Protochlorophyllide Reductase Research Articles

Developing and applying a virus-induced gene silencing system for functional genomics in walnut (Juglans regia L.) mediated by tobacco rattle virus.

Walnut (Juglans regia L.) is a high-value tree species planted worldwide, but the incomplete less developed genetic transformation system limits its gene function analysis. In this study, virus-induced gene silencing (VIGS) mediated by tobacco rattle virus (TRV) technology was applied to walnut seedlings to degrade the transcript of target gene. Different infiltration methods were used to explore the effects of infection mode, Agrobacterium cell density, silencing fragment length, and walnut cultivars. The results showed that spray infiltration of seedlings resulted in a photobleaching phenotype of the whole plant. Leaf injection was a more effective way of infiltration. The optimal combination was the Agrobacterium cell density at OD600 = 1.1 with target fragment = 255 bp for the treatment of walnut early-fruiting cultivar 'Xiangling.' This combination can reach up to 48 % of gene silencing efficiency. Based on this optimized VIGS system, silencing a walnut chlorophyll synthesis-related gene, JrPOR (Protochlorophyllide reductase), to further validate the system's effect. The results showed that the expression of JrPOR was significantly repressed, and the chlorophyll level of the silenced plants was significantly decreased compared with the control. The above results indicate that the walnut TRV-VIGS system has been successfully established and can be used for reverse genetic studies, providing an option for verifying gene function in walnut.

Microbial ecophysiology: Shedding light on the re-greening of chlorotic cyanobacteria

Cyanobacteria, key contributors to aquatic CO2 fixation, employ sophisticated acclimation strategies for survival under nitrogen-limited conditions. A new study reveals that red light influences the resuscitation of chlorotic cells through mechanisms involving the dark-operative protochlorophyllide reductase, which facilitates chlorophyll biosynthesis and re-greening under low-light conditions.

Red-light-dependent chlorophyll synthesis kindles photosynthetic recovery of chlorotic dormant cyanobacteria using a dark-operative enzyme

Chlorosis dormancy resulting from nitrogen starvation and its resuscitation upon available nitrogen contributes greatly to the fitness of cyanobacterial population under nitrogen-fluctuating environments. The reinstallation of the photosynthetic machinery is a key process for resuscitation from a chlorotic dormant state; however, the underlying regulatory mechanism is still elusive. Here, we reported that red light is essential for re-greening chlorotic Synechocystis sp. PCC 6803 (a non-diazotrophic cyanobacterium) after nitrogen supplement under weak light conditions. The expression of dark-operative protochlorophyllide reductase (DPOR) governed by the transcriptional factor RpaB was strikingly induced by red light in chlorotic cells, and its deficient mutant lost the capability of resuscitation from a dormant state, indicating DPOR catalyzing chlorophyll synthesis is a key step in the photosynthetic recovery of dormant cyanobacteria. Although light-dependent protochlorophyllide reductase is widely considered as a master switch in photomorphogenesis, this study unravels the primitive DPOR as a spark to activate the photosynthetic recovery of chlorotic dormant cyanobacteria. These findings provide new insight into the biological significance of DPOR in cyanobacteria and even some plants thriving in extreme environments.

Probing the effects of streptomycin on Brassica napus germination and assessing its molecular interactions using extensive molecular dynamics (MD) simulations

Antibiotics are chemical compounds that are used to treat and prevent disease in humans and animals. They have been used in animal feed for over 60 years and are widely used in industrial farming. Antibiotics can have negative environmental impacts, including the potential to contribute to the development of antibiotic-resistant organisms. They can enter the environment through various pathways, including the manufacturing process, the direct application of antibiotic-laden manure to fields, and through grazing animals. Antibiotics that are given to animals can be excreted from where they can enter soil and groundwater which enable their entry in plants. Streptomycin is an antibiotic that is used against a range of gram-positive and gram-negative bacteria, but its use has led to the development of antibiotic resistance in some pathogens. It has also been shown to have negative impacts on a range of plant species, including tobacco, tomato, and wheat. Although, the major effect of streptomycin on plant physiology have been studied, the molecular mechanisms at play are barely understood in plant body. In current study, we examined the impact of streptomycin on germination of Brassica napus and then using docking, MM-GBBSA and MD simulations identified key proteins that interact with streptomycin by performing rigorous computational screening of 106 different proteins. Our finding suggest that streptomycin might be interacting with acyl-CoA oxidases, protochlorophyllide reductase B and leucoanthocyanidin dioxygenase based on simulation and docking analysis.

Transcriptome Revealed the Effect of Shading on the Photosynthetic Pigment and Photosynthesis of Overwintering Tea Leaves

The physiological state of overwintering tea leaves is crucial for the growth and quality formation of spring tea shoots. Low temperatures in winter can easily cause damage to overwintering tea plants, leading to leaf chlorosis and abnormal physiological functions. Many pieces of research have shown that shading could promote chlorophyll (Chl) accumulation in tea leaves, but the impact on overwintering tea plants is not yet known. In this study, different shading rates (no-shading, S0%; 30% shading, S30%; 75% shading, S75%) were used to treat overwintering tea plants, which explored the effect of shading on the color and physiological functions of tea leaves. The results showed that Chl, carotenoid, and soluble sugar (SS) contents were S75% > S30% > S0%, and the net photosynthetic rate (Pn) was S75% > S30% > S0%. Transcriptome analysis showed that the genes involved in chlorophyll and carotenoid metabolism (such as protochlorophyllide reductase POR and zeaxanthin epoxidase ZEP) and photosynthesis (such as photosystem II P680 reaction center D2 protein PsbA and photosystem II CP47 chlorophyll apoprotein PsbB) were significantly up-regulated under shading. In addition, many differentially expressed genes (DEGs) were enriched in “starch and sucrose metabolism (ko00500)” and “anthocyanin biosynthesis (ko00942)” pathways. In summary, this study provided a theoretical basis and technical support for maintaining green leaves and normal physiological functions of overwintering tea plants.

The wheat leaf delayed virescence of mutant dv4 is associated with the abnormal photosynthetic and antioxidant systems

Chlorophyll (Chl) is a key pigment for wheat (Triticum aestivum L.) photosynthesis, consequently impacts grain yield. A wheat mutant named as delayed virescence 4 (dv4) was obtained from cultivar Guomai 301 (wild type, WT) treated with ethyl methane sulfonate (EMS). The seedling leaves of dv4 were shallow yellow, apparently were chlorophyll deficient. They started to turn green at the jointing stage and returned to almost ordinary green at the heading stage. Leaf transcriptome comparison of Guomai 301 and dv4 at the jointing stage showed that most differentially expressed genes (DEGs) of transcription and translation were highly expressed in dv4, one key gene nicotianamine aminotransferase A (NAAT-A) involved in the synthesis and metabolism pathways of tyrosine, methionine and phenylalanine was significantly lowly expressed. The expression levels of the most photosynthesis related genes, such as photosystem I (PS I), ATPase and light-harvesting chlorophyll protein complex-related homeotypic genes, and protochlorophyllide reductase A (PORA) were lower; but macromolecule degradation and hypersensitivity response (HR) related gene heat shock protein 82 (HSP82) was highly expressed. Compared to WT, the contents of macromolecules such as proteins and sugars were reduced; the contents of Chl a, Chl b, total Chl, and carotenoids in leaves of dv4 were significantly less at the jointing stage, while the ratio of Chl a / Chl b was the same as that of WT. The net photosynthetic rate, stomatal conductance and transpiration rate of dv4 were significantly lower. The H2O2 content were higher, while the contents of total phenol and malondialdehyde (MDA), antioxidant enzyme activities were lower in leaves of dv4. In conclusion, the reduced contents of macromolecules and photosynthetic pigments, the abnormal photosynthetic and antioxidant systems were closely related to the phenotype of dv4.

Manganese Deficiency Suppresses Growth and Photosynthetic Processes but Causes an Increase in the Expression of Photosynthetic Genes in Scots Pine Seedlings

Manganese deficiency is a serious plant nutritional disorder, resulting in the loss of crop productivity in many parts of the world. Despite the progress made in the study of angiosperms, the demand for Mn in gymnosperms and the physiological responses to Mn deficiency remain unexplored. We studied the influence of Mn deficiency for 24 weeks on Pinus sylvestris L. seedling growth, ion homeostasis, pigment contents, lipid peroxidation, chlorophyll fluorescence indices and the transcript levels of photosynthetic genes and genes involved in chlorophyll biosynthesis. It was shown that Mn-deficient plants demonstrated suppressed growth when the Mn content in the needles decreased below 0.34 µmol/g DW. The contents of photosynthetic pigments decreased when the Mn content in the needles reached 0.10 µmol/g DW. Mn deficiency per se did not lead to a decrease in the nutrient content in the organs of seedlings. Photoinhibition of PSII was observed in Mn-deficient plants, although this was not accompanied by the development of oxidative stress. Mn-deficient plants had an increased transcript abundance of genes (psbO, psbP, psbQ, psbA and psbC), encoding proteins directly associated with the Mn cluster also as other proteins involved in photosynthesis, whose activities do not depend on Mn directly. Furthermore, the transcript levels of the genes encoding the large subunit of Rubisco, light-dependent NADPH-protochlorophyllide oxidoreductase and subunits of light-independent protochlorophyllide reductase were also increased in Mn-deficient plants.

β-Cyclocitral Does Not Contribute to Singlet Oxygen-Signalling in Algae, but May Down-Regulate Chlorophyll Synthesis.

Light stress signalling in algae and plants is partially orchestrated by singlet oxygen (1O2), a reactive oxygen species (ROS) that causes significant damage within the chloroplast, such as lipid peroxidation. In the vicinity of the photosystem II reaction centre, a major source of 1O2, are two β-carotene molecules that quench 1O2 to ground-state oxygen. 1O2 can oxidise β-carotene to release β-cyclocitral, which has emerged as a 1O2-mediated stress signal in the plant Arabidopsis thaliana. We investigated if β-cyclocitral can have similar retrograde signalling properties in the unicellular alga Chlamydomonas reinhardtii. Using RNA-Seq, we show that genes up-regulated in response to exogenous β-cyclocitral included CAROTENOID CLEAVAGE DIOXYGENASE 8 (CCD8), while down-regulated genes included those associated with porphyrin and chlorophyll anabolism, such as tetrapyrrole-binding protein (GUN4), magnesium chelatases (CHLI1, CHLI2, CHLD, CHLH1), light-dependent protochlorophyllide reductase (POR1), copper target 1 protein (CTH1), and coproporphyrinogen III oxidase (CPX1). Down-regulation of this pathway has also been shown in β-cyclocitral-treated A. thaliana, indicating conservation of this signalling mechanism in plants. However, in contrast to A. thaliana, a very limited overlap in differential gene expression was found in β-cyclocitral-treated and 1O2-treated C. reinhardtii. Furthermore, exogenous treatment with β-cyclocitral did not induce tolerance to 1O2. We conclude that while β-cyclocitral may down-regulate chlorophyll synthesis, it does not seem to contribute to 1O2-mediated high light stress signalling in algae.

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.

Comparison of Metabolome and Functional Properties of Three Korean Cucumber Cultivars.

Cucumber (Cucumis sativus L.) is consumed worldwide and various cultivars have been developed to enhance fruit quality. However, few studies have comprehensively evaluated the quality of various cultivars. We carried out a metabolomics approach to study the three different cucumber cultivars (Chuichung, White Dadagi, and Mini) and their parts (peel and flesh) coupled with antioxidant activities. The amino acids, sugars, flavonoids, carotenoids, and chlorophylls were upregulated in Mini flesh; however, in the case of peel, they were highly expressed in Chuichung. The highest antioxidant activity was observed in the peel of Chuichung and flesh of Mini. Through correlation analysis between metabolites and antioxidant activity, apigenin and quercetin derivatives, chlorophyll a, chlorophyll b, lutein, α-carotene, and β-carotene were found to be significantly positively correlated with antioxidant activity. To understand the metabolism of these compounds, we performed a comprehensive pathway analysis using a metabolomics approach and analysis of associated gene expression. In secondary metabolism, the expression levels of carotenoid-related genes (15-cis-phytoene synthase and ζ-carotene desaturase) and chlorophyll-related genes (protochlorophyllide reductase and glutamyl-tRNA reductase) were consistent with the metabolome analysis data. Collectively, carotenoid and chlorophyll metabolism were upregulated in Chuichung peel and Mini flesh, which had the highest antioxidant activity in each part. These bioactive compounds can be used as biomarkers of commercial cucumber fruit quality. Accordingly, this study offers integrative insights into the quality of different cucumber cultivars and explores valuable metabolites and genes that are helpful in improving quality with functional properties.

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.

Multiomics Approach To Decipher the Origin of Chlorophyll Content in Virgin Olive Oil.

The color of virgin olive oils, ranging from intense green to brown-yellow, is one of the main selection factors for consumers and a quality criterion in specific legislations. Such coloration is due to their chlorophyll content and depends on the composition of the olive fruit. Through analytical chemistry (HPLC-hrMSn), biochemistry (enzymatic activity), and molecular biology (qRT-PCR) approaches, we have analyzed the origin of the differences in the chlorophyll content among several varieties of olive fruit throughout their ripening process. The higher chlorophyll biosynthetic capacity in olive fruits is due to the enzyme protochlorophyllide reductase, whereas chlorophyll degradation is accomplished through the stay-green and pheophytinase pathways. For the first time, the implication of chlorophyll dephytylase during the turnover of chlorophylls in fruit is shown to be responsible for the exclusive accumulation of dephytylated chlorophyll in Arbequina fruit. The multiomics results excluded the in vivo participation of chlorophyllase in chlorophyll degradation in olive fruits.

Arabidopsis transcription factor TCP4 represses chlorophyll biosynthesis to prevent petal greening

Green petals pose a challenge for pollinators to distinguish flowers from leaves, but they are valuable as a specialty flower trait. However, little is understood about the molecular mechanisms that underlie the development of green petals. Here, we report that CINCINNATA (CIN)-like TEOSINTE BRANCHED 1/CYCLOIDEA/PCF (TCP) proteins play key roles in the control of petal color. The septuple tcp2/3/4/5/10/13/17 mutant produced flowers with green petals due to chlorophyll accumulation. Expression of TCP4 complemented the petal phenotype of tcp2/3/4/5/10/13/17. We found that chloroplasts were converted into leucoplasts in the distal parts of wild-type petals but not in the proximal parts during flower development, whereas plastid conversion was compromised in the distal parts of tcp2/3/4/5/10/13/17 petals. TCP4 and most CIN-like TCPs were predominantly expressed in distal petal regions, consistent with the green–white pattern in wild-type petals and the petal greening observed in the distal parts of tcp2/3/4/5/10/13/17 petals. RNA-sequencing data revealed that most chlorophyll biosynthesis genes were downregulated in the white distal parts of wild-type petals, but these genes had elevated expression in the distal green parts of tcp2/3/4/5/10/13/17 petals and the green proximal parts of wild-type petals. We revealed that TCP4 repressed chlorophyll biosynthesis by directly binding to the promoters of PROTOCHLOROPHYLLIDE REDUCTASE (PORB), DIVINYL REDUCTASE (DVR), and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), which are known to promote petal greening. We found that the conversion of chloroplasts to leucoplasts and the green coloration in the proximal parts of petals appeared to be conserved among plant species. Our findings uncover a major molecular mechanism that underpins the formation of petal color patterns and provide a foundation for the breeding of plants with green flowers.

Wheat 2-Cys peroxiredoxin plays a dual role in chlorophyll biosynthesis and adaptation to high temperature.

The molecular mechanism of high-temperature stress (HTS) response, in plants, has so far been investigated using transcriptomics, while the dynamics of HTS-responsive proteome remain unexplored. We examined the adaptive responses of the resilient wheat cultivar 'Unnat Halna' and dissected the HTS-responsive proteome landscape. This led to the identification of 55 HTS-responsive proteins (HRPs), which are predominantly involved in metabolism and defense pathways. Interestingly, HRPs included a 2-cysteine peroxiredoxin (2CP), designated Ta2CP, presumably involved in stress perception and adaptation. Complementation of Ta2CP in yeast and heterologous expression in Arabidopsis demonstrated its role in thermotolerance. Both Ta2CP silencing and overexpression inferred the involvement of Ta2CP in plant growth and chlorophyll biosynthesis. We demonstrated that Ta2CP interacts with protochlorophyllide reductase b, TaPORB. Reduced TaPORB expression was found in Ta2cp-silenced plants, while upregulation was observed in Ta2CP-overexpressed plants. Furthermore, the downregulation of Ta2CP in Taporb-silenced plants and reduction of protochlorophyllide in Ta2cp-silenced plants suggested the key role of Ta2CP in chlorophyll metabolism. Additionally, the transcript levels of AGPase1 and starch were increased in Ta2cp-silenced plants. More significantly, HTS-treated Ta2cp-silenced plants showed adaptive responses despite increased reactive oxygen species and peroxide concentrations, which might help in rapid induction of high-temperature acclimation.

Photosystem Disorder Could be the Key Cause for the Formation of Albino Leaf Phenotype in Pecan.

Pecan is one of the most famous nut species in the world. The phenotype of mutants with albino leaves was found in the process of seeding pecan, providing ideal material for the study of the molecular mechanisms leading to the chlorina phenotype in plants. Both chlorophyll a and chlorophyll b contents in albino leaves (ALs) were significantly lower than those in green leaves (GLs). A total of 5171 differentially expression genes (DEGs) were identified in the comparison of ALs vs. GLs using high-throughput transcriptome sequencing; 2216 DEGs (42.85%) were upregulated and 2955 DEGs (57.15%) were downregulated. The expressions of genes related to chlorophyll biosynthesis (HEMA1, encoding glutamyl-tRNA reductase; ChlH, encoding Mg-protoporphyrin IX chelatase (Mg-chelatase) H subunit; CRD, encoding Mg-protoporphyrin IX monomethylester cyclase; POR, encoding protochlorophyllide reductase) in ALs were significantly lower than those in GLs. However, the expressions of genes related to chlorophyll degradation (PAO, encoding pheophorbide a oxygenase) in ALs were significantly higher than those in GLs, indicating that disturbance of chlorophyll a biosynthesis and intensification of chlorophyll degradation lead to the absence of chlorophyll in ALs of pecan. A total of 72 DEGs associated with photosynthesis pathway were identified in ALs compared to GLs, including photosystem I (15), photosystem II (19), cytochrome b6-f complex (3), photosynthetic electron transport (6), F-type ATPase (7), and photosynthesis-antenna proteins (22). Moreover, almost all the genes (68) mapped in the photosynthesis pathway showed decreased expression in ALs compared to GLs, declaring that the photosynthetic system embedded within the thylakoid membrane of chloroplast was disturbed in ALs of pecan. This study provides a theoretical basis for elucidating the molecular mechanism underlying the phenotype of chlorina seedlings of pecan.

In silico structural analysis and ligand-binding predictions of a few developmental stage specific-proteins during in vitro morphogenesis in Vanilla

Vanilla (Vanilla planifolia Jacks. ex Andrews) is an orchid. It is adored for vanillin, a unique flavouring principle, used in numerous products. The production of Vanilla is dependent on in vitro mass propagation. Since it is a recalcitrant plant, its callus-based regeneration is often enigmatic. The present study reports a few development-stage specific proteins, their structural analysis and ligand-binding predictions, which are essential for the undifferentiated callus of Vanilla during the transition to the differentiated state. Of these proteins, the up regulation of Light-independent protochlorophyllide reductase in the dark grown-callus and retaining the same when the callus was placed under light after subculture to regeneration medium possibly indicated that it helped the callus to acquire its autotrophic stature that is vital for its self-sustenance in the differentiated state. Additionally, the up regulation of Phloem protein 2 in the callus and regenerating callus is an important finding since translocation of solutes is of utmost importance for the formation of the conductive elements, which are necessary for the development of the organized structures for the undifferentiated tissue to convert into the differentiated state.

BEL1-LIKE HOMEODOMAIN4 regulates chlorophyll accumulation, chloroplast development, and cell wall metabolism in tomato fruit.

Tomato (Solanum lycopersicum) is a model plant for studying fruit development and ripening. In this study, we found that down-regulation of a tomato bell-like homeodomain 4 (SlBL4) resulted in a slightly darker-green fruit phenotype and increased accumulation of starch, fructose, and glucose. Analysis of chlorophyll content and TEM observations was consistent with these phenotypes, indicating that SlBL4 was involved in chlorophyll accumulation and chloroplast formation. Ripened fruit of SlBL4-RNAi plants had noticeably decreased firmness, larger intercellular spaces, and thinner cell walls than the wild-type. RNA-seq identified differentially expressed genes involved in chlorophyll metabolism, chloroplast development, cell wall metabolism, and carotenoid metabolism. ChIP-seq identified (G/A) GCCCA (A/T/C) and (C/A/T) (C/A/T) AAAAA (G/A/T) (G/A) motifs. SlBL4 directly inhibited the expression of protoporphyrinogen oxidase (SlPPO), magnesium chelatase H subunit (SlCHLD), pectinesterase (SlPE), protochlorophyllide reductase (SlPOR), chlorophyll a/b binding protein 3B (SlCAB-3B), and homeobox protein knotted 2 (TKN2). In contrast, it positively regulated the expression of squamosa promoter binding protein-like colorless non-ripening (LeSPL-CNR). Our results indicate that SlBL4 is involved in chlorophyll accumulation, chloroplast development, cell wall metabolism, and the accumulation of carotenoids during tomato fruit ripening, and provide new insights for the transcriptional regulation mechanism of BELL-mediated fruit growth and ripening.

Mannose regulates water balance, leaf senescence, and genes related to stress tolerance in white clover under osmotic stress

Mannose (MAN), an important monosaccharide, contributes to coping with abiotic stresses in plants. Objectives of this study were to examine whether exogenous MAN (30 mM) could significantly increase drought tolerance and further to reveal MAN-regulated tolerance mechanism in white clover under osmotic stress induced by 18 % (m/v) polyethylene glycol 6000 for 10 d in controlled growth chambers. Results show that the application of MAN significanlty alleviated stress damage and the inhibition of growth and photosynthesis in white clover under osmotic stress. The MAN-induced increase in endogenous MAN content and the accumulation of organic osmolytes (proline and water soluble sugars) could be responsible for a lower osmotic potential (OP) in white clover. The exogenous application of MAN also enhanced antioxidant enzyme (superoxide dismutase, peroxidase, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase) activities and maintained ascorbic acid content in white clover during osmotic stress. As concern chlorophyll (Chl) metabolism, the MAN-treated plants showed significantly higher transcription of genes involved in Chl synthesis Mg-chelatase and protochlorophyllide reductase and lower transcription of pheophorbide a oxygenase and chlorophyllase related to Chl degradation and also a senescence associated gene 101 than untreated plants. In addition, the MAN application increased transcription of SK2-, Y2K-, and Y2SK-type dehydrin genes, and dehydrin b in leaves of white clover under osmotic stress. These results indicate that MAN plays important roles in drought tolerance not only acting as a compatible solute for OP but also delaying leaf senescence through enhancing antioxidant metabolism, decreasing Chl degradation, and increasing transcription of dehydrin genes contributing to enhanced drought tolerance in white clover.

SlMYB72 Regulates the Metabolism of Chlorophylls, Carotenoids, and Flavonoids in Tomato Fruit.

Tomato (Solanum lycopersicum) fruit ripening is accompanied by the degradation of chlorophylls and the accumulation of carotenoids and flavonoids. Tomato SlMYB72 belongs to the R2R3 MYB subfamily, is located in the nucleus, and possesses transcriptional activator activity. Down-regulation of the SlMYB72 gene produced uneven-colored fruits; that is, dark green spots appeared on immature and mature green fruits, whereas yellow spots appeared on red fruits. Down-regulation of SlMYB72 increased chlorophyll accumulation, chloroplast biogenesis and development, and photosynthesis rate in fruits. This down-regulation decreased lycopene content, promoted β-carotene production and chromoplast development, and increased flavonoid accumulation in fruits. RNA sequencing analysis revealed that down-regulation of SlMYB72 altered the expression levels of genes involved in the biosynthesis of chlorophylls, carotenoids, and flavonoids. SlMYB72 protein interacted with the auxin response factor SlARF4. SlMYB72 directly targeted protochlorophyllide reductase, Mg-chelatase H subunit, and knotted1-like homeobox2 genes and regulated chlorophyll biosynthesis and chloroplast development. SlMYB72 directly bound to phytoene synthase, ζ-carotene isomerase, and lycopene β-cyclase genes and regulated carotenoid biosynthesis. SlMYB72 directly targeted 4-coumarate-coenzyme A ligase and chalcone synthase genes and regulated the biosynthesis of flavonoids and phenolic acid. The uneven color phenotype in RNA interference-SlMYB72 fruits was due to uneven silencing of SlMYB72 and uneven expression of chlorophyll, carotenoid, and flavonoid biosynthesis genes. In summary, this study identified important roles for SlMYB72 in the regulation of chlorophyll, carotenoid, and flavonoid metabolism and provided a potential target to improve fruit nutrition in horticultural crops.

Comparative Plastid Genomics of Cryptomonas Species Reveals Fine-Scale Genomic Responses to Loss of Photosynthesis.

Loss of photosynthesis is a recurring theme in eukaryotic evolution. In organisms that have lost the ability to photosynthesize, nonphotosynthetic plastids are retained because they play essential roles in processes other than photosynthesis. The unicellular algal genus Cryptomonas contains both photosynthetic and nonphotosynthetic members, the latter having lost the ability to photosynthesize on at least three separate occasions. To elucidate the evolutionary processes underlying the loss of photosynthesis, we sequenced the plastid genomes of two nonphotosynthetic strains, Cryptomonas sp. CCAC1634B and SAG977-2f, as well as the genome of the phototroph Cryptomonas curvata CCAP979/52. These three genome sequences were compared with the previously sequenced plastid genome of the nonphotosynthetic species Cryptomonas paramecium CCAP977/2a as well as photosynthetic members of the Cryptomonadales, including C. curvata FBCC300012D. Intraspecies comparison between the two C. curvata strains showed that although their genome structures are stable, the substitution rates of their genes are relatively high. Although most photosynthesis-related genes, such as the psa and psb gene families, were found to have disappeared from the nonphotosynthetic strains, at least ten pseudogenes are retained in SAG977-2f. Although gene order is roughly shared among the plastid genomes of photosynthetic Cryptomonadales, genome rearrangements are seen more frequently in the smaller genomes of the nonphotosynthetic strains. Intriguingly, the light-independent protochlorophyllide reductase comprising chlB, L, and N is retained in nonphotosynthetic SAG977-2f and CCAC1634B. On the other hand, whereas CCAP977/2a retains ribulose-1,5-bisphosphate carboxylase/oxygenase-related genes, including rbcL, rbcS, and cbbX, the plastid genomes of the other two nonphotosynthetic strains have lost the ribulose-1,5-bisphosphate carboxylase/oxygenase protein-coding genes.