Transformation of carotenoid biosynthetic genes using a micro-cross section method in kiwifruit (Actinidia deliciosa cv. Hayward)

Genetic transformation using a micro-cross section (MCS) technique was conducted to improve the carotenoid content in kiwifruit (Actinidia deliciosa cv. Hayward). The introduced carotenoid biosynthetic genes include geranylgeranyl diphosphate synthase (GGPS), phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), β-carotene hydroxylase (CHX), and phytoene synthase (PSY). The transformed explants were selected on half-strength MS medium containing 0.001 mg l⁻¹ of 2,4-D and 0.1 mg l⁻¹ of zeatin, either 5 mg l⁻¹ hygromycin or 25 mg l⁻¹ kanamycin, and 500 mg l⁻¹ cefotaxime. The genomic PCR, genomic Southern blot analysis, and RT-PCR were performed to confirm the integration and expression of the transgenes. The transformation efficiencies of either kanamycin- or hygromycin-resistant shoots ranged from 2.9 to 22.1% depending on the target genes, and from 2.9 to 24.2% depending on the reporter genes. The selection efficiencies ranged from 66.7 to 100% for the target genes and from 95.8 to 100% for the reporter genes. Changes of carotenoid content in the several PCR-positive plants were determined by UPLC analysis. As a result, transgenic plants expressing either GGPS or PSY increased about 1.2- to 1.3-fold in lutein or β-carotene content compared to non-transgenic plants. Our results suggest that the Agrobacterium-mediated transformation efficiency of kiwifruit can be greatly increased by this MCS method and that the carotenoid biosynthetic pathway can be modified in kiwifruit by genetic transformation. Our results further suggest that GGPS and PSY genes could be major target genes to increase carotenoid contents in kiwifruit.     

Effects of Light Quality and Norflurazon on the Formation of Plastid Pigments in Cotyledons of Pinus sylvestris

The effects of different light qualities and a special inhibitor of carotenoid biosynthesis on formation of plastid pigments of cotyledons of Pinus sylvestris were studied. The experimental results indicate: 1. The rate of synthesis of carotenoids in far-red light is relatively higher than that of chlorophylls, on the contrary in red light the rate of chlorophyll synthesis is higher. 2. When biosynthesis of carotenoids is inhibited, in white light the rate of total chlorophyll synthesis reduced with similar proportion. Accumulation of chlorophyll, however, is relatively much more than that of carotenoids. The highest molar ratio of chlorophyll/carotenoids is approximately 10.0. This implicates that chlorophyll and carotenoid synthesis proceed with certain independence. 3. After 4h exposure of strong white light of 9 day-old pine seedlings grown with 10-5 mol 1-1 norflurazon in farred light, contents of carotenoids and total, chlorophyll of cotyledons increase. Chlorophyll a biosynthesis promoted by light is higher than photooxidation of the pigment.     

光质和除草剂norflurazon对欧洲赤松子叶质体色素形成的影响

本文研究了不同光质和类胡萝卜素专一抑制剂 norflurazon 对欧洲赤松(Pinussylvestris)子叶叶绿体色素形成的影响,所得结果如下:1.在远红光下,类胡萝卜素相对合成速率大于叶绿素的合成速率,而在红光下恰恰相反。2.在白光下,当类胡萝卜素合成受抑制时,叶绿素的合成速率也有所降低。但是叶绿素相对积累量比类胡萝卜素大得多,叶绿素与类胡萝卜素的分子比增大到10:1,说明这两种色素的合成与积累有相当大的独立性。3.把连续在远红光下类胡萝卜素合成受抑制(含量为正常幼苗的30%)的松苗,移入强白光下4小时后,其总叶绿素和类胡萝卜素都有增加,表明只要有少量类胡萝卜素存在,光对色素合成的促进仍大于光氧化破坏。     

Overexpression of rice phytochrome A partially complements phytochrome B deficiency in Arabidopsis

The red/far-red reversible phytochromes play a central role in regulating the development of plants in relation to their light environment. Studies on the roles of different members of the phytochrome family have mainly focused on light-labile, phytochrome A and light-stable, phytochrome B. Although these two phytochromes often regulate identical responses, they appear to have discrete photosensory functions. Thus, phytochrome A predominantly mediates responses to prolonged far-red light, as well as acting in a non-red/far-red-reversible manner in controlling responses to light pulses. In contrast, phytochrome B mediates responses to prolonged red light and acts photoreversibly under light-pulse conditions. However, it has been reported that rice (Oryza sativa L.) phytochrome A operates in a classical red/far-red reversible fashion following its expression in transgenic tobacco plants. Thus, it was of interest to determine whether transgenic rice phytochrome A could substitute for loss of phytochrome B in phyB mutants of Arabidopsis thaliana (L.) Heynh. We have observed that ectopic expression of rice phytochrome A can correct the reduced sensitivity of phyB hypocotyls to red light and restore their response to end-of-day far-red treatments. The latter is widely regarded as a hallmark of phytochrome B action. However, although transgenic rice phytochrome A can correct other aspects of elongation growth in the phyB mutant it does not restore other responses to end-of-day far-red treatments nor does it restore responses to low red:far-red ratio. Furthermore, transgenic rice phytochrome A does not correct the early-flowering phenotype of phyB seedlings. Received: 12 July 1998 / Accepted: 13 August 1998     

Identification of novel members in sweet orange carotenoid biosynthesis gene families

Novel expressed and genomic members in sweet orange (Citrus sinensis [L.] Osbeck) carotenoid biosynthesis gene families have been identified through mining of an expressed sequence tags (ESTs) database and hybridization with a bacterial artificial chromosome (BAC) library. These new expressed members included one phytoene synthase (PSY), one phytoene desaturase (PDS), ten zeta-carotene desaturases (ZDS), one lycopene beta-cyclase (LCYB), one lycopene epsilon-cyclase (LCYE), four carotenoid beta-ring hydroxylases (CHYB), and one capsanthin/capsorubin synthase (CCS). Most unigenes with multiple ESTs, including the ones containing the known genes and these new members, were heterozygous, in which putative single nucleotide polymorphisms distinguished two alleles. According to digital gene expression profiling, fruit was the primary tissue where at least one member of each gene family was specifically or highly expressed. Digital expression levels varied among the members and tissues. According to Southern hybridization of the identified BAC clones, genomic members of the families were either clustered in a single BAC contig or distributed in several different contigs. PSY has four members in one contig, PDS two in one, ZDS 12 in three, LCYB 11 in three, LCYE three in two, CHYB eight in one, and CCS 14 in four, respectively. The number of the genomic members in most families tended to be more than that of the expressed members, suggesting that some genomic members may not be expressed or structurally functional. These new carotenoid gene members, along with much first-hand genomic information, can be used further for functional genomics and genetic mapping.     

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.     

Regulation and activation of phytoene synthase, a key enzyme in carotenoid biosynthesis, during photomorphogenesis

 During photomorphogenesis in higher plants, a coordinated increase occurs in the chlorophyll and carotenoid contents. The carotenoid level is under phytochrome control, as reflected by the light regulation of the mRNA level of phytoene synthase (PSY), the first enzyme in the carotenoid biosynthetic pathway. We investigated PSY protein levels, enzymatic activity and topological localization during photomorphogenesis. The results revealed that PSY protein levels and enzymatic activity increase during de-etiolation and that the enzyme is localized at thylakoid membranes in mature chloroplasts. However, under certain light conditions (e.g., far-red light) the increases in PSY mRNA and protein levels are not accompanied by an increase in enzymatic activity. Under those conditions, PSY is localized in the prolamellar body fraction in a mostly enzymatically inactive form. Subsequent illumination of dark-grown and/or in far-red light grown seedlings with white light causes the decay of these structures and a topological relocalization of PSY to developing thylakoids which results in its enzymatic activation. This light-dependent mechanism of enzymatic activation of PSY in carotenoid biosynthesis shares common features with the regulation of the NADPH:protochlorophyllide oxidoreductase, the first light-regulated enzyme in chlorophyll biosynthesis. The mechanism of regulation described here may contribute to ensuring a spatially and temporally coordinated increase in both carotenoid and chlorophyll contents. Received: 14 February 2000 / Accepted: 15 March 2000     

Phytochrome Control of Chlorophyll and Carotenoid Accumulation in Sorghum bicolor

Etiolated Sorghum bicolor seedlings manifested a significantmorphological response to short term irradiations by red andfar-red light and to a continuous far-red light. Accumulationof chlorophylls in white light and carotenoids in darkness isunder red/far-red reversible control as well as along with theeffectiveness of ‘High Irradiance Reaction’. Phytochromeis also found to eliminate the lag phase during the accumulationof chlorophylls and carotenoids in white light. (Received March 11, 1981; Accepted May 2, 1981)     

Biosynthesis of acyclic and cyclic carotenoids in higher plants and the control by phytochrome

Radish plants ( Raphanus sativus L. cv. Saxa treib) were grown in the presence of three different herbicides interfering with the biosynthesis of cyclic carotenoids. The herbicides caused an accumulation of acyclic biosynthetic intermediates. Plants were then irradiated using four different light programs in order to gain more insight into the first steps of carotenoid biosynthesis and their control by light and phytochrome. Plants grown in the dark in the presence of SAN 6706 or aminotriazole accumulated the acyclic intermediate phytoene, and those treated with J 852, the intermediates phytoene, phytofluene and zeta-carotene. In herbicide-treated plants short time irradiation with red light enhanced the formation of phytoene, phytofluene, zeta-carotene or lycopene, consistent with an effect of phytochrome on the early steps of carotenoid biosynthesis. Biosynthesis of cyclic carotenoids was also enhanced by red light in the untreated controls. In amitrole-treated plants formation of β-carotene, but not that of xanthophylls was stimulated by red light. In many cases neither the red light-induced biosynthesis of cyclic carotenoids nor the formation of acyclic intermediates could be prevented by a subsequent irradiation with far-red light. Similar enhancement as with red light was also obtained after treatment with far-red light only. Presented data may be taken as evidence that the biosynthesis and dehydrogenation of phytoene and the cyclization of lycopene are activated by a low threshold of active phytochrome. This may be further supported by the observation that far-red light itself stimulated carotenoid biosynthesis.     

ISOLATION AND CHARACTERIZATION OF THE PHYTOENE DESATURASE GENE AS A POTENTIAL SELECTIVE MARKER FOR GENETIC ENGINEERING OF THE ASTAXANTHIN‐PRODUCING GREEN ALGA CHLORELLA ZOFINGIENSIS (CHLOROPHYTA)1

Phytoene desaturase (PDS) is a rate‐limiting enzyme in carotenoid biosynthesis. Algal PDS is inhibited by some herbicides, leading to the bleaching of the cells due to destruction of chl. Specific point mutations in PDS confer resistance to the herbicide norflurazon, suggesting that mutated PDS could be used as a dominant selectable marker for genetic engineering of algae, for which very few selective markers are available. In this study, we report the isolation and characterization of the PDS gene from the astaxanthin‐producing green alga Chlorella zofingiensis Dönz. The open reading frame (ORF) of this PDS gene, interrupted by six introns, encoded a polypeptide of 558 amino acid residues. The deduced protein sequence showed significant homology to phytoene desaturases of algae, cyanobacteria, and higher plants. Expression of the PDS gene in Escherichia coli demonstrated that the enzyme was able to convert phytoene to ζ‐carotene. The PDS gene in Chlorella was shown to be up‐regulated by high light and glucose treatment. With a single amino acid change (L516R), the mutated PDS‐L516R was still active and exhibited ～36‐fold greater resistance to the bleaching herbicide norflurazon than the unaltered enzyme. Thus, the modified PDS gene could be a useful tool for genetic engineering of carotenoid biosynthesis in C. zofingiensis and perhaps also in other algae.     

Minor complexes at work: light-harvesting by carotenoids in the photosystem II antenna complexes CP24 and CP26

Plant photosynthesis relies on the capacity of chlorophylls and carotenoids to absorb light. One of the roles of carotenoids is to harvest green-blue light and transfer the excitation energy to the chlorophylls. The corresponding dynamics were investigated here for the first time, to our knowledge, in the CP26 and CP24 minor antenna complexes. The results for the two complexes differ substantially. In CP26 fast transfer (80 fs) occurs from the carotenoid S₂ state to chlorophylls a absorbing at 675 and 678 nm, whereas transfer from the hot S₁ state to the lowest energy chlorophylls is observed in <1 ps. In CP24, energy transfer from the S₂ state leads in 80 fs to the population of chlorophylls b and high-energy chlorophylls a absorbing at 670 nm, whereas the low-energy chlorophylls a are populated only in several picoseconds. The results suggest that CP26 has a structural and functional organization similar to that of LHCII, whereas CP24 differs substantially from the other Lhc complexes, especially regarding the lutein L1 binding domain. No energy transfer from the carotenoid S₁ state to chlorophylls was observed in either complex, suggesting that this state is energetically below the chlorophyll Qy state and therefore may play a role in the quenching of chlorophyll excitations.