The Inhibition Effect and Excessive Carbon Flux Resulting from Blocking Anthocyanin Biosynthesis Under Darkness in <Emphasis Type="Italic">Begonia semperflorens</Emphasis>

Similar to many plants, the leaves of Begonia semperflorens accumulate anthocyanins and turn red in autumn. This induction of anthocyanin biosynthesis in autumn has been attributed to low temperature, but the effects of light on this process are still under debate. In the present work, light was found to be necessary for anthocyanin biosynthesis under low temperature. When seedlings were exposed to light and low temperature, both upstream (phenylalanine ammonialyase and chalcone isomerase) and downstream [dihydroflavonol 4-reductase (DFR), flavonoid-3-O-glucosyltransferase (UFGT)] enzymes of the anthocyanin biosynthesis pathway were activated. However, when seedlings were exposed to low temperature in the dark, downstream enzymes (DFR and UFGT) were inhibited. The carbon flux caused by blocked anthocyanin biosynthesis in the dark-exposed plants channeled into flavonoid (for example, flavonol) and phenolic acid, but not lignin, biosynthesis.     

The expression level of anthocyanidin synthase determines the anthocyanin content of crabapple (<Emphasis Type="Italic">Malus</Emphasis> sp.) petals

In addition to contributing to the coloration of plant organs and their defense against herbivores, the consumption of anthocyanins in the human diet has a number of health benefits. Crabapple (Malus sp.) represents a valuable experimental model system to research the mechanisms and regulation of anthocyanin accumulation, in part due to the often vivid and varied petal and leaf coloration that is exhibited by various cultivars. The enzyme anthocyanidin synthase (ANS) plays a pivotal role in anthocyanin biosynthesis; however, the relationship between ANS expression and petal pigmentation has yet to be established in crabapple. To illuminate the mechanism of anthocyanin accumulation in crabapple petals, we evaluated the expression of two crabapple ANS allelic genes (McANS-1 and McANS-2) and the levels of anthocyanins in petals from cultivars with dark red (‘Royalty’) and white (‘Flame’) petals, as well as another (‘Radiant’) whose petals have an intermediate pink color. We determined that the expression of McANS in the three cultivars correlated with the variation of anthocyanin accumulation during different petal developmental stages. Furthermore, transgenic tobacco plants constitutively overexpressing one of the two McANS genes, McANS-1, had showed elevated anthocyanin accumulation and a deeper red coloration in their petals than those from untransformed control lines. In conclusion, we propose that McANS are responsible for anthocyanin accumulation during petal coloration in different crabapple cultivars.     

Silencing of <Emphasis Type="Italic">GbANS</Emphasis> reduces cotton resistance to <Emphasis Type="Italic">Verticillium dahliae</Emphasis> through decreased ROS scavenging during the pathogen invasion process

Anthocyanins are secondary metabolites that play important roles in plant adaption to adverse environments. The anthocyanin biosynthetic pathway is conserved in high plants. Previous studies revealed the significant role of anthocyanins in natural-colorized cotton. However, little is known about the involvement of anthocyanins in the interaction of cotton and pathogen. In this study, a pathogen-induced gene was isolated from Gossypium barbadense that encodes an anthocyanidin synthase protein (GbANS) with dioxygenase structures. GbANS was preferentially expressed in colored tissue. Silencing of GbANS significantly reduced the production of anthocyanins, as well as the cotton’s resistance to Verticillium dahliae. Biochemical studies revealed that GbANS-silenced cotton accumulated more hydrogen peroxide compared to control plants during the V. dahliae invasion process. This accumulation of hydrogen peroxide corresponded with increased cell death around the invasion sites, which in turn accelerated the V. dahliae infection. Taken together, we found that GbANS contributes to the biosynthesis of anthocyanins in cotton and anthocyanins positively regulate cotton’s resistance to V. dahliae.     

Content of Osmolytes and Flavonoids under Salt Stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Plants Defective in Jasmonate Signaling

The effects of the salt stress (200 mM NaCl) and exogenous jasmonic acid (JA) on levels of osmolytes and flavonoids in leaves of four-week-old Arabidopsis thaliana L. plants of the wild-type (WT) Columbia-0 (Col-0) and the mutant jin1 (jasmonate insensitive 1) with impaired jasmonate signaling were studied. The increase in proline content caused by the salt stress was higher in the Col-0 plants than in the mutant jin1. This difference was especially marked if the plants had been pretreated with exogenous 0.1 μM JA. The sugar content increased in response to the salt stress in the JA-treated WT plants but decreased in the jin1 mutant. Treatment with JA of the WT plants but not mutant defective in jasmonate signaling also enhanced the levels of anthocyanins and flavonoids absorbed in UV-B range in leaves. The presence of JA increased salinity resistance of the Col-0 plants, since the accumulation of lipid peroxidation products and growth inhibition caused by NaCl were less pronounced. Under salt stress, JA almost did not render a positive effect on the jin1 plants. It is concluded that the protein JIN1/MYC2 is involved in control of protective systems under salt stress.     

Dihydroflavonol 4-reductase cDNA from non-anthocyanin-producing species in the Caryophyllales

Two types of red pigment, anthocyanins and betacyanins, never occur together in the same plant. Although anthocyanins are widely distributed in higher plants as flower and fruit pigments, betacyanins have replaced anthocyanins in the Caryophyllales. We isolated cDNAs encoding dihydroflavonol 4-reductase (DFR), which is the first enzyme committed to anthocyanin biosynthesis in the flavonoid pathway, from Spinacia oleracea and Phytolacca americana, plants that belong to the Caryophyllales. The deduced amino acid sequence of Spinacia DFR and Phytolacca DFR revealed a high degree of homology with DFRs of anthocyanin-producing plants. The DFR of carnation, an exception in the Caryophyllales that synthesizes anthocyanin, showed the highest level of identity. In the phylogenetic tree, Spinacia DFR and Phytolacca DFR clustered with the DFRs of anthocyanin-synthesizing dicots. Recombinant Spinacia and Phytolacca DFRs expressed in Escherichia coli convert dihydroflavonol to leucoanthocyanidin. The expression and function of DFR in spinach and pokeweed are discussed in relation to the molecular evolution of red pigment biosynthesis in higher plants.     

Role of 5-aminolevulinic acid on growth,photosynthetic parameters and antioxidant enzyme activity in NaCl-stressed <Emphasis Type="Italic">Isatis indigotica</Emphasis> Fort.

5-aminolevulinic acid (ALA) is a key precursor for the biosynthesis of porphyrins such as heme and chlorophyll. ALA alleviates salinity stress damage in germinating seeds and improves seedling growth. Exogenous application of ALA at low concentrations has been shown to enhance salt tolerance in a number of plants. In the present study, we studied the effect of exogenous application of ALA on enhancing salt stress tolerance in Isatis indigotica Fort. (Anhui population as S1, Shanxi population as S2). A foliar application of 0, 12.5, 16.7, 25.0, and 50.0 mg/L ALA was given to the leaves of I. indigotica plants treated with 100 mmol/L NaCl. The fresh weight of leaves and roots; chlorophyll relative content (SPAD value); photosynthetic parameters, such as net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular carbon dioxide concentration (Ci) and water use efficiency of the treated plants were determined. The third leaf of each treated plant was used to determine the activities of antioxidant enzymes. Superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), glutamate synthase (GOGAT), nitrate reductase (NR) activities and the malondialdehyde (MDA) content increased in response to 100 mmol/L NaCl in both S1 and S2 plants. However, the fresh weight of leaf and root, chlorophyll relative content, Pn, Gs, Ci decreased in response to salt stress in both S1 and S2 plants. In all foliar application of ALA in S1 plants, the MDA content, and the activities of SOD and POD were the highest in response to 50.0 mg/L foliar application of ALA. GOGAT and NR activities were the highest in response to 16.7 mg/L foliar ALA. Chlorophyll content and Pn were the highest in S1 plants treated with by 25.0 mg/L ALA. In S2 plants, plant fresh weight, chlorophyll relative content, SOD, CAT, NR activities and Pn treated with 16.7 mg/L ALA were higher than that of the control (CK0). POD, MDA, GOGAT activities in S2 plants treated with 25.0 mg/L ALA were the highest among all treatments. Thus, our results showed that the optimal concentration of ALA (16.7 ~ 25.0 mmol/L) increases the activity of antioxidant enzymes, which in turn helps to abate the damage caused by salt stress in I. indigotica seedlings. Furthermore, ALA also results in an increase in chlorophyll content, Pn and the activities of GOGAT and NR.     

Temporal and spatial regulation of anthocyanin biosynthesis provide diverse flower colour intensities and patterning in <Emphasis Type="Italic">Cymbidium</Emphasis> orchid

Main conclusion

This study confirmed pigment profiles in different colour groups, isolated key anthocyanin biosynthetic genes and established a basis to examine the regulation of colour patterning in flowers of Cymbidium orchid. Cymbidium orchid (Cymbidium hybrida) has a range of flower colours, often classified into four colour groups; pink, white, yellow and green. In this study, the biochemical and molecular basis for the different colour types was investigated, and genes involved in flavonoid/anthocyanin synthesis were identified and characterised. Pigment analysis across selected cultivars confirmed cyanidin 3-O-rutinoside and peonidin 3-O-rutinoside as the major anthocyanins detected; the flavonols quercetin and kaempferol rutinoside and robinoside were also present in petal tissue. β-carotene was the major carotenoid in the yellow cultivars, whilst pheophytins were the major chlorophyll pigments in the green cultivars. Anthocyanin pigments were important across all eight cultivars because anthocyanin accumulated in the flower labellum, even if not in the other petals/sepals. Genes encoding the flavonoid biosynthetic pathway enzymes chalcone synthase, flavonol synthase, flavonoid 3′ hydroxylase (F3′H), dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) were isolated from petal tissue of a Cymbidium cultivar. Expression of these flavonoid genes was monitored across flower bud development in each cultivar, confirming that DFR and ANS were only expressed in tissues where anthocyanin accumulated. Phylogenetic analysis suggested a cytochrome P450 sequence as that of the Cymbidium F3′H, consistent with the accumulation of di-hydroxylated anthocyanins and flavonols in flower tissue. A separate polyketide synthase, identified as a bibenzyl synthase, was isolated from petal tissue but was not associated with pigment accumulation. Our analyses show the diversity in flower colour of Cymbidium orchid derives not from different individual pigments but from subtle variations in concentration and pattern of pigment accumulation.

     

Drastic anthocyanin increase in response to <Emphasis Type="Italic">PAP1</Emphasis> overexpression in <Emphasis Type="Italic">fls1</Emphasis> knockout mutant confers enhanced osmotic stress tolerance in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Key message

pap1 - D/fls1ko double mutant plants that produce substantial amounts of anthocyanin show tolerance to abiotic stress.

Abstract

Anthocyanins are flavonoids that are abundant in various plants and have beneficial effects on both plants and humans. Many genes in flavonoid biosynthetic pathways have been identified, including those in the MYB-bHLH-WD40 (MBW) complex. The MYB gene Production of Anthocyanin Pigment 1 (PAP1) plays a particularly important role in anthocyanin accumulation. PAP1 expression in many plant systems strongly increases anthocyanin levels, resulting in a dark purple color in many plant organs. In this study, we generated double mutant plants that harbor fls1ko in the pap1-D background (i.e., pap1-D/fls1ko plants), to examine whether anthocyanins can be further enhanced by blocking flavonol biosynthesis under PAP1 overexpression. We also wanted to examine whether the increased anthocyanin levels contribute to defense against osmotic stresses. The pap1-D/fls1ko mutants accumulated higher anthocyanin levels than pap1-D plants in both control and sucrose-treated conditions. However, flavonoid biosynthesis genes were slightly down-regulated in the pap1-D/fls1ko seedlings as compared to their expression in pap1-D seedlings. We also report the performance of pap1-D/fls1ko seedlings in response to plant osmotic stresses.

     

Effects of ALA on Photosynthesis,Antioxidant Enzyme Activity,and Gene Expression,and Regulation of Proline Accumulation in Tomato Seedlings Under NaCl Stress

This study examined the effects of 5-aminolevulinic acid (ALA) application on photosynthesis, activity and gene expression of key antioxidant enzymes, and on proline accumulation in tomato (Lycopersicon esculentum Mill. ‘Hezuo 903’) seedlings under NaCl stress. NaCl stress significantly decreased the net photosynthetic rates and inhibited the activity of photosystem II, whereas exogenous ALA application significantly restored the net photosynthetic rates, quantum yield of electron transport, and energy conversion efficiency of photosystem II of tomato under NaCl stress. Production of superoxide, hydrogen peroxide, and malondialdehyde strongly increased in response to NaCl stress, and these increases were significantly counteracted by ALA. ALA increased the activity of reactive oxygen species (ROS) scavenging antioxidant enzymes, including superoxide dismutase, catalase, ascorbate peroxidase, and peroxidase, and upregulated the expression of SOD, APX, and POD, genes that encode these enzymes in NaCl-treated plants. ALA simultaneously increased proline accumulation in tomato seedlings under NaCl stress by regulating the expression of genes that encode ALA biosynthetic enzymes and that control proline biosynthesis and metabolism, for example, expression of GluRS and GluTR was downregulated, accompanied by a significant increase in the expression of P5CS and decline in the expression of ProDH. ALA provided protection against NaCl stress by increasing photosynthetic capacity, regulating antioxidant enzyme gene expression and proline accumulation, and decreasing ROS accumulation and lipid peroxidation in tomato.     

Role of 5-aminolevulinic acid in the formation of winter rape resistance to sulfonylurea herbicides

Growing of winter rape (Brassica napus L.) plants for 7–8 days in the presence of a sulfonylurea herbicide Magnum (methsulfuron methyl; 200 and 500 mg/L) exerted an organ-specific influence on the seedlings: suppressed hypocotyl and root growth and increased the accumulation of fresh weight of cotyledonary leaves. Exogenous 5-aminolevulinic acid (ALA; 0.1, 1.0, and 10 mg/L) partially negated the adverse effect of Magnum on the length and fresh weight of roots and hypocotyls and stimulated the development of cotyledons. The herbicide suppressed the activity of ascorbate peroxidase (APX). The addition of ALA to the 200 mg/L herbicide solution caused a steady activation of APX as compared with the effect of herbicide alone. In plants grown on Magnum solutions, the activity of glutathione reductase (GR) rose. Positive effect of exogenous ALA was only observed when 500 mg/L herbicide was used (Magnum-500 type of treatment). In plants grown on the Magnum solution, the content of reduced and oxidized forms of glutathione rose. In contrasty, exogenous ALA reduced the total content of glutathione but in this case the ratio between its reduced and oxidized forms rose. In the presence of the herbicide, the content of anthocyans considerably decreased and upon the addition of exogenous ALA their levels rose. In rape plants treated with Magnum, the ability to generate superoxide anion radical was essentially the same as in control plants; however, the content of hydrogen peroxide (H₂O₂) therein rose. The addition of ALA to the herbicide reduced the ability to generate superoxide anion radical and the level of H₂O₂. The activity of acetolactate synthase remained on the control level when 200 mg/L Magnum was used, decreased in the plants of Magnum-500 treatment, and rose upon the addition of exogenous ALA to this type of treatment. It was concluded that ALA had a positive effect on the development of winter rape resistance to Magnum via stimulation of growth processes, activation of APX and GR, predominant elevation of the content of reduced glutathione and anthocyans, and partial recovery of acetolactate synthase activity, which on the whole caused a decrease in the H₂O₂ level and in the ability of plants to generate superoxide anion radical.     

Exogenous 5-aminolevulinic acid alleviates the detrimental effects of UV-B stress on lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis> L) seedlings

One of the abiotic stress factors affecting plant metabolism is ultraviolet-B (UV-B) radiation. 5-Aminolevulinic acid (ALA), a key precursor of porphyrin biosynthesis, promotes plant growth and crop yields. To investigate the alleviating effects of exogenous ALA on the damages caused by UV-B exposure, two different concentrations [10 ppm (ALA1) and 25 ppm (ALA2)] of ALA were applied to lettuce seedlings for 24 h and then they were exposed to 3.3 W m^?2 UV-B. Results showed that UV-B treatment significantly decreased chlorophyll a and b (Chl a and b) concentration, enhanced the activity of antioxidant enzymes, total phenolic concentration, soluble sugar contents, expression of phenylalanine ammonia lyase (PAL) and γ-tocopherol methyltransferase (γ-TMT) genes, the concentration of malondialdehyde (MDA), hydrogen peroxide (H₂O₂), and the rate of superoxide radical (\({\text{O}}_{2}^{ - }\)) generation in the lettuce seedlings when compared to the control. Pre-treatment with exogenous ALA significantly enhanced UV-B stress tolerance in lettuce seedlings by decreasing the reactive oxygen species. On the other hand, ALA application caused more increases in the PAL and γ-TMT gene expression, antioxidant enzymes activities, Chl a and b concentration, total phenolic content, antioxidant capacity and the concentrations of soluble sugars. Obtained results indicated that UV-B radiation exerts an adverse effect on lettuce seedlings, and some of the negative effects of UV-B radiation can be alleviated by exogenous ALA.     

Complementation of the Tomato anthocyanin without (aw) Mutant Using the Dihydroflavonol 4-Reductase Gene

We isolated the dihydroflavonol 4-reductase (DFR) gene from tomato (Lycopersicon esculentum) using a previously characterized cDNA as probe. Earlier studies had indicated that the DFR gene is present in tomato as a single gene located on chromosome 2 near the locus anthocyanin without (aw). Mutant alleles of the aw locus result in the complete absence of anthocyanin pigmentation throughout all stages of plant development. When the genomic DFR clone was introduced by Agrobacterium-mediated transformation into plants bearing the aw mutation, primary transgenic seedlings accumulated anthocyanins that could be observed while the plants were still in tissue culture and which continued to be observed as the plants matured. Progeny of self pollinated and backcrossed transgenic plants segregated for anthocyanin pigmentation, and Southern hybridization analyses indicated the presence of the DFR transgene exclusively in those plants with pigmentation. These data indicate that the aw locus likely corresponds to the structural gene for DFR and that DFR can be used as a visual, nondestructive, plant-derived marker gene for tomato.     

Transcriptome analysis reveals anthocyanin acts as a protectant in <Emphasis Type="Italic">Begonia semperflorens</Emphasis> under low temperature

Anthocyanins are natural bioactive pigments in plants that play important roles in many physiological functions. They are found in various tissues and can protect plants against different stress conditions. Anthocyanins are synthesized and accumulate in nutritional organs, which is crucial for plants to adapt to and resist adverse environmental conditions, including high exposure to light, ultraviolet light, low temperatures, drought, pests and disease. Some progress has been made in understanding the adaptability of anthocyanin to the external environment. Begonia semperflorens is an excellent model for studying the function and regulation of anthocyanin synthesis. To investigate the biosynthesis and regulation of anthocyanins, RNA sequencing techniques were employed to investigate anthocyanin biosynthesis induced by low temperature in B. semperflorens leaves. A total of 74,779 unigenes with a mean length of 1249 bp were assembled. Functional annotations were implemented using five protein databases. Differentially expressed genes involved in the process of anthocyanin biosynthesis were identified. This study represents the first report of a broad-scale gene expression study on B. semperflorens.