Increasing Heavy Metal Tolerance by the Exogenous Application of Organic Acids.

Several metals belong to a group of non-biodegradable inorganic constituents that, at low concentrations, play fundamental roles as essential micronutrients for the growth and development of plants. However, in high concentrations they can have toxic and/or mutagenic effects, which can be counteracted by natural chemical compounds called chelators. Chelators have a diversity of chemical structures; many are organic acids, including carboxylic acids and cyclic phenolic acids. The exogenous application of such compounds is a non-genetic approach, which is proving to be a successful strategy to reduce damage caused by heavy metal toxicity. In this review, we will present the latest literature on the exogenous addition of both carboxylic acids, including the Kreb's Cycle intermediates citric and malic acid, as well as oxalic acid, lipoic acid, and phenolic acids (gallic and caffeic acid). The use of two non-traditional organic acids, the phytohormones jasmonic and salicylic acids, is also discussed. We place particular emphasis on physiological and molecular responses, and their impact in increasing heavy metal tolerance, especially in crop species.

Carrot DcALFIN4 and DcALFIN7 Transcription Factors Boost Carotenoid Levels and Participate Differentially in Salt Stress Tolerance When Expressed in Arabidopsis thaliana and Actinidia deliciosa.

ALFIN-like transcription factors (ALs) are involved in several physiological processes such as seed germination, root development and abiotic stress responses in plants. In carrot (Daucus carota), the expression of DcPSY2, a gene encoding phytoene synthase required for carotenoid biosynthesis, is induced after salt and abscisic acid (ABA) treatment. Interestingly, the DcPSY2 promoter contains multiple ALFIN response elements. By in silico analysis, we identified two putative genes with the molecular characteristics of ALs, DcAL4 and DcAL7, in the carrot transcriptome. These genes encode nuclear proteins that transactivate reporter genes and bind to the carrot DcPSY2 promoter in yeast. The expression of both genes is induced in carrot under salt stress, especially DcAL4 which also responds to ABA treatment. Transgenic homozygous T3 Arabidopsis thaliana lines that stably express DcAL4 and DcAL7 show a higher survival rate with respect to control plants after chronic salt stress. Of note is that DcAL4 lines present a better performance in salt treatments, correlating with the expression level of DcAL4, AtPSY and AtDXR and an increase in carotenoid and chlorophyll contents. Likewise, DcAL4 transgenic kiwi (Actinidia deliciosa) lines show increased carotenoid and chlorophyll content and higher survival rate compared to control plants after chronic salt treatment. Therefore, DcAL4 and DcAL7 encode functional transcription factors, while ectopic expression of DcAL4 provides increased tolerance to salinity in Arabidopsis and Kiwi plants.

Unraveling the induction of phytoene synthase 2 expression by salt stress and abscisic acid in Daucus carota.

Phytoene synthase (PSY) is the first committed enzyme of the carotenoid biosynthesis pathway and the most important point of regulation. Carotenoids are precursors of abscisic acid (ABA), which mediates abiotic stress tolerance responses in plants. ABA activates the synthesis of its own precursors through induction of PSY expression. Carrot, a species that accumulates very high amounts of carotenoids in its reserve root, has two PSY paralog genes that are expressed differentially in the root. Here, we determined that DcPSY2 expression is induced by salt stress and ABA. A DcPSY2 promoter fragment was obtained and characterized. Bioinformatic analysis showed the presence of three ABA responsive elements (ABREs). Through overexpressing pPSY2:GFP in Nicotiana tabacum we determined that all three ABREs are necessary for the ABA response. In the carrot transcriptome, we identified three ABRE binding protein (DcAREB) transcription factor candidates that localized in the nucleus, but only one, DcAREB3, was induced under ABA treatment in carrot roots. We found that AREB transcription factors bind to the carrot DcPSY2 promoter and transactivate the expression of reporter genes. We conclude that DcPSY2 is involved in ABA-mediated salt stress tolerance in carrot through the binding of AREB transcription factors to its promoter.

Abnormal glycosphingolipid mannosylation triggers salicylic acid-mediated responses in Arabidopsis.

The Arabidopsis thaliana protein GOLGI-LOCALIZED NUCLEOTIDE SUGAR TRANSPORTER (GONST1) has been previously identified as a GDP-d-mannose transporter. It has been hypothesized that GONST1 provides precursors for the synthesis of cell wall polysaccharides, such as glucomannan. Here, we show that in vitro GONST1 can transport all four plant GDP-sugars. However, gonst1 mutants have no reduction in glucomannan quantity and show no detectable alterations in other cell wall polysaccharides. By contrast, we show that a class of glycosylated sphingolipids (glycosylinositol phosphoceramides [GIPCs]) contains Man and that this mannosylation is affected in gonst1. GONST1 therefore is a Golgi GDP-sugar transporter that specifically supplies GDP-Man to the Golgi lumen for GIPC synthesis. gonst1 plants have a dwarfed phenotype and a constitutive hypersensitive response with elevated salicylic acid levels. This suggests an unexpected role for GIPC sugar decorations in sphingolipid function and plant defense signaling. Additionally, we discuss these data in the context of substrate channeling within the Golgi.

Light-dependent changes in plastid differentiation influence carotenoid gene expression and accumulation in carrot roots.

Carrot is an important nutritional crop due to the high levels of pro-vitamin A carotenoids (ß-carotene and, to a lower extent, α-carotene) that accumulate in its storage root during secondary growth. In this work we show that in carrots, contrary to that reported for aerial organs of other plant species, light has a profound effect on root development by inhibiting root thickening, preventing the differentiation of chromoplasts and eventually repressing the expression of most genes required for the biosynthesis of ß-carotene and α-carotene and to a lesser extent genes for xanthophylls and apocarotenoids biosynthesis. We observed a correlation in the carotenoid profile and the patterns of gene expression during the development of root segments grown either in the light or in the dark, which suggests a transcriptional regulation for carotenoid synthesis during carrot root development. Furthermore, our work supports the conclusion that the differentiation of chromoplasts coincides with carotenoid accumulation during the later stages of development of underground storage roots.

Phylogenetic and biochemical evidence supports the recruitment of an ADP-glucose translocator for the export of photosynthate during plastid endosymbiosis.

The acquisition of photosynthesis by eukaryotic cells through enslavement of a cyanobacterium represents one of the most remarkable turning points in the history of life on Earth. In addition to endosymbiotic gene transfer, the acquisition of a protein import apparatus and the coordination of gene expression between host and endosymbiont genomes, the establishment of a metabolic connection was crucial for a functional endosymbiosis. It was previously hypothesized that the first metabolic connection between both partners of endosymbiosis was achieved through insertion of a host-derived metabolite transporter into the cyanobacterial plasma membrane. Reconstruction of starch metabolism in the common ancestor of photosynthetic eukaryotes suggested that adenosine diphosphoglucose (ADP-Glc), a bacterial-specific metabolite, was likely to be the photosynthate, which was exported from the early cyanobiont. However, extant plastid transporters that have evolved from host-derived endomembrane transporters do not transport ADP-Glc but simple phosphorylated sugars in exchange for orthophosphate. We now show that those eukaryotic nucleotide sugar transporters, which define the closest relatives to the common ancestor of extant plastid envelope carbon translocators, possess an innate ability for transporting ADP-Glc. Such an unexpected ability would have been required to establish plastid endosymbiosis.

Abiotic Stress in Crop Species: Improving Tolerance by Applying Plant Metabolites.

Reductions in crop yields brought about by abiotic stress are expected to increase as climate change, and other factors, generate harsher environmental conditions in regions traditionally used for cultivation. Although breeding and genetically modified and edited organisms have generated many varieties with greater abiotic stress tolerance, their practical use depends on lengthy processes, such as biological cycles and legal aspects. On the other hand, a non-genetic approach to improve crop yield in stress conditions involves the exogenous application of natural compounds, including plant metabolites. In this review, we examine the recent literature related to the application of different natural primary (proline, l-tryptophan, glutathione, and citric acid) and secondary (polyols, ascorbic acid, lipoic acid, glycine betaine, α-tocopherol, and melatonin) plant metabolites in improving tolerance to abiotic stress. We focus on drought, saline, heavy metal, and temperature as environmental parameters that are forecast to become more extreme or frequent as the climate continues to alter. The benefits of such applications are often evaluated by measuring their effects on metabolic, biochemical, and morphological parameters in a variety of crop plants, which usually result in improved yields when applied in greenhouse conditions or in the field. As this strategy has proven to be an effective way to raise plant tolerance to abiotic stress, we also discuss the prospect of its widespread implementation in the short term.

Daucus carota DcPSY2 and DcLCYB1 as Tools for Carotenoid Metabolic Engineering to Improve the Nutritional Value of Fruits.

Carotenoids are pigments with important nutritional value in the human diet. As antioxidant molecules, they act as scavengers of free radicals enhancing immunity and preventing cancer and cardiovascular diseases. Moreover, α-carotene and ß-carotene, the main carotenoids of carrots (Daucus carota) are precursors of vitamin A, whose deficiency in the diet can trigger night blindness and macular degeneration. With the aim of increasing the carotenoid content in fruit flesh, three key genes of the carotenoid pathway, phytoene synthase (DcPSY2) and lycopene cyclase (DcLCYB1) from carrots, and carotene desaturase (XdCrtI) from the yeast Xanthophyllomyces dendrorhous, were optimized for expression in apple and cloned under the Solanum chilense (tomatillo) polygalacturonase (PG) fruit specific promoter. A biotechnological platform was generated and functionally tested by subcellular localization, and single, double and triple combinations were both stably transformed in tomatoes (Solanum lycopersicum var. Microtom) and transiently transformed in Fuji apple fruit flesh (Malus domestica). We demonstrated the functionality of the S. chilense PG promoter by directing the expression of the transgenes specifically to fruits. Transgenic tomato fruits expressing DcPSY2, DcLCYB1, and DcPSY2-XdCRTI, produced 1.34, 2.0, and 1.99-fold more total carotenoids than wild-type fruits, respectively. Furthermore, transgenic tomatoes expressing DcLCYB1, DcPSY2-XdCRTI, and DcPSY2-XdCRTI-DcLCYB1 exhibited an increment in ß-carotene levels of 2.5, 3.0, and 2.57-fold in comparison with wild-type fruits, respectively. Additionally, Fuji apple flesh agroinfiltrated with DcPSY2 and DcLCYB1 constructs showed a significant increase of 2.75 and 3.11-fold in total carotenoids and 5.11 and 5.84-fold in ß-carotene, respectively whereas the expression of DcPSY2-XdCRTI and DcPSY2-XdCRTI-DcLCYB1 generated lower, but significant changes in the carotenoid profile of infiltrated apple flesh. The results in apple demonstrate that DcPSY2 and DcLCYB1 are suitable biotechnological genes to increase the carotenoid content in fruits of species with reduced amounts of these pigments.

Solanum lycopersicum (tomato) possesses mitochondrial and plastidial lipoyl synthases capable of increasing lipoylation levels when expressed in bacteria.

Lipoic acid (LA) and its reduced form (dihydrolipoic acid, DHLA) have unique antioxidant properties among such molecules. Moreover, after a process termed lipoylation, LA is an essential prosthetic group covalently-attached to several key multi-subunit enzymatic complexes involved in primary metabolism, including E2 subunits of pyruvate dehydrogenase (PDH). The metabolic pathway of lipoylation has been extensively studied in Escherichia coli and Arabidopsis thaliana in which protein modification occurs via two routes: de novo synthesis and salvage. Common to both pathways, lipoyl synthase (LIP1 in plants, LipA in bacteria, EC 2.8.1.8) inserts sulphur atoms into the molecule in a final, activating step. However, despite the detection of LA and DHLA in other plant species, including tomato (Solanum lycopersicum), no plant LIP1s have been characterised to date from species other than Arabidopsis. In this work, we present the identification and characterisation of two LIPs from tomato, SlLIP1 and SlLIP1p. Consistent with in silico data, both are widely-expressed, particularly in reproductive organs. In line with bioinformatic predictions, we determine that yellow fluorescent protein tagged versions of SlLIP1 and SlLIP1p are mitochondrially- and plastidially-localised, respectively. Both possess the molecular hallmarks and domains of well-characterised bacterial LipAs. When heterologously-expressed in an E. coli lipA mutant, both are capable of complementing specific growth phenotypes and increasing lipoylation levels of E2 subunits of PDH in vivo, demonstrating that they do indeed function as lipoyl synthases.

Understanding Alstroemeria pallida Flower Colour: Links between Phenotype, Anthocyanins and Gene Expression.

Flower colour is mainly due to the accumulation of flavonoids, carotenoids and betalains in the petals. Of these pigments, flavonoids are responsible for a wide variety of colours ranging from pale yellow (flavones, flavonols and flavanodiols) to blue-violet (anthocyanins). This character plays a crucial ecological role by attracting and guiding pollinators. Moreover, in the ornamental plants market, colour has been consistently identified as the main feature chosen by consumers when buying flowers. Considering the importance of this character, the aim of this study was to evaluate flower colour in the native Chilean geophyte Alstroemeria pallida, by using three different approaches. Firstly, the phenotype was assessed using both a colour chart and a colourimeter, obtaining CIELab parameters. Secondly, the anthocyanin content of the pigmented tepals was evaluated by high-performance liquid chromatography (HPLC), and finally, the expression of two key flavonoid genes, chalcone synthase (CHS) and anthocyanidin synthase (ANS) was analysed using real-time polymerase chain reaction (PCR). Visual evaluation of A. pallida flower colour identified 5 accessions, ranging from white (Royal Horticultural Society (RHS) N999D) to pink (RHS 68C). Moreover, this visual evaluation of the accessions correlated highly with the CIELab parameters obtained by colourimetry. An anthocyanidin corresponding to a putative 6-hydroxycyanidin was identified, which was least abundant in the white accession (RHS N999D). Although CHS was not expressed differentially between the accessions, the expression of ANS was significantly higher in the accession with pink flowers (RHS 68C). These results suggest a correlation between phenotype, anthocyanin content and ANS expression for determining flower colour of A. pallida, which could be of interest for further studies, especially those related to the breeding of this species with ornamental value.

Daucus carota as a novel model to evaluate the effect of light on carotenogenic gene expression.

Carotenoids are synthesized in prokaryotic and eukaryotic organisms. In plants and algae, these lipophilic molecules possess antioxidant properties acting as reactive oxygen species scavengers and exert functional roles in hormone synthesis, photosynthesis, photomorphogenesis and in photoprotection. During the past decade almost all carotenogenic genes have been identified as a result of molecular, genetic and biochemical approaches utilizing Arabidopsis thaliana as the model system. Studies carried out in leaves and fruits of A. thaliana and tomato determined that light regulates carotenoid biosynthesis preferentially through the modulation of carotenogenic gene transcription. In this work we showed for the first time that light induces accumulation of psy1, pds and zds2 transcripts in leaves of Daucus carota (carrot), a novel plant model. In addition, modified roots of carrots exposed to light accumulate zds1, whereas the pds gene is highly repressed, suggesting that some carotenogenic genes, which are expressed in roots, are regulated by light. Additionally, light negatively regulates the development of the modified carrot root in a reversible manner. Therefore, this suggests that light affects normal growth and carotenogenic gene expression in the modified root of carrot plants. The molecular insight gained into the light-regulated expression of carotenoid genes in this and other model systems will facilitate our understanding of the regulation of carotenoid biosynthesis to improve the prospects for the metabolic engineering of carotenoid production in plants.

Identification of a type II cystatin in Fragaria chiloensis: A proteinase inhibitor differentially regulated during achene development and in response to biotic stress-related stimuli.

The equilibrium between protein synthesis and degradation is key to maintaining efficiency in different physiological processes. The proteinase inhibitor cystatin regulates protease activities in different developmental and physiological contexts. Here we describe for the first time the identification and the biological function of the cysteine protease inhibitor cystatin of Fragaria chiloensis, FchCYS1. Based on primary sequence and 3D-structural homology modelling, FchCYS1 is a type II phytocystatin with high identity to other cystatins of the Fragaria genus. Both the papain-like and the legumain-like protease inhibitory domains are indeed functional, based on in vitro assays performed with Escherichia coli protein extracts containing recombinant FchCYS1. FchCYS1 is differentially-expressed in achenes of F. chiloensis fruits, with highest expression as the fruit reaches the ripened stage, suggesting a role in preventing degradation of storage proteins that will nourish the embryo during seed germination. Furthermore, FchCYS1 responds transcriptionally to the application of salicylic acid and to mechanical injury, strongly suggesting that FchCYS1 could be involved in the response against pathogen attack. Overall these results point to a role for FchCYS1 in diverse physiological processes in F. chiloensis.

Structural Modifications of Fructans in Aloe barbadensis Miller (Aloe Vera) Grown under Water Stress.

Aloe barbadensis Miller (Aloe vera) has a Crassulaceae acid metabolism which grants the plant great tolerance to water restrictions. Carbohydrates such as acemannans and fructans are among the molecules responsible for tolerating water deficit in other plant species. Nevertheless, fructans, which are prebiotic compounds, have not been described nor studied in Aloe vera, whose leaf gel is known to possess beneficial pharmaceutical, nutritional and cosmetic properties. As Aloe vera is frequently cultivated in semi-arid conditions, like those found in northern Chile, we investigated the effect of water deficit on fructan composition and structure. For this, plants were subjected to different irrigation regimes of 100%, 75%, 50% and 25% field capacity (FC). There was a significant increase in the total sugars, soluble sugars and oligo and polyfructans in plants subjected to water deficit, compared to the control condition (100% FC) in both leaf tips and bases. The amounts of fructans were also greater in the bases compared to the leaf tips in all water treatments. Fructans also increase in degree of polymerization with increasing water deficit. Glycosidic linkage analyses by GC-MS, led to the conclusion that there are structural differences between the fructans present in the leaves of control plants with respect to plants irrigated with 50% and 25% FC. Therefore, in non-stressed plants, the inulin, neo-inulin and neo-levan type of fructans predominate, while in the most stressful conditions for the plant, Aloe vera also synthesizes fructans with a more branched structure, the neofructans. To our knowledge, the synthesis and the protective role of neo-fructans under extreme water deficit has not been previously reported.

Exogenous application of pectin-derived oligosaccharides to grape berries modifies anthocyanin accumulation, composition and gene expression.

Anthocyanins are secondary metabolites synthesized in grape berry skins via the phenylpropanoid pathway, with functions ranging from skin coloration to protection against pathogens or UV light. Accumulation of these compounds is highly variable depending on genetics, environmental factors and viticultural practices. Besides their biological functions, anthocyanins improve wine quality, as a high anthocyanin content in berries has a positive impact on the color, total phenolic concentration and, ultimately, the price of wine. The present work studies the effect of the pre-veraison application of pectin derived oligosaccharides (PDO) on the synthesis and accumulation of these compounds, and associates the changes observed with the expression of key genes in the phenylpropanoid pathways. To this end, pre-veraison Cabernet Sauvignon bunches were treated with PDO to subsequently determine total anthocyanin content, the anthocyanin profile (by HPLC-DAD) and gene expression (by qRT-PCR), using Ethrel and water treatments for comparison. The results show that PDO were as efficient as Ethrel in generating a significant rise in total anthocyanin content at 30 days after treatment (dat), compared with water treatments (1.32, 1.48 and 1.02 mg e.Mv-3G/g FW respectively) without any undesirable effect on berry size, soluble solids, tartaric acid concentration or pH. In addition, a significant alteration in the anthocyanin profile was observed. Specifically, a significant increase in the relative concentration of malvidin was observed for both PDO and Ethrel treatments, compared with water controls (52.8; 55.0 and 48.3%, respectively), with a significant rise in tri-hydroxylated forms and a fall in di-hydroxylated anthocyanins. The results of gene expression analyses suggest that the increment in total anthocyanin content is related to a short term increase in phenylalanine ammonia-lyase (PAL) expression, mediated by a decrease in MYB4A expression. A longer term increase in UDP-glucose flavonoid 3-O-glucosyltransferase (UFGT) expression, probably mediated by a rise in MYBA1 was also observed. Regarding the anthocyanin profile, despite the increase observed in MYB5A expression in PDO and Ethrel treatments, no changes in flavonoid 3'-hydroxylase (F-3'-H); flavonoid 3'5'-hydroxylase (F-3'5'-H) or O-methyltransferase (OMT) could be related with the profile modifications described. Overall, this study highlights that application of PDO is a novel means of altering specific grape berry anthocyanins, and could be a means of positively influencing wine quality without the addition of agrochemicals.

FcLDP1, a Gene Encoding a Late Embryogenesis Abundant (LEA) Domain Protein, Responds to Brassinosteroids and Abscisic Acid during the Development of Fruits in Fragaria chiloensis.

White Chilean strawberries (Fragaria chiloensis) are non-climacteric fruits, with an exotic color and aroma. In order to discover genes involved in the development of these fruits, we identified a fragment of a gene encoding a late embryogenesis abundant domain protein, FcLDP1, that was expressed in early stages of fruit development, particularly in receptacles. Hormones play key roles in regulating the development of non-climacteric fruits. We show that the brassinosteroid content of the white strawberry varies during development. Additionally, FcLDP1 as well as the closest ortholog in the woodland strawberry, F. vesca (FvLDP1) possess multiple brassinosteroid, as well as abscisic acid (ABA) response motifs in the promoter region, consistent with the response of transiently expressed FcLDP1 promoter-GFP fusions to these hormones, and the rise in FcLDP1 transcript levels in white strawberry fruits treated with brassinosteroids or ABA. These findings suggest that both hormones regulate FcLDP1 expression during the development of white strawberries.

Boron stress response and accumulation potential of the extremely tolerant species Puccinellia frigida.

Phytoremediation is a promising technology to tackle boron toxicity, which restricts agricultural activities in many arid and semi-arid areas. Puccinellia frigida is a perennial grass that was reported to hyperaccumulate boron in extremely boron-contaminated sites. To further investigate its potential for phytoremediation, we determined its response to boron stress under controlled conditions (hydroponic culture). Also, as a first step towards understanding the mechanisms underlying its extreme tolerance, we evaluated the presence and expression of genes related with boron tolerance. We found that P. frigida grew normally even at highly toxic boron concentrations in the medium (500mg/L), and within its tissues (>5000mg/kg DW). We postulate that the strategies conferring this extreme tolerance involve both restricting boron accumulation and an internal tolerance mechanism; this is consistent with the identification of putative genes involved in both mechanisms, including the expression of a possible boron efflux transporter. We also found that P. frigida hyperaccumulated boron over a wide range of boron concentrations. We propose that P. frigida could be used for boron phytoremediation strategies in places with different soil characteristics and boron concentrations. Further studies should pave the way for the development of clean and low-cost solutions to boron toxicity problems.

VvGONST-A and VvGONST-B are Golgi-localised GDP-sugar transporters in grapevine (Vitis vinifera L.).

Plant nucleotide-sugar transporters (NSTs) are responsible for the import of nucleotide-sugar substrates into the Golgi lumen, for subsequent use in glycosylation reactions. NSTs are specific for either GDP- or UDP-sugars, and almost all transporters studied to date have been isolated from Arabidopsis thaliana L. In order to determine the conservation of the import mechanism in other higher plant species, here we report the identification and characterisation of VvGONST-A and VvGONST-B from grapevine (Vitis vinifera L. cv. Thompson Seedless), which are the orthologues of the GDP-sugar transporters GONST3 and GONST4 in Arabidopsis. Both grapevine NSTs possess the molecular features characteristic of GDP-sugar transporters, including a GDP-binding domain (GXL/VNK) towards the C-terminal. VvGONST-A and VvGONST-B expression is highest at berry setting and decreases throughout berry development and ripening. Moreover, we show using green fluorescent protein (GFP) tagged versions and brefeldin A treatments, that both are localised in the Golgi apparatus. Additionally, in vitro transport assays after expression of both NSTs in tobacco leaves indicate that VvGONST-A and VvGONST-B are capable of transporting GDP-mannose and GDP-glucose, respectively, but not a range of other UDP- and GDP-sugars. The possible functions of these NSTs in glucomannan synthesis and/or glycosylation of sphingolipids are discussed.

Polyol specificity of recombinant Arabidopsis thaliana sorbitol dehydrogenase studied by enzyme kinetics and in silico modeling.

Polyols are enzymatically-produced plant compounds which can act as compatible solutes during periods of abiotic stress. Nicotinamide adenine dinucleotide(+)-dependent SORBITOL DEHYDROGENASE (SDH, E. C. 1.1.1.14) from Arabidopsis thaliana L. sorbitol dehydrogenase (AtSDH) is capable of oxidizing several polyols including sorbitol, ribitol, and xylitol. In the present study, enzymatic assays using recombinant AtSDH demonstrated a higher specificity constant for xylitol compared to sorbitol and ribitol, all of which are C2 (S) and C4 (R) polyols. Enzyme activity was reduced by preincubation with ethylenediaminetetraacetic acid, indicating a requirement for zinc ions. In humans, it has been proposed that sorbitol becomes part of a pentahedric coordination sphere of the catalytic zinc during the reaction mechanism. In order to determine the validity of this pentahedric coordination model in a plant SDH, homology modeling, and Molecular Dynamics simulations of AtSDH ternary complexes with the three polyols were performed using crystal structures of human and Bemisia argentifolii (Genn.) (Hemiptera: Aleyrodidae) SDHs as scaffolds. The results indicate that the differences in interaction with structural water molecules correlate very well with the observed enzymatic parameters, validate the proposed pentahedric coordination of the catalytic zinc ion in a plant SDH, and provide an explanation for why AtSDH shows a preference for polyols with a chirality of C2 (S) and C4 (R).

Levels of lycopene ß-cyclase 1 modulate carotenoid gene expression and accumulation in Daucus carota.

Plant carotenoids are synthesized and accumulated in plastids through a highly regulated pathway. Lycopene ß-cyclase (LCYB) is a key enzyme involved directly in the synthesis of α-carotene and ß-carotene through the cyclization of lycopene. Carotenoids are produced in both carrot (Daucus carota) leaves and reserve roots, and high amounts of α-carotene and ß-carotene accumulate in the latter. In some plant models, the presence of different isoforms of carotenogenic genes is associated with an organ-specific function. D. carota harbors two Lcyb genes, of which DcLcyb1 is expressed in leaves and storage roots during carrot development, correlating with an increase in carotenoid levels. In this work, we show that DcLCYB1 is localized in the plastid and that it is a functional enzyme, as demonstrated by heterologous complementation in Escherichia coli and over expression and post transcriptional gene silencing in carrot. Transgenic plants with higher or reduced levels of DcLcyb1 had incremented or reduced levels of chlorophyll, total carotenoids and ß-carotene in leaves and in the storage roots, respectively. In addition, changes in the expression of DcLcyb1 are accompanied by a modulation in the expression of key endogenous carotenogenic genes. Our results indicate that DcLcyb1 does not possess an organ specific function and modulate carotenoid gene expression and accumulation in carrot leaves and storage roots.

Sorbitol dehydrogenase is a cytosolic protein required for sorbitol metabolism in Arabidopsis thaliana.

Sorbitol is converted to fructose in Rosaceae species by SORBITOL DEHYDROGENASE (SDH, EC 1.1.1.14), especially in sink organs. SDH has also been found in non-Rosaceae species and here we show that the protein encoded by At5g51970 in Arabidopsis thaliana (L.) Heynh. possesses the molecular characteristics of an SDH. Using a green fluorescent protein-tagged version and anti-SDH antisera, we determined that SDH is cytosolically localized, consistent with bioinformatic predictions. We also show that SDH is widely expressed, and that SDH protein accumulates in both source and sink organs. In the presence of NAD+, recombinant SDH exhibited greatest oxidative activity with sorbitol, ribitol and xylitol as substrates; other sugar alcohols were oxidized to a lesser extent. Under standard growth conditions, three independent sdh- mutants developed as wild-type. Nevertheless, all three exhibited reduced dry weight and primary root length compared to wild-type when grown in the presence of sorbitol. Additionally, under short-day conditions, the mutants were more resistant to dehydration stress, as shown by a reduced loss of leaf water content when watering was withheld, and a greater survival rate on re-watering. This evidence suggests that limitations in the metabolism of sugar alcohols alter the growth of Arabidopsis and its response to drought.