Research progress on differentiation and regulation of plant chromoplasts.

Carotenoids, natural tetraterpenoids found abundantly in plants, contribute to the diverse colors of plant non-photosynthetic tissues and provide fragrance through their cleavage products, which also play crucial roles in plant growth and development. Understanding the synthesis, degradation, and storage pathways of carotenoids and identifying regulatory factors represents a significant strategy for enhancing plant quality. Chromoplasts serve as the primary plastids responsible for carotenoid accumulation, and their differentiation is linked to the levels of carotenoids, rendering them a subject of substantial research interest. The differentiation of chromoplasts involves alterations in plastid structure and protein import machinery. Additionally, this process is influenced by factors such as the ORANGE (OR) gene, Clp proteases, xanthophyll esterification, and environmental factors. This review shows the relationship between chromoplast and carotenoid accumulation by presenting recent advances in chromoplast structure, the differentiation process, and key regulatory factors, which can also provide a reference for rational exploitation of chromoplasts to enhance plant quality.

Comparative transcriptomic and plastid development analysis sheds light on the differential carotenoid accumulation in kiwifruit flesh.

Carotenoids are colorful lipophilic isoprenoids synthesized in all photosynthetic organisms which play roles in plant growth and development and provide numerous health benefits in the human diet (precursor of Vitamin A). The commercially popular kiwifruits are golden yellow-fleshed (Actinidia chinensis) and green fleshed (A. deliciosa) cultivars which have a high carotenoid concentration. Understanding the molecular mechanisms controlling the synthesis and sequestration of carotenoids in Actinidia species is key to increasing nutritional value of this crop via breeding. In this study we analyzed fruit with varying flesh color from three Actinidia species; orange-fleshed A. valvata (OF), yellow-fleshed A. polygama (YF) and green-fleshed A. arguta (GF). Microscopic analysis revealed that carotenoids accumulated in a crystalline form in YF and OF chromoplasts, with the size of crystals being bigger in OF compared to YF, which also contained globular substructures in the chromoplast. Metabolic profiles were investigated using ultra-performance liquid chromatography (UPLC), which showed that ß-carotene was the predominant carotenoid in the OF and YF species, while lutein was the dominant carotenoid in the GF species. Global changes in gene expression were studied between OF and GF (both tetraploid) species using RNA-sequencing which showed higher expression levels of upstream carotenoid biosynthesis-related genes such as DXS, PSY, GGPPS, PDS, ZISO, and ZDS in OF species compared to GF. However, low expression of downstream pathway genes was observed in both species. Pathway regulatory genes (OR and OR-L), plastid morphology related genes (FIBRILLIN), chlorophyll degradation genes (SGR, SGR-L, RCCR, and NYC1) were upregulated in OF species compared to GF. This suggests chlorophyll degradation (primarily in the initial ripening stages) is accompanied by increased carotenoid production and localization in orange flesh tissue, a contrast from green flesh tissue. These results suggest a coordinated change in the carotenoid pathway, as well as changes in plastid type, are responsible for an orange phenotype in certain kiwifruit species.

Nutritional Enrichment of Plant Leaves by Combining Genes Promoting Tocopherol Biosynthesis and Storage.

The enrichment of plant tissues in tocochromanols (tocopherols and tocotrienols) is an important biotechnological goal due to their vitamin E and antioxidant properties. Improvements based on stimulating tocochromanol biosynthesis have repeatedly been achieved, however, enhancing sequestering and storage in plant plastids remains virtually unexplored. We previously showed that leaf chloroplasts can be converted into artificial chromoplasts with a proliferation of plastoglobules by overexpression of the bacterial crtB gene. Here we combined coexpression of crtB with genes involved in tocopherol biosynthesis to investigate the potential of artificial leaf chromoplasts for vitamin E accumulation in Nicotiana benthamiana leaves. We show that this combination improves tocopherol levels compared to controls without crtB and confirm that VTE1, VTE5, VTE6 and tyrA genes are useful to increase the total tocopherol levels, while VTE4 further leads to enrichment in α-tocopherol (the tocochromanol showing highest vitamin E activity). Additionally, we show that treatments that further promote plastoglobule formation (e.g., exposure to intense light or dark-induced senescence) result in even higher improvements in the tocopherol content of the leaves. An added advantage of our strategy is that it also results in increased levels of other related plastidial isoprenoids such as carotenoids (provitamin A) and phylloquinones (vitamin K1).

Isolation and characterization of sub-plastidial fractions from carotenoid rich fruits.

Carotenoid biosynthesis and sequestration in higher plants occurs in the plastid organelle. Among diverse germplasm collections displaying natural variation for carotenoids and outputs from metabolic engineering experiments it has become clear that plastid type and numbers can have important implications on the quantitative composition of carotenoids accumulating. Therefore, it is important to characterize these organelles to fully evaluate the potential of the germplasm to enhance carotenoids and create nutrient dense fruits and vegetables. In this article the procedures used to isolate sub-plastidial structures from carotenoid-rich Solanaceae fruits (tomato and Capsicum) are described.

Phytoene synthase 2 in tomato fruits remains functional and contributes to abscisic acid formation.

In ripening tomato fruits, the leaf-specific carotenoids biosynthesis mediated by phytoene synthase 2 (PSY2) is replaced by a fruit-specific pathway by the expression of two chromoplast-specific genes: phytoene synthase 1 (PSY1) and lycopene-ß-cyclase (CYCB). Though both PSY1 and PSY2 genes express in tomato fruits, the functional role of PSY2 is not known. To decipher whether PSY2-mediated carotenogenesis operates in ripening fruits, we blocked the in vivo activity of lycopene-ß-cyclases in fruits of several carotenoids and ripening mutants by CPTA (2-(4-Chlorophenylthio)triethylamine hydrochloride), an inhibitor of lycopene-ß-cyclases. The CPTA-treatment induced accumulation of lycopene in leaves, immature-green and ripening fruits. Even in psy1 mutants V7 and r that are deficient in fruit-specific carotenoid biosynthesis, CPTA triggered lycopene accumulation but lowered the abscisic acid level. Differing from fruit-specific carotenogenesis, CPTA-treated V7 and r mutant fruits accumulated lycopene but not phytoene and phytofluene. The lack of phytoene and phytofluene accumulation was reminiscent of PSY2-mediated leaf-like carotenogenesis, where phytoene and phytofluene accumulation is never seen. The lycopene accumulation was associated with the partial transformation of chloroplasts to chromoplasts bearing thread-like structures. Our study uncovers the operation of a parallel carotenogenesis pathway mediated by PSY2 that provides precursors for abscisic acid biosynthesis in ripening tomato fruits.

The esterification of xanthophylls in Solanum lycopersicum (tomato) chromoplasts; the role of a non-specific acyltransferase.

The esterification of carotenoids has been associated with high-level accumulation, greater stability and potentially improved dietary bioavailability. Engineering the formation of ketocarotenoids into tomato fruit has resulted in the esterification of these non-endogenous metabolites. A genotype of tomato was created that contains; (i) the mutant pale yellow petal (pyp)1-1 allele, which is responsible for the absence of carotenoid esters in tomato flowers and (ii) the heterologous enzymes for ketocarotenoid formation. Analysis of the resulting progeny showed altered quantitative and qualitative differences in esterified carotenoids. For example, in ripe fruit tissues, in the presence of the pyp mutant allele, non-endogenous ketocarotenoid esters were absent while their free forms accumulated. These data demonstrate the involvement of the PYP gene product in the esterification of diverse xanthophylls.

Inhibition of Carotenoid Biosynthesis by CRISPR/Cas9 Triggers Cell Wall Remodelling in Carrot.

Recent data indicate that modifications to carotenoid biosynthesis pathway in plants alter the expression of genes affecting chemical composition of the cell wall. Phytoene synthase (PSY) is a rate limiting factor of carotenoid biosynthesis and it may exhibit species-specific and organ-specific roles determined by the presence of psy paralogous genes, the importance of which often remains unrevealed. Thus, the aim of this work was to elaborate the roles of two psy paralogs in a model system and to reveal biochemical changes in the cell wall of psy knockout mutants. For this purpose, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas9) proteins (CRISPR/Cas9) vectors were introduced to carotenoid-rich carrot (Daucus carota) callus cells in order to induce mutations in the psy1 and psy2 genes. Gene sequencing, expression analysis, and carotenoid content analysis revealed that the psy2 gene is critical for carotenoid biosynthesis in this model and its knockout blocks carotenogenesis. The psy2 knockout also decreased the expression of the psy1 paralog. Immunohistochemical staining of the psy2 mutant cells showed altered composition of arabinogalactan proteins, pectins, and extensins in the mutant cell walls. In particular, low-methylesterified pectins were abundantly present in the cell walls of carotenoid-rich callus in contrast to the carotenoid-free psy2 mutant. Transmission electron microscopy revealed altered plastid transition to amyloplasts instead of chromoplasts. The results demonstrate for the first time that the inhibited biosynthesis of carotenoids triggers the cell wall remodelling.

Carotenoid Biosynthesis and Plastid Development in Plants: The Role of Light.

Light is an important cue that stimulates both plastid development and biosynthesis of carotenoids in plants. During photomorphogenesis or de-etiolation, photoreceptors are activated and molecular factors for carotenoid and chlorophyll biosynthesis are induced thereof. In fruits, light is absorbed by chloroplasts in the early stages of ripening, which allows a gradual synthesis of carotenoids in the peel and pulp with the onset of chromoplasts' development. In roots, only a fraction of light reaches this tissue, which is not required for carotenoid synthesis, but it is essential for root development. When exposed to light, roots start greening due to chloroplast development. However, the colored taproot of carrot grown underground presents a high carotenoid accumulation together with chromoplast development, similar to citrus fruits during ripening. Interestingly, total carotenoid levels decrease in carrots roots when illuminated and develop chloroplasts, similar to normal roots exposed to light. The recent findings of the effect of light quality upon the induction of molecular factors involved in carotenoid synthesis in leaves, fruit, and roots are discussed, aiming to propose consensus mechanisms in order to contribute to the understanding of carotenoid synthesis regulation by light in plants.

Building the Synthetic Biology Toolbox with Enzyme Variants to Expand Opportunities for Biofortification of Provitamin A and Other Health-Promoting Carotenoids.

Carotenoids are a large class of structures that are important in human health and include both provitamin A and nonprovitamin A compounds. Vitamin A deficiency is a global health problem that can be alleviated by enriching provitamin A carotenoids in a range of food crops. Suitable plants for biofortification are those with high levels of the provitamin A biosynthetic precursor, lycopene, which is enzymatically converted by lycopene ß-cyclase (LCYB) to ß-carotene, a provitamin A carotenoid. Crops, such as citrus, naturally accumulate high levels of provitamin A and other health-promoting carotenoids. Such plants may have useful genes to expand the synthetic biology toolbox for producing a range of phenotypes, including both high provitamin A crops and crops with unique compositions of health-promoting carotenoids. To examine enzyme variants having different activity levels, we introduced two citrus LCYB alleles into tomato, a plant with fruit rich in lycopene. Overexpression in tomato of the stronger allele of the citrus chromoplast-specific lycopene ß-cyclase (CsLCYb2a) produced "golden" transgenic tomato fruits with 9.3-fold increased levels of ß-carotene at up to 1.5 mg/g dry weight. The use of the weaker allele, CsLCYb2b, also led to enhanced levels of ß-carotene but in the context of a more heterogeneous composition of carotenoids. From a synthetic biology standpoint, these allelic differences have value for producing cultivars with unique carotenoid profiles. Overexpression of the citrus LCYB genes was accompanied by increased expression of other genes encoding carotenoid biosynthetic enzymes and increased size and number of chromoplasts needed to sequester the elevated levels of carotenoids in the transgenic tomato fruits. The overexpression of the citrus LCYB genes also led to a pleiotropic effect on profiles of phytohormones and primary metabolites. Our findings show that enzyme variants are essential synthetic biology parts needed to create a wider range of metabolic engineering products. In this case, strong and weak variants of LCYB proved useful in creating dietary sources to alleviate vitamin A deficiency or, alternatively, to create crops with a heterogeneous composition including provitamin A and healthful, nonprovitamin A carotenoids.

The effect of low ascorbic acid content on tomato fruit ripening.

MAIN CONCLUSION: The oxidant/antioxidant balance affects the ripening time of tomato fruit. Ripening of tomato fruit is associated with several modifications such as loss of cell wall firmness and transformation of chloroplasts to chromoplasts. Besides a peak in H2O2, reactive oxygen species (ROS) are observed at the transition stage. However, the role of different components of oxidative stress metabolism in fruit ripening has been scarcely addressed. Two GDP-L-galactose phosphorylase (GGP) Solanum lycopersicum L. cv Micro-Tom mutants which have fruit with low ascorbic acid content (30% of wild type) were used in this work to unravel the participation of ascorbic acid and H2O2 in fruit maturation. Both GGP mutants show delayed fruit maturation with no peak of H2O2; treatment with ascorbic acid increases its own concentration and accelerates ripening only in mutants to become like wild type plants. Unexpectedly, the treatment with ascorbic acid increases H2O2 synthesis in both mutants resembling what is observed in wild type fruit. Exogenous supplementation with H2O2 decreases its own synthesis delaying fruit maturation in plants with low ascorbic acid content. The site of ROS production is localized in the chloroplasts of fruit of all genotypes as determined by confocal microscopy analysis. The results presented here demonstrate that both ascorbic acid and H2O2 actively participate in tomato fruit ripening.

Influence of high hydrostatic pressure pretreatment on properties of vacuum-freeze dried strawberry slices.

Effects of high hydrostatic pressure (HHP) (50, 100, 150, 200 and 250 MPa) pretreatment on water mobility and distribution, drying duration, microstructure, color, cell wall fraction and tissue structure of strawberry slices were investigated. HHP significantly increased water mobility of the strawberry slices, resulting in the reduction of drying duration by 9-24%. As the pretreatment pressure was increased, redness value and anthocyanin content continuously increased, soluble pectin (SBP) content increased and then decreased, while the contents of protopectin (PTP) and cellulose decreased. After the HHP pretreatment, chromoplasts and moisture was distributed more uniformly in the strawberry slices. Microscopy images showed the formation of microscopic holes or channels in the matrix and the breakdown of tissue structure by HHP. Results suggested HHP pretreatment disrupted the integrity of the fresh strawberry which enhanced the drying efficiency and migration of the chromoplasts during the vacuum-freeze drying process.

Light Microscopy and Raman Imaging of Carotenoids in Plant Cells In Situ and in Released Carotene Crystals.

Light microscopy with a bright field mode offers an easy and fast examination of plant specimen for carotenoid presence in its cells. Using basic techniques such as hand sectioned or squashed preparations, carotenoid-rich chromoplasts can be identified without applying any staining procedure and their localization within the cell, their shape and number can be assessed. More detailed information can be obtained by using Raman spectroscopy which is suitable for the analysis of carotenoids due to their unique Raman spectra and allows semiquantification of their contents. Raman imaging (mapping) can be additionally used to show the distribution of carotenoids within the sample. Raman spectra can be taken from extracted carotenoids but can be also obtained directly from plant tissues or cells as Raman measurements are nondestructive for the sample. Here we describe preparations of intact tissue samples, monolayer cell samples, isolated protoplasts as well as carotene crystals released from chromoplasts that are suitable for subsequent observations using light microscopy and for analysis using Raman spectroscopy.

Comparative transcriptomics and weighted gene co-expression correlation network analysis (WGCNA) reveal potential regulation mechanism of carotenoid accumulation in Chrysanthemum × morifolium.

The variation of flower color of chrysanthemum (Chrysanthemum×morifolium) is extremely rich, and carotenoids, which are mainly stored in the plastid, are important pigments that determine the color of chrysanthemum. However, the genetic regulation of the carotenoid metabolism pathway in this species still remains unclear. In this study, a pink chrysanthemum cultivar, 'Jianliuxiang Pink', and its three bud sport mutants (including white, yellow and red color mutants, 'Jianliuxiang White', 'Jianliuxiang Yellow' and 'Jianliuxiang Red', respectively) were used as experimental materials to analyze the dynamic changes of carotenoid components and plastid ultrastructure at different developmental stages of ray florets. We found that the carotenoid components and plastid ultrastructure of the four color cultivars in the early developmental stage of the chrysanthemum capitulum (S1) were almost identical, and the carotenoids mainly included violaxanthin, lutein and ß-carotene, which exist in proplastids and immature chloroplasts. With the development of capitulum, the chloroplasts in 'Jianliuxiang White' and 'Jianliuxiang Pink' were degraded, and the protoplasts did not transform but rather formed vesicles that accumulated trace amounts of carotenoids. The proplastids and chloroplasts in 'Jianliuxiang Yellow' and 'Jianliuxiang Red' were all transformed into chromoplasts and consist of lutein as well as lutein's isomer and derivatives. Using comparative transcriptomics combined with gene expression analysis, we found that CmPg-1, CmPAP10, and CmPAP13, which were involved in chromoplast transformation, CmLCYE, which was involved in carotenoid biosynthesis, and CmCCD4a-2, which was involved in carotenoid degradation, were differentially expressed between four cultivars, and these key genes therefore should affect the accumulation of carotenoids in chrysanthemum. In addition, six transcription factors, CmMYB305, CmMYB29, CmRAD3, CmbZIP61, CmAGL24, CmNAC1, were screened using weighted gene co-expression correlation network analysis (WGCNA) combined with correlative analysis to determine whether they play an important role in carotenoid accumulation by regulating structural genes related to the carotenoid metabolism pathway and plastid development. This study analyzed dynamic changes of carotenoid components and plastid ultrastructure of the four bud mutation cultivars of chrysanthemum and identified structural genes and transcription factors that may be involved in carotenoid accumulation. The above results laid a solid foundation for further analysis of the regulatory mechanism of the carotenoid biosynthesis pathway in chrysanthemum.

Different colored Chrysanthemum × morifolium cultivars represent distinct plastid transformation and carotenoid deposit patterns.

Carotenoids are the most important pigments determining the color of C. × morifolium; however, it is still unknown whether the changes of plastid ultrastructure affect carotenoids accumulation. In this study, we compared the change of carotenoid composition, content, and the plastid ultrastructures in the different developmental stages of capitulum among fourteen C. × morifolium cultivars from seven color groups. We found that the carotenoids and plastids detected at the early stage of capitulum development were similar in all cultivars, including violaxanthin, lutein, and ß-carotene, which were present in proplastids and immature chloroplasts. Immature chloroplasts were degraded completely, forming loosely broken plastids during the development of the capitulum in white and pink cultivars. Meanwhile, a number of lipid vesicles appeared at proplastids, which resulted in only trace amounts of carotenoid accumulation in these cultivars. For yellow, orange, red, and brown cultivars, a great number of chromoplasts were found, which contained diverse ultrastructures, such as plastoglobules, tubules, and lipid droplets; these chromoplasts were derived from proplastids or chloroplasts. Compared with the early stage of capitulum development, these cultivars accumulated large amounts of carotenoids, primarily including lutein, lutein derivatives, and their isomers. In green cultivars, proplastids and immature chloroplasts were completely transformed into mature chloroplasts. These chloroplasts mainly contained violaxanthin, lutein, ß-carotene, and two new components, (9Z)-lutein and (9'Z)-lutein, compared with carotenoid components presented in proplastids and immature chloroplasts. This research will be helpful for understanding the mechanisms of carotenoid metabolism of C. × morifolium. Furthermore, we found that two different chromoplast transformation patterns could be present in the same tissue cell, which contributed to the research on plastid differentiation and development in higher plants.

Control of plastidial metabolism by the Clp protease complex.

Plant metabolism is strongly dependent on plastids. Besides hosting the photosynthetic machinery, these endosymbiotic organelles synthesize starch, fatty acids, amino acids, nucleotides, tetrapyrroles, and isoprenoids. Virtually all enzymes involved in plastid-localized metabolic pathways are encoded by the nuclear genome and imported into plastids. Once there, protein quality control systems ensure proper folding of the mature forms and remove irreversibly damaged proteins. The Clp protease is the main machinery for protein degradation in the plastid stroma. Recent work has unveiled an increasing number of client proteins of this proteolytic complex in plants. Notably, a substantial proportion of these substrates are required for normal chloroplast metabolism, including enzymes involved in the production of essential tetrapyrroles and isoprenoids such as chlorophylls and carotenoids. The Clp protease complex acts in coordination with nuclear-encoded plastidial chaperones for the control of both enzyme levels and proper folding (i.e. activity). This communication involves a retrograde signaling pathway, similarly to the unfolded protein response previously characterized in mitochondria and endoplasmic reticulum. Coordinated Clp protease and chaperone activities appear to further influence other plastid processes, such as the differentiation of chloroplasts into carotenoid-accumulating chromoplasts during fruit ripening.

Study of commercial quality parameters, sugars, phenolics, carotenoids and plastids in different tomato varieties.

The aim of this study was to assess commercial quality parameters, sugars, phenolics, carotenoids and plastid in diverse and little studied tomato varieties to gain insight into their commercial and functional quality and reveal possible noticeable differences. Five cherry tomato varieties and six common (i.e., non-cherry) tomatoes were evaluated. The highest levels of lycopene were detected in 'Tigerella' and 'Byelsa', and those of phytoene in 'Orange', those of phenolics in 'Green Zebra', all of them common tomatoes. The levels of sugars in both groups of tomatoes were comparable. Interesting differences in plastid carotenoid-accumulating sub-structures as a function of the carotenoid profile were observed. Given the importance of chromoplasts in the deposition of carotenoids in plants and their release during digestion, this information can be valuable in investigations on the regulation of the biosynthesis and the bioavailability of tomato carotenoids.

Microstructural and histochemical characteristics of Lycium barbarum L. fruits used in folk herbal medicine and as functional food.

Lycium barbarum L. fruits, referred to as functional food, have long been used in traditional and folk herbal medicine due to their therapeutic properties. The fruit microstructure was analysed using light, scanning and transmission electron microscopes. The distribution of bioactive compounds in drupe tissues was assessed with histochemical and fluorescence assays. The analysis of the microstructure has shown that the fruit is covered by a skin with an amorphous cuticle and a layer of amorphous epicuticular waxes on the surface. The skin is composed of a single-layered epidermis with thickened walls and one layer of hypodermis with slightly thickened periclinal walls. The pericarp cells contain different types of chromoplasts, which most often contained exhibited reticulotubules/fibrils of carotenoid pigments and phytoferritine deposits. The results of the histochemical assays demonstrated that the secondary metabolites with high phytotherapeutic importance were located in all layers of the pericarp and seeds and, specifically, in the drupe exocarp and endocarp. The phytochemicals were represented by polysaccharides (LBP), lipid compounds (carotenoids, essential oils, sesquiterpenes, steroids), polyphenols (tannins and flavonoids), and alkaloids. This study, which is the first report of the microstructure and localisation of bioactive compounds in wolfberries, is a valuable complement of phytochemical analyses and can be helpful for enhancement of the therapeutic effect of the fruit as well as preliminary assessment of the medicinal potential in the search for new pharmaceuticals. Detailed anatomical studies are crucial for exploration of determinants of fruit quality and useful for identification of diagnostic taxonomic traits.

Non-photosynthetic plastids as hosts for metabolic engineering.

Using plants as hosts for production of complex, high-value compounds and therapeutic proteins has gained increasing momentum over the past decade. Recent advances in metabolic engineering techniques using synthetic biology have set the stage for production yields to become economically attractive, but more refined design strategies are required to increase product yields without compromising development and growth of the host system. The ability of plant cells to differentiate into various tissues in combination with a high level of cellular compartmentalization represents so far the most unexploited plant-specific resource. Plant cells contain organelles called plastids that retain their own genome, harbour unique biosynthetic pathways and differentiate into distinct plastid types upon environmental and developmental cues. Chloroplasts, the plastid type hosting the photosynthetic processes in green tissues, have proven to be suitable for high yield protein and bio-compound production. Unfortunately, chloroplast manipulation often affects photosynthetic efficiency and therefore plant fitness. In this respect, plastids of non-photosynthetic tissues, which have focused metabolisms for synthesis and storage of particular classes of compounds, might prove more suitable for engineering the production and storage of non-native metabolites without affecting plant fitness. This review provides the current state of knowledge on the molecular mechanisms involved in plastid differentiation and focuses on non-photosynthetic plastids as alternative biotechnological platforms for metabolic engineering.

Raman, AFM and SNOM high resolution imaging of carotene crystals in a model carrot cell system.

Three non-destructive and complementary techniques, Raman imaging, Atomic Force Microscopy and Scanning Near-field Optical Microscopy were used simultaneously to show for the first time chemical and structural differences of carotenoid crystals. Spectroscopic and microscopic scanning probe measurements were applied to the released crystals or to crystals accumulated in a unique, carotenoids rich callus tissue growing in vitro that is considered as a new model system for plant carotenoid research. Three distinct morphological crystal types of various carotenoid composition were identified, a needle-like, rhomboidal and helical. Raman imaging using 532 and 488 nm excitation lines provided evidence that the needle-like and rhomboidal crystals had similar carotenoid composition and that they were composed mainly of ß-carotene accompanied by α-carotene. However, the presence of α-carotene was not identified in the helical crystals, which had the characteristic spatial structure. AFM measurements of crystals identified by Raman imaging revealed the crystal topography and showed the needle-like and rhomboidal crystals were planar but they differed in all three dimensions. Combining SNOM and Raman imaging enabled indication of carotenoid rich structures and visualised their distribution in the cell. The morphology of identified subcellular structures was characteristic for crystalline, membraneous and tubular chromoplasts that are plant organelles responsible for carotenoid accumulation in cells.