Chlorophyll Synthase Research Articles

The transcription factor RppA regulates chlorophyll and carotenoid biosynthesis to improve photoprotection in cyanobacteria.

Chlorophyll is an essential photosynthetic pigment but also a strong photosensitizer. Excessive free chlorophyll and its precursors can cause oxidative damage to photosynthetic organisms. Cyanobacteria are the oldest oxygenic photosynthetic organisms and the ancestors of the chloroplast. Owing to their complex habitats, cyanobacteria require precise regulation of chlorophyll synthesis to respond to environmental factors, especially changes in light. Chlorophyll synthase, encoded by chlG, is the enzyme catalyzing the final step of chlorophyll biosynthesis, which is closely related to photosynthesis biogenesis. However, the transcriptional regulation on chlG remains unclear. Here, the transcription factor, regulator of photosynthesis and photopigment-related gene expression A (RppA) was identified to bind to the chlG promoter by screening a yeast one-hybrid library in the cyanobacterium Synechocystis sp. PCC 6803. The rppA knock-out mutant showed a phenotype of slow growth and severe oxidative damage under dark-light transition conditions. The up-regulated transcriptional expression of chlG was significantly higher and more chlorophyll and its precursors accumulated in the rppA knock-out mutant than those in the wild-type strain during the transition from darkness to light, indicating RppA represses the expression of chlG in Synechocystis. Meanwhile, RppA could synchronously promote the transcription of carotenoids biosynthesis-related genes to enhance carotenoids synthesis during the dark-light transition. These results reveal synergistic regulation of chlorophyll and carotenoids biosynthesis in cyanobacteria in response to frequent dark-light transitions, which slows down chlorophyll biosynthesis while promoting carotenoids biosynthesis to avoid oxidative damage caused by excessive reactive oxygen species accumulation.

Genes for the type-I reaction center and galactolipid synthesis are required for chlorophyll a accumulation in a purple photosynthetic bacterium.

Anoxygenic photosynthesis is diversified into two classes: chlorophototrophy based on a bacterial type-I or type-II reaction center (RC). Whereas the type-I RC contains both bacteriochlorophyll and chlorophyll, type-II RC-based phototrophy relies only on bacteriochlorophyll. However, type-II phototrophic bacteria theoretically have the potential to produce chlorophyll a by the addition of an enzyme, chlorophyll synthase, because the direct precursor for the enzyme, chlorophyllide a, is produced as an intermediate of BChl a biosynthesis. In this study, we attempted to modify the type-II proteobacterial phototroph Rhodovulum sulfidophilum to produce chlorophyll a by introducing chlorophyll synthase, which catalyzes the esterification of a diterpenoid group to chlorophyllide a thereby producing chlorophyll a. However, the resulting strain did not accumulate chlorophyll a, perhaps due to absence of endogenous chlorophyll a-binding proteins. We further heterologously incorporated genes encoding the type-I RC complex to provide a target for chlorophyll a. Heterologous expression of type-I RC subunits, chlorophyll synthase, and galactolipid synthase successfully afforded detectable accumulation of chlorophyll a in Rdv. sulfidophilum. This suggests that the type-I RC can work to accumulate chlorophyll a and that galactolipids are likely necessary for the type-I RC assembly. The evolutionary acquisition of type-I RCs could be related to prior or concomitant acquisition of galactolipids and chlorophylls.

Effects of aluminum (Al) stress on the isoprenoid metabolism of two Citrus species differing in Al-tolerance

Isoprenoid metabolism and its derivatives took part in photosynthesis, growth regulation, signal transduction, and plant defense to biotic and abiotic stresses. However, how aluminum (Al) stress affects the isoprenoid metabolism and whether isoprenoid metabolism plays a vital role in the Citrus plants in coping with Al stress remain unclear. In this study, we reported that Al–treatment–induced alternation in the volatilization rate of monoterpenes (α–pinene, β–pinene, limonene, α–terpinene, γ–terpinene and 3–carene) and isoprene were different between Citrus sinensis (Al-tolerant) and C. grandis (Al-sensitive) leaves. The Al-induced decrease of CO2 assimilation, maximum quantum yield of primary PSII photochemistry (Fv/Fm), the lower contents of glucose and starch, and the lowered activities of enzymes involved in the mevalonic acid (MVA) pathway and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway might account for the different volatilization rate of isoprenoids. Furthermore, the altered transcript levels of genes related to isoprenoid precursors and/or derivatives metabolism, such as geranyl diphosphate (GPP) synthase (GPPS) in GPP biosynthesis, geranylgeranyl diphosphate synthase (GGPPS), chlorophyll synthase (CHS) and GGPP reductase (GGPPR) in chlorophyll biosynthesis, limonene synthase (LS) and α-pinene synthase (APS) in limonene and α–pinene synthesis, respectively, might be responsible for the different contents of corresponding products in C. grandis and C. sinensis. Our data suggested that isoprenoid metabolism was involved in Al tolerance response in Citrus, and the alternation of some branches of isoprenoid metabolism could confer different Al-tolerance to Citrus species.

A pipeline for identification of causal mutations in barley identifies Xantha-j as the chlorophyll synthase gene.

Thousands of barley (Hordeum vulgare L.) mutants have been isolated over the last century, and many are stored in gene banks across various countries. In the present work, we developed a pipeline to efficiently identify causal mutations in barley. The pipeline is also efficient for mutations located in centromeric regions. Through bulked segregant analyses using whole genome sequencing of pooled F2 seedlings, we mapped 2 mutations and identified a limited number of candidate genes. We applied the pipeline on F2 mapping populations made from xan-j.59 (unknown mutation) and xan-l.82 (previously known). The Xantha-j (xan-j) gene was identified as encoding chlorophyll synthase, which catalyzes the last step in the chlorophyll biosynthetic pathway: the addition of a phytol moiety to the propionate side chain of chlorophyllide. Key amino acid residues in the active site, including the binding sites of the isoprenoid and chlorophyllide substrates, were analyzed in an AlphaFold2-generated structural model of the barley chlorophyll synthase. Three allelic mutants, xan-j.19, xan-j.59, and xan-j.64, were characterized. While xan-j.19 is a 1 base pair deletion and xan-j.59 is a nonsense mutation, xan-j.64 causes an S212F substitution in chlorophyll synthase. Our analyses of xan-j.64 and treatment of growing barley with clomazone, an inhibitor of chloroplastic isoprenoid biosynthesis, suggest that binding of the isoprenoid substrate is a prerequisite for the stable maintenance of chlorophyll synthase in the plastid. We further suggest that chlorophyll synthase is a sensor for coordinating chlorophyll and isoprenoid biosynthesis.

LED White Light Treatment Delays Postharvest Senescence of ‘Zaosu’ Pear Fruit with Inhibited Chlorophyll Degradation

To investigate the effects of LED white light treatment (LWT) on the storage quality of postharvest ‘Zaosu’ pears, as well as its role in maintaining fruit greenness and delaying senescence, pear fruits were treated with intermittent irradiation using LED white light for 12 h per day, with a light source distance of 30 ± 1 cm and a photon flux density of 151 μmoL/(m2·s) at 25 °C. The results showed that LWT preserved the postharvest quality of ‘Zaosu’ pear fruit by reducing weight loss and ascorbic acid degradation and promoting the ratio of sugar and organic acid. Meanwhile, LWT also substantially inhibited the respiration intensity and ethylene production during the storage process. Accordingly, the expressions of genes related to ethylene biosynthesis and signaling were reduced in LWT fruit. Notably, LWT retarded the decrease in chlorophyll content of fruit by increasing the activities of enzymes associated with chlorophyll synthase. Additionally, LWT also suppressed the chlorophyll degradation-related enzymes and their gene expressions in pear peel. These findings suggest that a moderate light irradiation can delay the de-green progress and benefit post-harvest storage of ‘Zaosu’ pear.

Chlorophyll a Synthesis in Rhodobacter sphaeroides by Chlorophyll Synthase of Nicotiana tabacum.

The production of phytylated chlorophyll a (Chl aP) in Rhodobacter sphaeroides, which uses phytylated bacteriochlorophyll a (BChl aP), is the first step in expanding the light absorption spectra. Unlike the chlorophyll synthase (ChlG) of the Synechocystis sp. PCC6803, ChlGs of angiosperms, including Arabidopsis thaliana, Nicotiana tabacum, Avena sativa, and Oryza sativa, showed bacteriochlorophyll synthase activity and resistance to inhibition by bacteriochlorophyllide a (BChlide a), geranylgeranylated BChl a (BChl aGG), and BChl aP, collectively called bacteriochlorins. Among the angiosperm ChlGs, N. tabacum ChlG had the highest bacteriochlorophyll synthase activity and resistance to inhibition by bacteriochlorins. Expression of N. tabacum chlG in R. sphaeroides resulted in the formation of free Chl aP in the presence of BChl aP during photoheterotrophic growth, even though reactive oxygen species were generated.

Overexpression of the Wheat TaPsb28 Gene Enhances Drought Tolerance in Transgenic Arabidopsis

Psb28 is a soluble protein in the photosystem II (PSII) complex, but its role in the drought stress response of wheat remains unclear. Here, we functionally characterized the TaPsb28 gene, which positively regulates drought tolerance in wheat. When the full-length 546-bp TaPsb28 cDNA was transferred into Arabidopsis thaliana, it was located in the guard cell chloroplast around the stroma. Overexpression of TaPsb28 conferred drought tolerance, as exhibited by the increases in the survival rate. Transgenic plants maintained lower MDA content and higher chlorophyll content by inducing chlorophyll synthase (ChlG) gene transcription. The content of abscisic acid (ABA) and zeatin increased significantly in wild-type (WT) plants under drought stress, and the transcriptional expression levels of RD22, dihydroflavonol 4-reductase (DFR) and anthocyanin reductase (ANR) genes were induced, thus enhancing the contents of endogenous cyanidin, delphinidin, and proanthocyanidins. However, in transgenic plants, although anthocyanins were further aggregated, the ABA increase was inhibited, zeatin was restored to the control level under drought stress, and stomatal closure was promoted. These findings indicate ABA and zeatin have opposite synergistic effects in the process of drought tolerance caused by TaPsb28 because only after the effect of zeatin is alleviated can ABA better play its role in promoting anthocyanin accumulation and stomatal closure, thus enhancing the drought tolerance of transgenic plants. The results suggest that overexpression of TaPsb28 exerts a positive role in the drought response by influencing the functional metabolism of endogenous hormones. The understanding acquired through the research laid a foundation for further in-depth investigation of the function of TaPsb28 in drought resistance in wheat, especially its relationship with anthocyanidin accumulation.

Identification of Genes and Metabolic Pathways Involved in Resin Yield in Masson Pine by Integrative Analysis of Transcriptome, Proteome and Biochemical Characteristics.

Masson pine (Pinus massoniana L.) is one of the most important resin-producing tree species in southern China. However, the molecular regulatory mechanisms of resin yield are still unclear in masson pine. In this study, an integrated analysis of transcriptome, proteome, and biochemical characteristics from needles of masson pine with the high and common resin yield was investigated. The results showed that chlorophyll a (Chl a), chlorophyll b (Chl b), total chlorophyll (Chl C), carotenoids (Car), glucose (Glu), gibberellin A9 (GA9), gibberellin A15 (GA15), and gibberellin A53 (GA53) were significantly increased, whereas fructose (Fru), jasmonic acid (JA), jasmonoyl-L-isoleucine (JA-ILE), gibberellin A1 (GA1), gibberellin A3 (GA3), gibberellin A19 (GA19), and gibberellin A24 (GA24) were significantly decreased in the high resin yield in comparison with those in the common one. The integrated analysis of transcriptome and proteome showed that chlorophyll synthase (chlG), hexokinase (HXK), sucrose synthase (SUS), phosphoglycerate kinase (PGK), dihydrolipoamide dehydrogenase (PDH), dihydrolipoamide succinyltransferase (DLST), 12-oxophytodienoic acid reductase (OPR), and jasmonate O-methyltransferases (JMT) were consistent at the transcriptomic, proteomic, and biochemical levels. The pathways of carbohydrate metabolism, terpenoid biosynthesis, photosynthesis, and hormone biosynthesis may play crucial roles in the regulation of resin yield, and some key genes involved in these pathways may be candidates that influence the resin yield. These results provide insights into the molecular regulatory mechanisms of resin yield and also provide candidate genes that can be applied for the molecular-assisted selection and breeding of high resin-yielding masson pine.

Genetic and Biochemical Investigation of Seed Fatty Acid Accumulation in Arabidopsis.

As a vegetable oil, consisting principally of triacylglycerols, is the major storage form of photosynthetically-fixed carbon in oilseeds which are of significant agricultural and industrial value. Photosynthesis in chlorophyll-containing green seeds, along with photosynthesis in leaves and other green organs, generates ATP and reductant (NADPH and NADH) needed for seed fatty acid production. However, contribution of seed photosynthesis to fatty acid accumulation in seeds have not been well-defined. Here, we report the contribution of seed-photosynthesis to fatty acid production by probing segregating green (photosynthetically-competent) and non-green or yellow (photosynthetically-non-competent) seeds in siliques of an Arabidopsis chlorophyll synthase mutant. Using this mutant, we found that yellow seeds lacking photosynthetic capacity reached 80% of amounts of oil in green seeds at maturity. Combining this with studies using shaded siliques, we determined that seed-photosynthesis accounts for 20% and silique and leaf/stem photosynthesis each account for ~40% of the ATP and reductant for seed oil production. Transmission electron microscopy (TEM) and pyridine nucleotides and ATP analyses revealed that seed photosynthesis provides ATP and reductant for oil production mostly during early development, as evidenced by delayed oil accumulation in non-green seeds. Transcriptomic analyses suggests that the oxidative pentose phosphate pathway could be the source of carbon, energy and reductants required for fatty acid synthesis beyond the early stages of seed development.

Chlorophyll dephytylase 1 and chlorophyll synthase: a chlorophyll salvage pathway for the turnover of photosystems I and II.

Chlorophyll (Chl) is made up of the tetrapyrrole chlorophyllide and phytol, a diterpenoid alcohol. The photosynthetic protein complexes utilize Chl for light harvesting to produce biochemical energy for plant development. However, excess light and adverse environmental conditions facilitate generation of reactive oxygen species, which damage photosystems I and II (PSI and PSII) and induce their turnover. During this process, Chl is released, and is thought to be recycled via dephytylation and rephytylation. We previously demonstrated that Chl recycling in Arabidopsis under heat stress is mediated by the enzymes chlorophyll dephytylase 1 (CLD1) and chlorophyll synthase (CHLG) using chlg and cld1 mutants. Here, we show that the mutants with high CLD1/CHLG ratio, by different combinations of chlg-1 (a knock-down mutant) and the hyperactive cld1-1 alleles, develop necrotic leaves when grown under long- and short-day, but not continuous light conditions, owing to the accumulation of chlorophyllide in the dark. Combination of chlg-1 with cld1-4 (a knock-out mutant) leads to reduced chlorophyllide accumulation and necrosis. The operation of CLD1 and CHLG as a Chl salvage pathway was also explored in the context of Chl recycling during the turnover of Chl-binding proteins of the two photosystems. CLD1 was found to interact with CHLG and the light-harvesting complex-like proteins OHP1 and LIL3, implying that auxiliary factors are required for this process.

Genome-wide association identifies a missing hydrolase for tocopherol synthesis in plants

The tocopherol biosynthetic pathway, encoded by VTE genes 1 through 6, is highly conserved in plants but most large effect quantitative trait loci for seed total tocopherols (totalT) lack VTE genes, indicating other activities are involved. A genome-wide association study of Arabidopsis seed tocopherols showed five of seven significant intervals lacked VTE genes, including the most significant, which mapped to an uncharacterized, seed-specific, envelope-localized, alpha/beta hydrolase with esterase activity, designated AtVTE7. Atvte7 null mutants decreased seed totalT 55% while a leaky allele of the maize ortholog, ZmVTE7, decreased kernel and leaf totalT 38% and 49%, respectively. Overexpressing AtVTE7 or ZmVTE7 partially or fully complemented the Atvte7 seed phenotype and increased leaf totalT by 3.6- and 6.9-fold, respectively. VTE7 has the characteristics of an esterase postulated to provide phytol from chlorophyll degradation for tocopherol synthesis, but bulk chlorophyll levels were unaffected in vte7 mutants and overexpressing lines. Instead, levels of specific chlorophyll biosynthetic intermediates containing partially reduced side chains were impacted and strongly correlated with totalT. These intermediates are generated by a membrane-associated biosynthetic complex containing protochlorophyllide reductase, chlorophyll synthase, geranylgeranyl reductase (GGR) and light harvesting-like 3 protein, all of which are required for both chlorophyll and tocopherol biosynthesis. We propose a model where VTE7 releases prenyl alcohols from chlorophyll biosynthetic intermediates, which are then converted to the corresponding diphosphates for tocopherol biosynthesis.

Iron oxide nanoparticles and selenium supplementation improve growth and photosynthesis by modulating antioxidant system and gene expression of chlorophyll synthase (CHLG) and protochlorophyllide oxidoreductase (POR) in arsenic-stressed Cucumis melo

Current research reveals the positive role of iron oxide nanoparticles (IONPs) and selenium (Se) in extenuation of arsenic (As) induced toxicity in Cucumis melo. C. melo plants grown in As spiked soil (20 mg kg−1 As) showed reduced growth, chlorophyll (Chl) content, photosynthetic rate, stomatal conductivity and transpiration. On the other hand, the alone applications of IONPs or Se improved growth and physiochemical parameters of C. melo plants. Additionally, exogenous application IONPs and Se synergistically improved the activity of antioxidative enzymes and glyoxalase system in C. melo plants. In addition, the collective treatment of IONPs and Se reduced As uptake, enhanced rate of photosynthesis and increased gas exchange attributes of C. melo plants under As stress. Interactive effect of IONPs and Se regulated reduced glutathione (GSH), oxidized glutathione (GSSG) and ascorbate (AsA) content in C. melo plants exposed to As-contaminated Soil. IONPs and Se treatment also regulated expression of respiratory burst oxidase homologue D (RBOHD) gene, chlorophyll synthase (CHLG) and protochlorophyllide oxidoreductase (POR). Therefore, the combined treatment of IONPs and Se may enhance the growth of crop plants by alleviating As stress.

The terminal enzymes of (bacterio)chlorophyll biosynthesis.

(Bacterio)chlorophylls are modified tetrapyrroles that are used by phototrophic organisms to harvest solar energy, powering the metabolic processes that sustain most of the life on Earth. Biosynthesis of these pigments involves enzymatic modification of the side chains and oxidation state of a porphyrin precursor, modifications that differ by species and alter the absorption properties of the pigments. (Bacterio)chlorophylls are coordinated by proteins that form macromolecular assemblies to absorb light and transfer excitation energy to a special pair of redox-active (bacterio)chlorophyll molecules in the photosynthetic reaction centre. Assembly of these pigment–protein complexes is aided by an isoprenoid moiety esterified to the (bacterio)chlorin macrocycle, which anchors and stabilizes the pigments within their protein scaffolds. The reduction of the isoprenoid ‘tail’ and its addition to the macrocycle are the final stages in (bacterio)chlorophyll biosynthesis and are catalysed by two enzymes, geranylgeranyl reductase and (bacterio)chlorophyll synthase. These enzymes work in conjunction with photosynthetic complex assembly factors and the membrane biogenesis machinery to synchronize delivery of the pigments to the proteins that coordinate them. In this review, we summarize current understanding of the catalytic mechanism, substrate recognition and regulation of these crucial enzymes and their involvement in thylakoid biogenesis and photosystem repair in oxygenic phototrophs.

Metabolic Pathways Involved in the Drought Stress Response of Nitraria tangutorum as Revealed by Transcriptome Analysis

Drought resistance in plants is controlled by multiple genes. To identify the genes that mediate drought stress responses and to assess the associated metabolic pathways in the desert shrub Nitraria tangutorum, we conducted a transcriptome analysis of plants under control (maximum field capacity) and drought (20% of the maximum field capacity) conditions. We analyzed differentially expressed genes (DEGs) of N. tangutorum and their enrichment in the KEGG metabolic pathways database, and explored the molecular biological mechanisms underlying the answer to its drought tolerance. Between the control and drought groups, 119 classified metabolic pathways annotated 3047 DEGs in the KEGG database. For drought tolerance, nitrate reductase (NR) gene expression was downregulated, indicating that NR activity was decreased to improve drought tolerance. In ammonium assimilation, drought stress inhibited glutamine formation. Protochlorophyllide reductase (1.3.1.33) expression was upregulated to promote chlorophyll a synthesis, whereas divinyl reductase (1.3.1.75) expression was downregulated to inhibit chlorophyll-ester a synthesis. The expression of the chlorophyll synthase (2.5.1.62) gene was downregulated, which affected the synthesis of chlorophyll a and b. Overall, drought stress appeared to improve the ability to convert chlorophyll b into chlorophyll a. Our data serve as a theoretical foundation for further elucidating the growth regulatory mechanism of desert xerophytes, thereby facilitating the development and cultivation of new, drought-resistant genotypes for the purpose of improving desert ecosystems.

Full-length transcriptome analysis of Adiantum flabellulatum gametophyte.

Ferns are important components of plant communities on earth, but their genomes are generally very large, with many redundant genes, making whole genome sequencing of ferns prohibitively expensive and time-consuming. This means there is a significant lack of fern reference genomes, making molecular biology research difficult. The gametophytes of ferns can survive independently, are responsible for sexual reproduction and the feeding of young sporophytes, and play an important role in the alternation of generations. For this study, we selected Adiantum flabellulatum as it has both ornamental and medicinal value and is also an indicator plant of acidic soil. The full-length transcriptome sequencing of its gametophytes was carried out using PacBio three-generation sequencing technology. A total of 354,228 transcripts were obtained, and 231,705 coding sequences (CDSs) were predicted, including 5,749 transcription factors (TFs), 2,214 transcription regulators (TRs) and 4,950 protein kinases (PKs). The transcripts annotated by non-redundant protein sequence database (NR), Kyoto encyclopedia of genes and genomes (KEGG), eukaryotic ortholog groups (KOG), Swissprot, protein family (Pfma), nucleotide sequence database (NT) and gene ontology (GO) were 251,501, 197,474, 193,630, 194,639, 195,956, 113,069 and 197,883, respectively. In addition, 138,995 simple sequence repeats (SSRs) and 111,793 long non-coding RNAs (lncRNAs) were obtained. We selected nine chlorophyll synthase genes for qRT-PCR, and the results showed that the full-length transcript sequences and the annotation information were reliable. This study can provide a reference gene set for subsequent gene expression quantification.

Defining date palm leaf pruning line in bearing status by tracking physiological markers and expression of senescence-related genes

The physiological and molecular markers were evaluated for defining leaf pruning line in bearing status of date palm (Phoenix dactylifera cv. Mazafati). Our results showed that the higher expression of the chlorophyllase (Chlase) gene was concomitant with lower expression of chlorophyll synthase (ChlG) and oxygen-evolving enhancer protein (PSBO) genes in the 6-year-old leaf line. In addition, the 6-year-old leaf line resulted in a lower Hill reaction rate, chlorophyll a/b ratio, photosystem Ⅱ quantum efficiency, and photosynthesis rate. Also, lower expression and activities of catalase (CAT), ascorbate peroxidase (APX), and glutathione peroxidase (GPX) was associated with higher malondialdehyde (MDA) and hydrogen peroxide (H2O2) accumulation in 6-year-old leaf line. These results suggested that the physiological maturity termination, senescence stage initiation, and source to sink transition could occur in 6-year-old leaves. Furthermore, delayed 5-year-old leaf pruning line adjustment in consecutive years could increase quantitative and qualitative yield loss, decrease water use efficiency, and promote alternate bearing and fruit abscission, which is considered a common problem of date growing areas. Further, the senescence-related markers in the alternate bearing trees pruned in pruning line 5 showed a tendency to normal trees. The five-year-old pruning line also resulted in the balance of bunching and loading the alternate bearing trees.

Physiological response and transcriptome analysis of Prunus mume to early salt stress

AbstractSoil salinisation is an important factor limiting the cultivation and distribution of Prunus mume. Therefore, identifying the regulation mechanism of salt tolerance of P. mume will promote its cultivation and molecular breeding. In this study, the garden tree species P. mume ‘Meiren’ was investigated, with a soil NaCl content (w/w) of 0.3%. Photosynthetic gas exchange parameters, relative electrical conductivity, malondialdehyde, osmoregulation substances, and antioxidant enzyme activity were measured in the early (3d), middle (10d), and late (30d) stages of stress. Salt treatment was applied for 0 h, 6 h, 24 h, and 72 h; following this, the gene library was constructed from the leaves. The results showed that the CBL-CIPK, mitogen-activated protein kinase, calcium-dependent protein kinase, and abscisic acid signalling pathways were involved in salt stress signal transduction of P. mume. Furthermore, bHLH, WRPK, ERF, and MYB were identified as potential key transcription factor families of salt tolerance in P. mume. Combined with determined physiological responses, the results showed that early salt stress inhibited the activities of RuBisCO, chlorophyll synthase, glutamyl tRNA reductase, divinyl reductase, and magnesium chelatase; resultantly, this led to a reduction in the photosynthetic rate. Also, it was found that P5CS, SS, LEA, and dehydrin regulated the synthesis of proline, soluble sugar, and macromolecular protein to alleviate osmotic stress. POD gene plays an important role in scavenging reactive oxygen species due to its regulation of POD activity. The findings of this research reveal the internal mechanism of the physiological response and provide a foundation for the construction of a genetic regulatory network in response to salt stress in P. mume.

Enhancement of phycoerythrin productivity in Porphyridium purpureum using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 ribonucleoprotein system

Genetic manipulation of the Porphyridium sp. may increase the production of phycoerythrin. Since phycobiliproteins capture and transfer energy to both photosystems (PS I and PS II), it was hypothesized that the gene mutation involved increases phycoerythrin synthesis. The gene encoding chlorophyll synthase (CHS1) was selected as chlorophyll synthase plays an important role in photosynthesis, mediating the final process of chlorophyll synthesis. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 ribonucleoprotein (CRISPR/Cas9 RNP) delivery system was used to generate the chlorophyll synthase loss-of-function mutants (Δchs1). Independent Δchs1 showed no differences in the growth and production of sulfated polysaccharide compared to control. Phycoerythrin contents of the two independent mutants substantially increased regardless of light source. This study provides a novel applicability for the CRISPR/Cas9 RNP method in red microalgae toward a bio-product of interest. The obtained mutants could serve as potential producers of phycoerythrin if Porphyridium is selected as a natural source.

Allelopathy and underlying mechanism of Karenia mikimotoi on the diatom Thalassiosira pseudonana under laboratory condition

Karenia mikimotoi is an important toxin-producing dinoflagellate, frequently forming blooms along the coast of China. It has been shown that K. mikimotoi has allelopathic effects on other organisms, which benefited it over other microalgal species in competition. However, the underlying allelopathy mechanisms remain largely unclear. Thalassiosira pseudonana, a worldwide-distributed planktonic diatom, is extensively used as a model in diatom physiology studies. Here, we investigated the responses of T. pseudonana to K. mikimotoi allelopathy when they were co-cultured under laboratory condition. We found that there were reciprocal inhibitory effects between K. mikimotoi and T. pseudonana, and the cell-free filtrate of K. mikimotoi also deeply suppressed the growth of T. pseudonana, corroborating that K. mikimotoi allelopathy mainly accounted for the inhibition of K. mikimotoi against T. pseudonana. After exposed to K. mikimotoi allelopathy, the photosynthetic efficiency, contents of neutral lipid and malondialdehyde (MDA), as well as superoxide dismutase (SOD) activity in T. pseudonana were significantly changed. Meanwhile, the expression of genes involved in photosynthesis, oxidative phosphorylation, detoxifying system and nutrients uptake including PsbO, chlorophyll synthase (ChlG), risp1, ferredoxin-NADP+ reductase (PetH), ABCB1, nitrate transporters 1 (NRT1), phosphate transporter (PHT), alkaline phosphatase (ALP) and ascorbate peroxidase (APX) in T. pseudonana dramatically altered. These results suggested that K. mikimotoi could highly affect the efficiency of photosynthesis and energy metabolism of T. pseudonana, and also inhibit the nutrients uptake and assimilation. Our findings might provide insight information for the K. mikimotoi allelopathy.

Xanthophyll carotenoids stabilise the association of cyanobacterial chlorophyll synthase with the LHC-like protein HliD

Chlorophyll synthase (ChlG) catalyses a terminal reaction in the chlorophyll biosynthesis pathway, attachment of phytol or geranylgeraniol to the C17 propionate of chlorophyllide. Cyanobacterial ChlG forms a stable complex with high light-inducible protein D (HliD), a small single-helix protein homologous to the third transmembrane helix of plant light-harvesting complexes (LHCs). The ChlG-HliD assembly binds chlorophyll, β-carotene, zeaxanthin and myxoxanthophyll and associates with the YidC insertase, most likely to facilitate incorporation of chlorophyll into translated photosystem apoproteins. HliD independently coordinates chlorophyll and β-carotene but the role of the xanthophylls, which appear to be exclusive to the core ChlG-HliD assembly, is unclear. Here we generated mutants of Synechocystis sp. PCC 6803 lacking specific combinations of carotenoids or HliD in a background with FLAG- or His-tagged ChlG. Immunoprecipitation experiments and analysis of isolated membranes demonstrate that the absence of zeaxanthin and myxoxanthophyll significantly weakens the interaction between HliD and ChlG. ChlG alone does not bind carotenoids and accumulation of the chlorophyllide substrate in the absence of xanthophylls indicates that activity/stability of the 'naked' enzyme is perturbed. In contrast, the interaction of HliD with a second partner, the photosystem II assembly factor Ycf39, is preserved in the absence of xanthophylls. We propose that xanthophylls are required for the stable association of ChlG and HliD, acting as a 'molecular glue' at the lateral transmembrane interface between these proteins; roles for zeaxanthin and myxoxanthophyll in ChlG-HliD complexation are discussed, as well as the possible presence of similar complexes between LHC-like proteins and chlorophyll biosynthesis enzymes in plants.