts M/Ni@C (M = Ru, Pt, Pd, Ir, and Rh) were synthesized by ethylene glycol reduction of metal M supported on encapsulated Ni@C. Compared with catalysts based on other M metals, Ru/Ni@C catalysts exhibited extraordinary efficiency with 91.4% selectivity for Ala synthesis from LA (63.7% yield of Ala and 69.7% conversion of LA). The results of experiments and catalyst characterization indicated that the doping of M metals could improve the dehydrogenation ability of catalysts, as well as the ability of NH3 adsorption, facilitating the reaction towards Ala. Overall, this study provides an efficient chemo-catalytic way for the production of Ala from biomass-derived substrates.

Modular control of multiple pathways of Corynebacterium glutamicum for 5-aminolevulinic acid production

5-aminolevulinic acid (ALA) has broad potential applications in the medical, agricultural and food industries. Several strategies have been implemented successfully to try to improve ALA synthesis. Nonetheless, the low yield has got in the way of large-scale bio-manufacture of 5-ALA. In this study, we explored strain engineering strategies for high‐level 5‐ALA production in Corynebacterium glutamicum F343 using the C4 pathway. Initially, the glutamate dehydrogenase-encoding gene gdhA was deleted to reduce glutamate yield. Then the C4 pathway was introduced in the gdhA mutant strain F2-A (∆gdhA + hemA), resulting in a 5-ALA yield of up to 3.2 g/L. Furthermore, the accumulations of downstream metabolites such as heme, porphobilinogen, and protoporphyrin IX, were decreased. After evaluating the mechanisms of this synthetic pathway by RNA-Seq, the results showed that genes involved in both the C5 pathway and heme pathways were down-regulated in strain F2-A (∆gdhA + hemA). Interestingly, upstream genes of succinyl-CoA in the tricarboxylic acid (TCA) cycle, such as icd, lpdA, were up-regulated, while its downstream genes, including sucC, sucD, sdhB, sdhA, sdhCD, were down-regulated. These changes amplify the sources of succinyl-CoA and reduce its expenditure, before pulling the carbon flux to produce 5-ALA. Furthermore, the down-regulation of most genes of the heme pathway could reduce the drainage of 5‐ALA, which further enhance its accumulation. To alleviate competition between glyoxylate and the TCA cycle, the isocitrate dehydrogenase-encoding gene aceA was also knocked out, resulting in 3.86 g/L of 5‐ALA. Finally, the fermentation conditions were optimized, resulting in a maximum 5-ALA yield of 5.6 g/L. Overall, the blocking of the glutamate synthesis pathway could be a powerful strategy to re-allocate the carbon flux to produce 5-ALA. It could also enable the efficient synthesis of other TCA derivatives in C. glutamicum.

Nutritional Factors That Affect the Formation of 5-Aminolevulinic Acid, a Key Intermediate of Heme Biosynthesis.

5-Aminolevulinic acid (ALA) is a key intermediate of heme biosynthesis, which is an essential component of the respiratory chain. Therefore, nutrients that affect ALA biosynthesis eventually affect ATP production, which is the basis of mitochondrial function. Although the effects of various non-nutrient components that affect ALA after biosynthesis have been reported, there are few reports on the effects of dietary amino acids/protein on ALA formation and the effects of dietary vitamins that are involved in amino acid metabolism. In mitochondria, ALA is synthesized from succinyl-CoA and glycine by the pyridoxal phosphate-dependent enzyme ALA synthase [EC 2.3.1.37]. In this study, the effects of dietary amino acids/protein and vitamins on the amount of ALA synthesized were investigated using mice, rats, and cultured cells. Amounts of ALA in plasma and urine, and porphyrins in plasma increased with increasing protein intake. Vitamin B1 insufficiency did not affect ALA synthesis. Vitamin B6 insufficiency increased the amount of ALA synthesized, while niacin deficiency markedly reduced ALA synthesis. Thus, for heme synthesis, an essential biological substance for life, the amounts of amino acids, as well as the pathways metabolizing amino acids to glycine and succinyl-CoA are very important. Specifically, it is important that niacin is associated with the formation of glycine and succinyl-CoA from amino acids.

In vivo functional analysis of the structural domains of FLUORESCENT (FLU).

The control of chlorophyll (Chl) synthesis in angiosperms depends on the light-operating enzyme protochlorophyllide oxidoreductase (POR). The interruption of Chl synthesis during darkness requires suppression of the synthesis of 5-aminolevulinic acid (ALA), the first precursor molecule specific for Chl synthesis. The inactivation of glutamyl-tRNA reductase (GluTR), the first enzyme in tetrapyrrole biosynthesis, accomplished the decreased ALA synthesis by the membrane-bound protein FLUORESCENT (FLU) and prevents overaccumulation of protochlorophyllide (Pchlide) in the dark. We set out to elucidate the molecular mechanism of FLU-mediated inhibition of ALA synthesis, and explored the role of each of the three structural domains of mature FLU, the transmembrane, coiled-coil and tetratricopeptide repeat (TPR) domains, in this process. Efforts to rescue the FLU knock-out mutant with truncated FLU peptides revealed that, on its own, the TPR domain is insufficient to inactivate GluTR, although tight binding of the TPR domain to GluTR was detected. A truncated FLU peptide consisting of transmembrane and TPR domains also failed to inactivate GluTR in the dark. Similarly, suppression of ALA synthesis could not be achieved by combining the coiled-coil and TPR domains. Interaction studies revealed that binding of GluTR and POR to FLU is essential for inhibiting ALA synthesis. These results imply that all three FLU domains are required for the repression of ALA synthesis, in order to avoid the overaccumulation of Pchlide in the dark. Only complete FLU ensures the formation of a membrane-bound ternary complex consisting at least of FLU, GluTR and POR to repress ALA synthesis.

Transcriptomic analysis of \u03b1-linolenic acid content and biosynthesis in Paeonia ostii fruits and seeds

BackgroundPaeonia ostii is a potentially important oilseed crop because its seed yield is high, and the seeds are rich in α-linolenic acid (ALA). However, the molecular mechanisms underlying ALA biosynthesis during seed kernel, seed testa, and fruit pericarp development in this plant are unclear. We used transcriptome data to address this knowledge gap.ResultsGas chromatograph-mass spectrometry indicated that ALA content was highest in the kernel, moderate in the testa, and lowest in the pericarp. Therefore, we used RNA-sequencing to compare ALA synthesis among these three tissues. We identified 227,837 unigenes, with an average length of 755 bp. Of these, 1371 unigenes were associated with lipid metabolism. The fatty acid (FA) biosynthesis and metabolism pathways were significantly enriched during the early stages of oil accumulation in the kernel. ALA biosynthesis was significantly enriched in parallel with increasing ALA content in the testa, but these metabolic pathways were not significantly enriched during pericarp development. By comparing unigene transcription profiles with patterns of ALA accumulation, specific unigenes encoding crucial enzymes and transcription factors (TFs) involved in de novo FA biosynthesis and oil accumulation were identified. Specifically, the bell-shaped expression patterns of genes encoding SAD, FAD2, FAD3, PDCT, PDAT, OLE, CLE, and SLE in the kernel were similar to the patterns of ALA accumulation in this tissue. Genes encoding BCCP, BC, KAS I– III, and FATA were also upregulated during the early stages of oil accumulation in the kernel. In the testa, the upregulation of the genes encoding SAD, FAD2, and FAD3 was followed by a sharp increase in the concentrations of ALA. In contrast, these genes were minimally expressed (and ALA content was low) throughout pericarp development.ConclusionsWe used three tissues with high, moderate, and low ALA concentrations as an exemplar system in which to investigate tissue-specific ALA accumulation mechanisms in P. ostii. The genes and TFs identified herein might be useful targets for future studies of ALA accumulation in the tree peony. This study also provides a framework for future studies of FA biosynthesis in other oilseed plants.

Cold acclimation can specifically inhibit chlorophyll biosynthesis in young leaves of Pakchoi

BackgroundLeaf color is an important trait in breeding of leafy vegetables. Y-05, a pakchoi (Brassica rapa ssp. chinensis) cultivar, displays yellow inner (YIN) and green outer leaves (GOU) after cold acclimation. However, the mechanism of this special phenotype remains elusive.ResultsWe assumed that the yellow leaf phenotype of Y-05 maybe caused by low chlorophyll content. Pigments measurements and transmission electron microscopy (TEM) analysis showed that the yellow phenotype is closely related with decreased chlorophyll content and undeveloped thylakoids in chloroplast. Transcriptomes and metabolomes sequencing were next performed on YIN and GOU. The transcriptomes data showed that 4887 differentially expressed genes (DEGs) between the YIN and GOU leaves were mostly enriched in the chloroplast- and chlorophyll-related categories, indicating that the chlorophyll biosynthesis is mainly affected during cold acclimation. Together with metabolomes data, the inhibition of chlorophyll biosynthesis is contributed by blocked 5-aminolevulinic acid (ALA) synthesis in yellow inner leaves, which is further verified by complementary and inhibitory experiments of ALA. Furthermore, we found that the blocked ALA is closely associated with increased BrFLU expression, which is indirectly altered by cold acclimation. In BrFLU-silenced pakchoi Y-05, cold-acclimated leaves still showed green phenotype and higher chlorophyll content compared with control, meaning silencing of BrFLU can rescue the leaf yellowing induced by cold acclimation.ConclusionsOur findings suggested that cold acclimation can indirectly promote the expression of BrFLU in inner leaves of Y-05 to block ALA synthesis, resulting in decreased chlorophyll content and leaf yellowing. This study revealed the underlying mechanisms of leaves color change in cold-acclimated Y-05.

Isolation and functional analyses of PvFAD2 and PvFAD3 involved in the biosynthesis of polyunsaturated fatty acids from Sacha Inchi (Plukenetia volubilis)

The development of ω-3 fatty acid-rich vegetable oils is essential to enrich the production of functional foods. Sacha Inchi (Plukenetia volubilis L.) is a unique oilseed crop with much potential. Its seeds contain rich polyunsaturated fatty acids (PUFAs), especially linoleic acid (LA, C18:2) and α-linolenic acid (ALA, C18:3). Endoplasmic reticulum -located ω-6 and ω-3 fatty acid desaturases (FAD) are responsible for the biosynthesis of LA and ALA, respectively, in plant seeds. Here, we isolated two full-length FAD genes from Sacha Inchi, named PvFAD2 and PvFAD3, which encoded predicted amino acid residues of 384 and 379 in protein, respectively. Protein sequence and subcellular localization analysis revealed that they were located in the endoplasmic reticulum (ER). Heterologous expression in Saccharomyces cerevisiae confirmed that PvFAD2 and PvFAD3 could catalyze LA and ALA synthesis, respectively. The stability and catalytic efficiency of the PvFAD3 protein may be closely related to temperature. In transgenic tobacco, using seed-specific expression promoters, PvFAD2 and PvFAD3 significantly promotes the production of LA (from 68% to 70.5%) and ALA (from 0.7% to 3.1%) in seed oil. These results show that PvFAD2 and PvFAD3 do, indeed, function as crucial enzymes for PUFAs biosynthesis, and provide a key gene source for the sustainable production of lipids with tailored fatty acid compositions via genetic engineering in other oil crops.

Upgrading industrial effluent for agricultural reuse: effects of digestate concentration and wood vinegar dosage on biosynthesis of plant growth promotor.

Emphasis on water reuse in agricultural sector receives a renewed interest to close the loop in circular economy, especially in dry and water-stressed regions. In this work, wastewater from cooperative smoked sheet rubber factory and the effluent (digestate) from its treatment system (anaerobic digester) were used as medium to grow purple non-sulfur bacteria (PNSB), Rhodopseudomonas palustris strain PP803, with wood vinegar supplement at mid-log growth phase to stimulate the release of 5-aminolevulinic acid (ALA), a plant growth promotor. Wastewater-to-digestate ratios (D:W) represented by soluble chemical oxygen demand (SCOD) were found to influence both the growth of R. palustris and synthesis of ALA. The highest ALA release of 16.02 ± 0.75μM and the biomass accumulation of 1302 ± 78mg/L were obtained from the medium SCOD of 4953mg/L. Although retarding biomass accumulation by 28-36%, wood vinegar (WV) addition was proven to improve ALA release by 40%. Result suggested that SCOD of 3438mg/L (75:25 D:W) contained sufficient carbon source for PNSB growth and was chosen to subsequently run the photo-bioreactor (PBR) to sustain R. palustris PP803 cells production. In continuous PBR operation, PNSB proliferation suffered from the low organic concentration in PBR at low organic loading. An organic loading increase to 1.21gCOD/Lday was found to attain highest biomass concentration and longest PNSB dominant period over microalgea. In this study, a real-time monitoring protocol of PNSB and microalgae was specifically developed based on image color analysis at acceptable accuracy (R2 = 0.94). In the final assay, verification of the PBR-grown inoculant was conducted and ALA release efficiency was discussed under various wood vinegar dosages and dosing frequencies. This work has advanced our understandings closer to practical field application.

Fatty acid composition and oil content of seeds from perilla (Perilla frutescens (L.) var. frutescens) germplasm of Republic of Korea

Perilla (Perilla frutescens (L.) var. frutescens) is an oilseed crop that produces a large amount of α-linolenic acid (ALA, 18:3), an ω-3 fatty acid in seeds. Perilla is also a fragrant leafy herb with great health benefits. To develop value-added perilla crop, we surveyed the fatty acid composition and oil content of 87 germplasms accessions maintained at Republic of Korea. Our results show that ALA is the major fatty acid, ranging from 47 to 64%, followed by linoleic acid (LA, 18:2, an ω-6 fatty acid) from 10 to 24% and the oleic acid (OA, 18:1, an ω-9 fatty acid) from 9 to 20%. The seed oil content among accessions ranges from 17 to 42.7%. To access if the different fatty acid profile and oil content among germplasms are associated with changes in key genes, we compare the sequences encoding fatty acid desaturase 2 (FAD2) and fatty acid desaturase 3 (FAD3) responsible for LA and ALA synthesis, respectively. Besides, we examine the sequence of diacylglycerol acyltransferase 1 (DGAT1), a key enzyme in determining oil content. We do not find major changes in FAD2 or FAD3 associated with different fatty acid profiles. We discover a change in DGAT1 that might associate a low oil content in seeds.

Production of 5-aminolevulinic Acid by Recombinant Streptomyces coelicolor Expressing hemA from Rhodobacter sphaeroides

Over the past two decades, intensive efforts have been made to construct recombinant Escherichia coli or Corynebacterium glutamicum by engineering C4 or C5 pathways to improve microbial production of 5-aminolevulinic acid (ALA), which has medical application for photodynamic cancer therapy and tumor diagnosis. In this study, we explored the feasibility of enhanced production of ALA by expressing C4 pathway enzyme, ALA synthase, in Streptomyces coelicolor, and medium optimization. The hemA from Rhodobacter sphaeroides was successfully integrated into the chromosome of Streptomyces coelicolor by conjugal transformation, and recombinant Streptomyces cells expressed well the foreign hemA. Glucose promoted ALA synthesis, and yeast extract showed a strong positive effect on ALA production. Optimization of casamino acid, peptone, malt extract, glycine, and succinic acid increased the product titer. In flask cultures, cell growth and ALA production of recombinant Streptomyces were 2.3 and 3.0-fold higher, respectively, in optimal medium than those of control. The maximum ALA, 137 mg/L, was obtained at 28 h in bioreactor culture, in which 3.1-fold higher cell mass and 2.9-fold greater volumetric productivity were achieved, compared to those in flask cultures.