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Processes vital to life such as respiration and photosynthesis critically depend on the availability of tetrapyrroles including hemes and chlorophylls. tRNA‐dependent catalysis generally is associated with protein biosynthesis. An exception is the reduction of glutamyl‐tRNA to glutamate‐1‐semialdehyde by the enzyme glutamyl‐tRNA reductase. This reaction is the indispensable initiating step of tetrapyrrole biosynthesis in plants and most prokaryotes. The crystal structure of glutamyl‐tRNA reductase from the archaeon Methanopyrus kandleri in complex with the substrate‐like inhibitor glutamycin at 1.9 Å resolution reveals an extended yet planar V‐shaped dimer. The well defined interactions of the inhibitor with the active site support a thioester‐mediated reduction process. Modeling the glutamyl‐tRNA onto each monomer reveals an extensive protein–tRNA interface. We furthermore propose a model whereby the large void of glutamyl‐tRNA reductase is occupied by glutamate‐1‐semialdehyde‐1,2‐mutase, the subsequent enzyme of this pathway, allowing for the efficient synthesis of 5‐aminolevulinic acid, the common precursor of all tetrapyrroles.

Background Golden leaf in autumn is a prominent feature of deciduous tree species like Ginkgo biloba L., a landscape tree widely cultivated worldwide. However, little was known about the molecular mechanisms of leaf yellowing, especially its dynamic regulatory network. Here, we performed a suite of comparative physiological and dynamic transcriptional analyses on the golden-leaf cultivar and the wild type (WT) ginkgo to investigate the underlying mechanisms of leaf yellowing across different seasons. Results In the present study, we used the natural bud mutant cultivar with yellow leaves “Wannianjin” (YL) as materials. Physiological analysis revealed that higher ratios of chlorophyll a to chlorophyll b and carotenoid to chlorophyll b caused the leaf yellowing of YL. On the other hand, dynamic transcriptome analyses showed that genes related to chlorophyll metabolism played key a role in leaf coloration. Genes encoding non-yellow coloring 1 ( NYC 1), NYC1-like ( NOL ), and chlorophyllase ( CLH ) involved in the degradation of chlorophyll were up-regulated in spring. At the summer stage, down-regulated HEMA encoding glutamyl-tRNA reductase functioned in chlorophyll biosynthesis, while CLH involved in chlorophyll degradation was up-regulated, causing a lower chlorophyll accumulation. In carotenoid metabolism, genes encoding zeaxanthin epoxidase ( ZEP ) and 9-cis-epoxy carotenoid dioxygenase ( NCED ) showed significantly different expression levels in the WT and YL. Moreover, the weighted gene co-expression network analysis (WGCNA) suggested that the most associated transcriptional factor, which belongs to the AP2/ERF-ERF family, was engaged in regulating pigment metabolism. Furthermore, quantitative experiments validated the above results. Conclusions By comparing the golden-leaf cultivar and the wide type of ginkgo across three seasons, this study not only confirm the vital role of chlorophyll in leaf coloration of YL but also provided new insights into the seasonal transcriptome landscape and co-expression network. Our novel results pinpoint candidate genes for further wet-bench experiments in tree species.

Key messageAn 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 an important 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.

Key messageRNA-seq was used to analyze the transcriptional changes in sugar beet (Beta vulgaris L.) triggered by alkaline solution to elucidate the molecular mechanism underlying alkaline tolerance in sugar beet. Several differentially expressed genes related to stress tolerance were identified. Our results provide a valuable resource for the breeding of new germplasms with high alkaline tolerance.Alkalinity is a highly stressful environmental factor that limits plant growth and production. Sugar beet own the ability to acclimate to various abiotic stresses, especially salt and alkaline stress. Although substantial previous studies on response of sugar beet to saline stress has been conducted, the expressions of alkali-responsive genes in sugar beet have not been comprehensively investigated. In this study, we conducted transcriptome analysis of leaves in sugar beet seedlings treated with alkaline solutions for 0 day (control, C), 3 days (short-term alkaline treatment, ST) and 7 days (long-term alkaline treatment, LT). The clean reads were obtained and assembled into 25,507 unigenes. Among them, 975 and 383 differentially expressed genes (DEGs) were identified in the comparison groups ST_vs_C and LT_vs_C, respectively. Gene ontology (GO) analysis revealed that oxidation–reduction process and lipid metabolic process were the most enriched GO term among the DEGs in ST_vs_C and LT_vs_C, respectively. According to Kyoto Encyclopedia of Genes and Genomes pathway, carbon fixation in photosynthetic organisms pathway were significantly enriched under alkaline stress. Besides, expression level of genes encoding d-3-phosphoglycerate dehydrogenase 1, glutamyl-tRNA reductase 1, fatty acid hydroperoxide lyase, ethylene-insensitive protein 2, metal tolerance protein 11 and magnesium-chelatase subunit ChlI, etc., were significantly altered under alkaline stress. Additionally, among the DEGs, 136 were non-annotated genes and 24 occurred with differential alternative splicing. Our results provide a valuable resource on alkali-responsive genes and should benefit the improvement of alkaline stress tolerance in sugar beet.

Background Chlorophylls (Chls) are magnesium-containing tetrapyrrole macromolecules responsible for the green color in plants. The Chl metabolic pathway has been intensively studied and nearly all the enzymes involved in the pathway have been identified and characterized. Synthesis and activity of these enzymes are tightly regulated in tissue- and developmental stage–specific manners. Leaves contain substantial amounts of Chls because Chls are indispensable for photosynthesis. In contrast, petals generally contain only trace amounts of Chls, which if present would mask the bright petal color. Limited information is available about the mechanisms that control such tissue-specific accumulation of Chls. Results To identify the regulatory steps that control Chl accumulation, we compared gene expression in petals and leaves of chrysanthemum cultivars with different Chl levels. Microarray and quantitative real-time PCR analyses showed that the expression levels of Chl biosynthesis genes encoding glutamyl-tRNA reductase, Mg-protoporphyrin IX chelatase, Mg-protoporphyrin IX monomethylester cyclase, and protochlorophyllide oxidoreductase were well associated with Chl content: their expression levels were lower in white petals than in green petals, and were highest in leaves. Among Chl catabolic genes, expression of STAY-GREEN , encoding Mg-dechelatase, which is a key enzyme controlling Chl degradation, was considerably higher in white and green petals than in leaves. We searched for transcription factor genes whose expression was well related to Chl level in petals and leaves and found three such genes encoding MYB113, CONSTANS-like 16, and DREB and EAR motif protein. Conclusions From our transcriptome analysis, we assume that a low rate of Chl biosynthesis and a high rate of Chl degradation lead to the absence of Chls in white chrysanthemum petals. We identified several candidate transcription factors that might affect Chl accumulation in chrysanthemum petals. Functional analysis of these transcription factors will provide a basis for future molecular studies of tissue-specific Chl accumulation.

ObjectivesTo investigate the level of 5-aminolevulinic acid (5-ALA), a key precursor of heme, and expression of heme-peroxidase on the regulation of heme synthesis in E. coli.MethodsA transporter gene (eamA) was knocked out, and glutamyl-tRNA reductase gene (hemA) for 5-ALA synthesis and a dye-decolorizing peroxidase gene (DyP) were overexpressed.ResultsKnockout of eamA caused decrease of 5-ALA secretion, indicating EamA participates in 5-ALA transportation. Overexpression of hemA elevated intracellular 5-ALA and heme levels. However, overexpression of hemA in eamA knockout mutant led to decrease of intracellular heme content and down-regulation of the transcription of heme synthetic gene hemL by ~ 5.2-fold. When overexpressing both hemA and DyP in the mutant, hemL was up-regulated suggesting the binding of heme to DyP released the feedback repression of hemL.ConclusionHemL expression is heme-mediated and the approach of intracellular immobilization of free heme by overexpression of heme-peroxidase benefits the understanding and application of heme regulation.

Premise Light is critical in the ability of plants to accumulate chlorophyll. When exposed to far‐red (FR) light and then grown in white light in the absence of sucrose, wild‐type seedlings fail to green in a response known as the FR block of greening (BOG). This response is controlled by phytochrome A through repression of protochlorophyllide reductase‐encoding (POR) genes by FR light coupled with irreversible plastid damage. Sigma (SIG) factors are nuclear‐encoded proteins that contribute to plant greening and plastid development through regulating gene transcription in chloroplasts and impacting retrograde signaling from the plastid to nucleus. SIGs are regulated by phytochromes, and the expression of some SIG factors is reduced in phytochrome mutant lines, including phyA. Given the association of phyA with the FR BOG and its regulation of SIG factors, we investigated the potential regulatory role of SIG factors in the FR BOG response. Methods We examined FR BOG responses in sig mutants, phytochrome‐deficient lines, and mutant lines for several phy‐associated factors. We quantified chlorophyll levels and examined expression of key BOG‐associated genes. Results Among six sig mutants, only the sig6 mutant significantly accumulated chlorophyll after FR BOG treatment, similar to the phyA mutant. SIG6 appears to control protochlorophyllide accumulation by contributing to the regulation of tetrapyrrole biosynthesis associated with glutamyl‐tRNA reductase (HEMA1) function, select phytochrome‐interacting factor genes (PIF4 and PIF6), and PENTA1, which regulates PORA mRNA translation after FR exposure. Conclusions Regulation of SIG6 plays a significant role in plant responses to FR exposure during the BOG response.

Tetrapyrrole biosynthesis in plants, algae, and most bacteria starts from the NADPH-dependent reduction of glutamyl-tRNA by glutamyl-tRNA reductase (GluTR). The GluTR-catalyzed reaction is the rate-limiting step, and GluTR is the target of multiple posttranslational regulations, such as heme feedback inhibition, for the tetrapyrrole biosynthetic pathway. A recently identified GluTR regulator, GluTR binding protein (GluBP), has been shown to spatially organize tetrapyrrole synthesis by distributing GluTR into different suborganellar locations. Here we report the complex structure of GluTR–GluBP from Arabidopsis thaliana . The dimeric GluBP binds symmetrically to the catalytic domains of the V-shaped GluTR dimer via its C-terminal domain. A substantial conformational change of the GluTR NADPH-binding domain is observed, confirming the postulated rotation of the NADPH-binding domain for hydride transfer from NADPH to the substrate. Arg146, “guarding the door” for metabolic channeling, adopts alternative conformations, which may represent steps involved in substrate recognition and product release. A coupled enzyme assay shows that GluBP stimulates GluTR catalytic efficiency with an approximate threefold increase of the 5-aminolevulinic acid formation rate. In addition, the GluTR activity can be inhibited by heme in a concentration-dependent way regardless of the presence of GluBP. A structural alignment indicates that GluBP belongs to a heme-binding family involved in heme metabolism. We propose a catalytic mechanism model for GluTR, through which photosynthetic organisms can achieve precise regulation of tetrapyrrole biosynthesis.

5-Aminolevulinic acid (ALA) is the universal precursor for tetrapyrrole biosynthesis and is synthesized in plants in three enzymatic steps: ligation of glutamate (Glu) to tRNA GlU by glutamyl-tRNA synthetase, reduction of activated Glu to Glu-1-semialdehyde by glutamyl-tRNA reductase (GluTR), and transamination to ALA by Glu 1-semialdehyde aminotransferase. ALA formation controls the metabolic flow into the tetrapyrrole biosynthetic pathway. GluTR is proposed to be the key regulatory enzyme that is tightly controlled at transcriptional and posttranslational levels. We identified a GluTR binding protein (GluTRBP; previously called PROTON GRADIENT REGULATION7) that is localized in chloroplasts and part of a 300-kD protein complex in the thylakoid membrane. Although the protein does not modulate activity of ALA synthesis, the knockout of GluTRBP is lethal in Arabidopsis thaliana, whereas mutants expressing reduced levels of GluTRBP contain less heme. GluTRBP expression correlates with a function in heme biosynthesis. It is postulated that GluTRBP contributes to subcompartmentalized ALA biosynthesis by maintaining a portion of GluTR at the plastid membrane that funnels ALA into the heme biosynthetic pathway. These results regarding GluTRBP support a model of plant ALA synthesis that is organized in two separate ALA pools in the chloroplast to provide appropriate substrate amounts for balanced synthesis of heme and chlorophyll.

Greenish leaf variation has been reported widely as a trait of great interest in wheat for improving photosynthesis. Zhongkenuomai No.1 (ZKNM1), a mutant with recoverable leaf spots, was regarded as a suitable material for studying chlorophyll synthesis-related mechanisms. In this study, transcriptome and DNA methylation analyses were conducted in ZKNM1 leaves to determine the transcriptional regulatory mechanism of leaf spot development. Ultimately, 890 differentially expressed genes (DEGs) were discovered, with chlorophyll biosynthesis pathway genes downregulated and chlorophyll degradation pathway genes upregulated, possibly acting as a double block to chlorophyll accumulation. Among them, HEMA1s (Glutamyl-tRNA reductase family proteins) and PORAs (protochlorophyllide oxidoreductase A) were the most important controlled genes. Furthermore, a genome-wide methylation analysis indicates that a hypermethylated region is present 1690 bp upstream of the transcriptional start sites in spot tissues (SPs), and 131 DNA methylation-mediated DEGs were identified, one of which encoded a putative resistance gene (TraesCS1A02G009500) and was a hub gene in interaction network modules. In the sample groups with leaf spots (SPs), this gene may be involved in the photosynthetic processes. The findings indicated that dynamic variations in DNA methylation play key roles in gene regulation to govern leaf spot development.