The sub-cellular localisation of the potato (Solanum tuberosum L.) carotenoid biosynthetic enzymes, CrtRb2 and PSY2

Carotenoids are isoprenoids with important biological roles both for plants and animals. The yellow flesh colour of potato (Solanum tuberosum L.) tubers is a quality trait dependent on the types and levels of carotenoids that accumulate. The carotenoid biosynthetic pathway is well characterised, facilitating the successful engineering of carotenoid content in numerous crops including potato. However, a clear understanding concerning the factors regulating carotenoid accumulation and localisation in plant storage organs, such as tubers, is lacking. In the present study, the localisation of key carotenoid biosynthetic enzymes was investigated, as one of the unexplored factors that could influence the accumulation of carotenoids in potato tubers. Stable transgenic potato plants were generated by over-expressing β-CAROTENE HYDROXYLASE 2 (CrtRb2) and PHYTOENE SYNTHASE 2 (PSY2) genes, fused to red fluorescent protein (RFP). Gene expression and carotenoid levels were both significantly increased, confirming functionality of the fluorescently tagged proteins. Confocal microscopy studies revealed different sub-organellar localisations of CrtRb2-RFP and PSY2-RFP within amyloplasts. CrtRb2 was detected in small vesicular structures, inside amyloplasts, whereas PSY2 was localised in the stroma of amyloplasts. We conclude that it is important to consider the location of biosynthetic enzymes when engineering the carotenoid metabolic pathway in storage organs such as tubers.     

An update on carotenoid biosynthesis in algae: phylogenetic evidence for the existence of two classes of phytoene synthase

Carotenoids play crucial roles in structure and function of the photosynthetic apparatus of bacteria, algae, and higher plants. The entry-step reaction to carotenoid biosynthesis is catalyzed by the phytoene synthase (PSY), which is structurally and functionally related in all organisms. A comparative genomic analysis regarding the PSY revealed that the green algae Ostreococcus and Micromonas possess two orthologous copies of the PSY genes, indicating an ancient gene duplication event that produced two classes of PSY in algae. However, some other green algae (Chlamydomonas reinhardtii, Chlorella vulgaris, and Volvox carteri), red algae (Cyanidioschyzon merolae), diatoms (Thalassiosira pseudonana and Phaeodactylum tricornutum), and higher plants retained only one class of the PSY gene whereas the other gene copy was lost in these species. Further, similar to the situation in higher plants recent gene duplications of PSY have occurred for example in the green alga Dunaliella salina/bardawil. As members of the PSY gene families in some higher plants are differentially regulated during development or stress, the discovery of two classes of PSY gene families in some algae suggests that carotenoid biosynthesis in these algae is differentially regulated in response to development and environmental stress as well.     

Characterizing the growth of <Emphasis Type="Italic">Sarcoptes scabiei</Emphasis> infrapopulations

The plant stress hypothesis posits that a herbivore’s reproductive success increases when it feeds on stressed plants, while the plant vigor hypothesis predicts that a herbivore preferentially feeds on more vigorous plants. We examined these opposing hypotheses by growing spider mites (Tetranychus urticae) on the leaves of stressed and healthy (vigorous) cucumber plants. Host plants were grown under controlled conditions at low, moderate, and high concentrations of NaCl (to induce salinity stress), at low, moderate, and high fertilizer concentrations (to support growth), and without these additions (control). The effects of these treatments were evaluated by measuring fresh and dry plant biomass, carotenoid and chlorophyll content, antioxidant enzyme activity, and concentrations of PO₄^3?, K⁺, and Na⁺ in plant tissues. The addition of low concentrations of fertilizer increased dry mass, protein, and carotenoid content relative to controls, suggesting a beneficial effect on plants. The highest NaCl treatment (2560 mg L^?1) resulted in increased Na⁺ and protein content relative to control plants, as well as reduced PO₄^3?, K⁺, and chlorophyll levels and reduced catalase and ascorbate peroxidase enzyme activity levels. Analysis of life table data of T. urticae mites raised on leaves from the aforementioned plant groups showed the intrinsic rate of increase (r) for mites was 0.167 day^?1 in control specimens, 0.125 day^?1 for mites reared on plants treated with a moderate concentration of fertilizer (10 mL L^?1), and was highest (0.241 day^?1) on plants grown under moderate salinity conditions (1920 mg L^?1 NaCl). Reproductive success of T. urticae did not differ on plants watered with a moderate concentration of NaCl or a high concentration of fertilizer. The moderately-stressed plants formed a favorable environment for the development and reproduction of spider mites, supporting the plant stress hypothesis.     

Enhancement of carotenoid biosynthesis in the green microalga Dunaliella salina with light-emitting diodes and adaptive laboratory evolution

There is a particularly high interest to derive carotenoids such as β-carotene and lutein from higher plants and algae for the global market. It is well known that β-carotene can be overproduced in the green microalga Dunaliella salina in response to stressful light conditions. However, little is known about the effects of light quality on carotenoid metabolism, e.g., narrow spectrum red light. In this study, we present UPLC-UV-MS data from D. salina consistent with the pathway proposed for carotenoid metabolism in the green microalga Chlamydomonas reinhardtii. We have studied the effect of red light-emitting diode (LED) lighting on growth rate and biomass yield and identified the optimal photon flux for D. salina growth. We found that the major carotenoids changed in parallel to the chlorophyll b content and that red light photon stress alone at high level was not capable of upregulating carotenoid accumulation presumably due to serious photodamage. We have found that combining red LED (75 %) with blue LED (25 %) allowed growth at a higher total photon flux. Additional blue light instead of red light led to increased β-carotene and lutein accumulation, and the application of long-term iterative stress (adaptive laboratory evolution) yielded strains of D. salina with increased accumulation of carotenoids under combined blue and red light.     

Enhancement of phenylpropanoid enzymes and lignin in Phalaenopsis orchid and their influence on plant acclimatisation at different levels of photosynthetic photon flux

Effects of three levels of photosynthetic photon flux (PPF: 60, 160 and 300 μmol m⁻²s⁻¹) were investigated in one-month-old Phalaenopsis plantlets acclimatised ex vitro. Optimal growth, chlorophyll and carotenoid concentations, and a high carotenoid:chlorophyll a ratio were obtained at 160 μmol m⁻²s⁻¹, while net CO₂ assimilation (A), stomatal conductance (g), transpiration rate (E) and leaf temperature peaked at 300 μmol m⁻²s⁻¹, indicating the ability of the plants to grow ex vitro. Adverse effects of the highest PPF were reflected in loss of chlorophyll, biomass, non-protein thiol and cysteine, but increased proline. After acclimatisation, glucose-6-phosphate dehydrogenase, shikimate dehydrogenase, phenylalanine ammonia-lyase (PAL) and cinnamyl alcohol dehydrogenase (CAD) increased, as did lignin. Peroxidases (POD), which play an important role in lignin synthesis, were induced in acclimatised plants. Polyphenol oxidase (PPO) and β-glucosidase (β-GS) activities increased to a maximum in acclimatised plants at 300 μmol m⁻²s⁻¹. A positive correlation between PAL, CAD activity and lignin concentration was observed, especially at 160 and 300 μmol m⁻²s⁻¹. The study concludes that enhancement of lignin biosynthesis probably not only adds rigidity to plant cell walls but also induces defence against radiation stress. A PPF of 160 μmol m⁻²s⁻¹was suitable for acclimatisation when plants were transferred from in vitro conditions.     

A high-resolution linkage map of the vicinity of the rice submergence tolerance locus Sub1

Resistance to submergence stress is an important breeding objective in areas where rice cultivars are subjected to complete inundation for a week or more. The present study was conducted to develop a high-resolution map of the region surrounding the submergence tolerance gene Sub1 in rice, which derives from the Indian cultivar FR13A. Submergence screening of 8-day-old plants of F₃ families kept for 14 days submerged in 60 cm of water allowed an accurate classification of Sub1 phenotypes. Bulked segregant analysis was used to identify AFLP markers linked to Sub1. A population of 2950 F₂ plants segregating for Sub1 was screened with two RFLP markers flanking the Sub1 locus, 2.4 and 4.9 cM away. Submergence tolerance was measured in the recombinant plants, and AFLP markers closely linked to Sub1 were mapped. Two AFLP markers cosegregated with Sub1 in this large population, and other markers were localized within 0.2 cM of Sub1. The high-resolution map should serve as the basis for map-based cloning of this important locus, as it will permit the identification of BAC clones spanning the region. Received: 15 December 1999 / Accepted: 18 February 2000     

Genetic architecture controlling variation in grain carotenoid composition and concentrations in two maize populations

Key message

Genetic control of maize grain carotenoid profiles is coordinated through several loci distributed throughout three secondary metabolic pathways, most of which exhibit additive, and more importantly, pleiotropic effects.

Abstract

The genetic basis for the variation in maize grain carotenoid concentrations was investigated in two F_2:3 populations, DEexp × CI7 and A619 × SC55, derived from high total carotenoid and high β-carotene inbred lines. A comparison of grain carotenoid concentrations from population DEexp × CI7 grown in different environments revealed significantly higher concentrations and greater trait variation in samples harvested from a subtropical environment relative to those from a temperate environment. Genotype by environment interactions was significant for most carotenoid traits. Using phenotypic data in additive, environment-specific genetic models, quantitative trait loci (QTL) were identified for absolute and derived carotenoid traits in each population, including those specific to the isomerization of β-carotene. A multivariate approach for these correlated traits was taken, using carotenoid trait principal components (PCs) that jointly accounted for 97 % or more of trait variation. Component loadings for carotenoid PCs were interpreted in the context of known substrate-product relationships within the carotenoid pathway. Importantly, QTL for univariate and multivariate traits were found to cluster in close proximity to map locations of loci involved in methyl-erythritol, isoprenoid and carotenoid metabolism. Several of these genes, including lycopene epsilon cyclase, carotenoid cleavage dioxygenase1 and beta-carotene hydroxylase, were mapped in the segregating populations. These loci exhibited pleiotropic effects on α-branch carotenoids, total carotenoid profile and β-branch carotenoids, respectively. Our results confirm that several QTL are involved in the modification of carotenoid profiles, and suggest genetic targets that could be used for the improvement of total carotenoid and β-carotene in future breeding populations.     

Structural insights into a key carotenogenesis related enzyme phytoene synthase of <Emphasis Type="Italic">P. falciparum</Emphasis>: a novel drug target for malaria

Carotenoids represent a diverse group of pigments derived from the common isoprenoid precursors and fulfill a variety of critical functions in plants and animals. Phytoene synthase (PSY), a transferase enzyme that catalyzes the first specific step in carotenoid biosynthesis plays a central role in the regulation of a number of essential functions mediated via carotenoids. PSYs have been deeply investigated in plants, bacteria and algae however in apicomplexans it is poorly studied. In an effort to characterize PSY in apicomplexans especially the malaria parasite Plasmodium falciparum (P. falciparum), a detailed bioinformatics analysis is undertaken. We have analysed the Phylogenetic relationship of PSY also referred to as octaprenyl pyrophosphate synthase (OPPS) in P. falciparum with other taxonomic groups. Further, we in silico characterized the secondary and tertiary structures of P. falciparum PSY/OPPS and compared the tertiary structures with crystal structure of Thermotoga maritima (T. maritima) OPPS. Our results evidenced the resemblance of P. falciparum PSY with the active site of T. maritima OPPS. Interestingly, the comparative structural analysis revealed an unconserved unique loop in P. falciparum OPPS/PSY. Such structural insights might contribute novel accessory functions to the protein thus, offering potential drug targets.     

Dynamic changes in photosynthesis and chlorophyll fluorescence in Nicotiana tabacum infested by Bemisia tabaci (Middle East–Asia Minor 1) nymphs

Bemisia tabaci Middle East–Asia Minor 1 (MEAM1) is a worldwide pest. To determine whether MEAM1 nymphs produce the same symptoms in different host plants, we measured the plant growth and chlorophyll content of tobacco and cotton plants that were infested by MEAM1 nymphs. Furthermore, to investigate the spatial and temporal changes in photosynthesis caused by MEAM1 nymphs, the net photosynthetic rate (Pn) and chlorophyll a fluorescence of local and systemic tobacco leaves were assayed at 8, 11, 14, and 20 days after MEAM1 adult removal, which represent the stages of 1st, 2nd, 3rd, and 4th instar nymphs, respectively. The results showed that MEAM1 nymph infestation reduced the plant height and internode length of tobacco at 14 and 20 days, as well as the dry weight of infested and systemic tobacco leaves. However, MEAM1 nymph infestation did not affect the plant height or internode length of cotton. Also, the dry weight and chlorophyll and carotenoid content of infested and systemic leaves of cotton plants were not influenced by MEAM1 nymph infestation. However, the contents of chlorophyll a and b and carotenoids in infested tobacco leaves decreased over time; the chlorophyll a content of systemic tobacco leaves decreased at 11, 14, and 20 days. The chlorophyll and carotenoid contents in infested and systemic leaves of cotton plants were not influenced by MEAM1 nymph infestation. In addition, the Pn of infested tobacco leaves decreased at 14 and 20 days, while the Pn in systemic tobacco leaves decreased after 11 days. The greatest decrease in performance index on absorption basis (PI _ABS ) of infested and systemic tobacco leaves occurred on day 14. The fluorescence intensity at 2 ms (J peak) and 300 μs (K peak) increased on day 14, which indicates that 3rd instar nymphs caused serious damage to the primary photochemical reactions and donor side of PSII. These results suggest that MEAM1 nymph infestation had different effects on tobacco and cotton plants. The infestation caused spatial and temporal changes in photosynthesis in tobacco plants. The lower chlorophyll a content may have been related to the lower net photosynthetic rate of systemic and infested tobacco leaves. The decreased stability of the oxygen-evolving complex and the reaction center of PSII and the decrease in electron transport were the main reasons for the decrease in the level of photosynthesis in tobacco leaves caused by MEAM1 nymphs during various stages of infestation.     

Carotenoid Crystal Formation in Arabidopsis and Carrot Roots Caused by Increased Phytoene Synthase Protein Levels

Background

As the first pathway-specific enzyme in carotenoid biosynthesis, phytoene synthase (PSY) is a prime regulatory target. This includes a number of biotechnological approaches that have successfully increased the carotenoid content in agronomically relevant non-green plant tissues through tissue-specific PSY overexpression. We investigated the differential effects of constitutive AtPSY overexpression in green and non-green cells of transgenic Arabidopsis lines. This revealed striking similarities to the situation found in orange carrot roots with respect to carotenoid amounts and sequestration mechanism.

Methology/Principal Findings

In Arabidopsis seedlings, carotenoid content remained unaffected by increased AtPSY levels although the protein was almost quantitatively imported into plastids, as shown by western blot analyses. In contrast, non-photosynthetic calli and roots overexpressing AtPSY accumulated carotenoids 10 and 100-fold above the corresponding wild-type tissues and contained 1800 and 500 µg carotenoids per g dry weight, respectively. This increase coincided with a change of the pattern of accumulated carotenoids, as xanthophylls decreased relative to β-carotene and carotene intermediates accumulated. As shown by polarization microscopy, carotenoids were found deposited in crystals, similar to crystalline-type chromoplasts of non-green tissues present in several other taxa. In fact, orange-colored carrots showed a similar situation with increased PSY protein as well as carotenoid levels and accumulation patterns whereas wild white-rooted carrots were similar to Arabidopsis wild type roots in this respect. Initiation of carotenoid crystal formation by increased PSY protein amounts was further confirmed by overexpressing crtB, a bacterial PSY gene, in white carrots, resulting in increased carotenoid amounts deposited in crystals.

Conclusions

The sequestration of carotenoids into crystals can be driven by the functional overexpression of one biosynthetic enzyme in non-green plastids not requiring a chromoplast developmental program as this does not exist in Arabidopsis. Thus, PSY expression plays a major, rate-limiting role in the transition from white to orange-colored carrots.     

Genetic mapping of a major codominant QTL associated with β-carotene accumulation in watermelon

The common flesh color of commercially grown watermelon is red due to the accumulation of lycopene. However, natural variation in carotenoid composition that exists among heirloom and exotic accessions results in a wide spectrum of flesh colors. We previously identified a unique orange flesh watermelon accession (NY0016) that accumulates mainly β-carotene and no lycopene. We hypothesized this unique accession could serve as a viable source for increasing provitamin A content in watermelon. Here we characterize the mode of inheritance and genetic architecture of this trait. Analysis of testcrosses of NY0016 with yellow and red fruited lines indicated a codominant mode of action as F₁ fruits exhibited a combination of carotenoid profiles from both parents. We combined visual color phenotyping with genotyping-by-sequencing of an F_2:3 population from a cross of NY0016 by a yellow fruited line, to map a major locus on chromosome 1, associated with β-carotene accumulation in watermelon fruit. The QTL interval is approximately 20 cM on the genetic map and 2.4 Mb on the watermelon genome. Trait-linked marker was developed and used for validation of the QTL effect in segregating populations across different genetic backgrounds. This study is a step toward identification of a major gene involved in carotenoid biosynthesis and accumulation in watermelon. The codominant inheritance of β-carotene provides opportunities to develop, through marker-assisted breeding, β-carotene-enriched red watermelon hybrids.     

Mapping quantitative trait loci associated with blush in peach [Prunus persica (L.) Batsch]

Blush is an important trait for marketing peaches. The red skin pigmentation develops through the flavonoid and anthocyanin pathways, and both genetic and environmental stimuli, and their interaction, control the regulation of these pathways. The molecular basis of blush development in peach is yet to be understood. An F₂ blush population (ZC²) derived from a cross between two peach cultivars with contrasting phenotypes for blush, “Zin Dai” (～30 % red) and “Crimson Lady” (～100 % red), was used for linkage map construction and quantitative trait loci (QTLs) mapping. The segregating population was phenotyped for blush for 4 years using a visual rating scale and quantified using a colorimeter (L*, a*, and b*) 1 year. The ZC² population was genotyped with the IPSC 9 K peach single-nucleotide polymorphism (SNP) array v1, and a high-density ZC² genetic linkage map was constructed. The map covers genetic a distance of ～452.51 cM with an average marker spacing of 2.38 cM/marker. Four QTLs were detected: one major QTL on LG3 (Blush.Pp.ZC-3.1) and three minor QTLs on LG 4 and 7 (Blush.Pp.ZC-4.1; Blush.Pp.ZC-4.2; Blush.Pp.ZC-7.1), indicating the presence of major and minor genes involved in blush development. Candidate genes involved in skin and flesh coloration of peach (PprMYB10), cherry (PavMYB10), and apple (MdMYB1/MdMYBA/MdMYB10) are located within the interval of the major QTL on LG3, suggesting the same genetic control for color development in the Rosaceae family. Marker-assisted selection (MAS) for blush is discussed.     

In the presence of red light,cucumber and possibly other host plants lose their attractability to the melon thrips <Emphasis Type="Italic">Thrips palmi</Emphasis> (Thysanoptera: Thripidae)

The melon thrips, Thrips palmi Karny (Thysanoptera: Thripidae), is a serious agricultural pest of many crops. Previous studies have shown that red light decreases the number of Thrips palmi in greenhouses. In order to understand how red light affects T. palmi, we examined the behavioral responses to host plants that were irradiated with a red light-emitting diode panel (660 nm) in an environment with natural or fluorescent (normal-white) light. When T. palmi were allowed to move freely around in the experimental arena, we found that fewer individuals were attracted to plants irradiated by red light than to plants under normal light illumination. We then used a sticky trap of green coloration to exclude olfactory and visual stimuli associated with the host plants in order to test binary choice behavior in T. palmi. The number of thrips attracted to the green sticky trap irradiated with red light was approximately half of that without red light irradiation. This is the first study to show that an addition of red light can change the behavior of insects, leading to an avoidance of green targets in an environment of normal illumination.     

Molecular and chemical characterization of plants regenerated from Ri-mediated hairy root cultures of Rauwolfia serpentina

Hairy root lines were induced from leaf explants of Rauwolfia serpentina known to contain high levels of reserpine (0.0882 % DW) content. Out of five high yielding hairy root lines, three (R1, R14 and R15) exhibited spontaneous regeneration of shoots after 6–8 weeks in liquid B5 medium. Excised regenerated shoots underwent robust shoot proliferation when cultured on Murashige and Skoog (MS) medium supplemented with 0.1 mg/l naphthanleneacetic acid (NAA) and 1.0 mg/l 6-benzyladenine. When shoots were transferred to a root induction medium, consisting of MS basal medium and 1.0 mg/l NAA, all rooted within 2–3 weeks. Of a total of 45 plants developed from three different hairy root lines, 30 were successfully acclimatized and transferred to the green house. Almost 90 % of these plants grown in the green house showed no observed phenotypic differences, while 10 % were stunted and grew poorly, in comparison to non-transformed plants. Phenotypic assessment of regenerated plants for plant length, number of nodes and intermodal lengths, number of leaves per node, leaf color, leaf size, number of flowering shoots, flower size, fruit size, lateral root branching and root biomass was conducted. Polymerase chain reaction and Southern blot hybridization revealed that all plants derived from hairy roots carried the Ri T_L-DNA fragment. Moreover for plants derived from transgenic hairy root line R14, presence of more than a single transgene copy number was observed, and this might have contributed to observed abnormal phenotypes. Analysis of reserpine content revealed that roots of regenerated plants had similar levels (0.0889 % DW) to those of their corresponding hairy roots.     

Carotenoid and lipid production by the autotrophic microalga Chlorella protothecoides under nutritional, salinity, and luminosity stress conditions

Today microalgae represent a viable alternative source for high-value products. The specie Chlorella protothecoides (Cp), heterotrophically grown, has been widely studied and provides a high amount of lutein and fatty acids (FA) and has a good profile for biodiesel production. This work studies carotenoid and FA production by autotrophic grown Cp. Cp was grown until the medium’s nitrogen was depleted, then diluted in NaCl solution, resulting in nutritional, luminosity, and salinity stresses. Different NaCl concentrations were tested (10, 20, 30 g/L) at two different dilutions. After dilution, a color shifting from green to orange-red was noticed, showing carotenoid production. The best production of both carotenoids and FA was attained with a 20 g/L NaCl solution. The total carotenoid content was 0.8 % w/w (canthaxanthin (23.3 %), echinenone (14.7 %), free astaxanthin (7.1 %), and lutein/zeaxanthin (4.1 %)). Furthermore, the total lipid content reached 43.4 % w/w, with a FA composition of C18:1 (33.64 %), C16:0 (23.30 %), C18:2 (11.53 %), and less than 12 % of C18:3, which is needed to fulfill the biodiesel quality specifications (EN 14214).