Tissue-specific transcriptional regulation and metabolite accumulation in tomato (Solanum lycopersicum L.).

Tomato is an excellent model for studying fruit development, ripening, and other secondary metabolic pathways such as carotenoid biosynthetic pathway, flavonoid pathway, and many more. Tomato fruit development and ripening occurs under tight genetic control and involves the expression of thousands of genes affecting fruit quality and accumulation of pigments and metabolites. Here, we have described the development of a microarray platform that has allowed establishment of a framework for quantification of the expression of large number of genes and transcription factors possibly regulating various secondary metabolic pathways in tomato. To unravel the molecular mechanisms of fruit development and ripening, a tomato 60-mer oligonucleotide 44 K microarray along with the custom array for many genes and transcription factors was designed and validated in the fruit and leaf tissues. Comparative profiling of gene expression studies has allowed us to identify a large number of differentially expressed genes and transcription factors. Gene ontology revealed the involvement of these genes in various biological, cellular, and molecular processes like isoprenoid, terpenoid, pigment, ethylene biosynthesis, phytohormone signaling, and fruit ripening. Further, correlation, as well as differential expression studies, has revealed that several transcription factors like RIN, AGAMOUS, TAGL1, MYB, MADS-box etc. could be the possible regulators of various secondary metabolic pathways. The present study has identified various metabolites, their biosynthetic pathways and genes which may possibly be controlled by different transcription factors. The present findings have laid a base for understanding the transcriptional and metabolic shifts which occur in parallel during programmed fruit ripening and developmental processes in tomato.

Identification and in silico characterization of cis-acting elements of genes involved in carotenoid biosynthesis in tomato.

Carotenoids, the widespread and structurally diverse class of pigments, accumulate in the fruits of tomato plants in a tissue specific manner. The carotenoid biosynthetic pathway genes have been cloned and characterized in tomato and other plants, however, its regulation is still obscure. We collected and analyzed forty different accessions of tomato for the present study. HPLC analysis revealed differential accumulation of major carotenoids (lycopene and ß-carotene) in the ripe fruit tissue. In order to understand the underlying regulatory mechanisms in carotenoid biosynthesis and accumulation, we sequenced the cis-acting elements i.e. promoter, 5' and 3' untranslated regions of the carotenoid pathway genes, in all accessions, followed by their in silico validation. Major differences observed in the CAAT Box, Opaque-2 Box and L-box in the promoters of carotenoid isomerase and lycopene-beta cyclase genes, respectively, along with the variations in musashi binding element of 5' untranslated regions of the carotenoid isomerase gene, suggest their differential role in regulating the carotenogenesis process in tomato. The binding sites for various transcription factors namely RIN, AGAMOUS, CRY, RAP2.2 and PIF1 on the promoters of important carotenoid pathway genes were predicted in silico. We propose that expression of carotenoid genes and also the formation of protein product in ripe tomato fruits, is regulated efficiently by the binding of these transcription factors at selected sites in the promoter region. Finally, the differential expression of the above-mentioned genes in different developmental tissues supports the possible involvement of promoters and untranslated regions in carotenoid biosynthesis and accumulation process. The present study has generated significant information concerning regulatory players involved in the carotenoid biosynthesis in tomato.

Hydrogen peroxide signaling integrates with phytohormones during the germination of magnetoprimed tomato seeds.

Seeds of tomato were magnetoprimed at 100 mT for 30 min followed by imbibition for 12 and 24 h, respectively, at 20 °C, to examine the biochemical and molecular changes involved in homeostasis of hydrogen peroxide (H2O2) and its signaling associated with hormone interactions for promoting vigor. The relative transcript profiles of genes involved in the synthesis of H2O2 like Cu-amine oxidase (AO), receptor for activated C kinase 1 (RACK1) homologue (ArcA2) and superoxide dismutase (SOD1 and SOD9) increased in magnetoprimed tomato seeds as compared to unprimed ones with a major contribution (21.7-fold) from Cu-amine oxidase. Amongst the genes involved in the scavenging of H2O2 i.e, metallothionein (MT1, MT3 and MT4), catalase (CAT1) and ascorbate peroxidase (APX1 and APX2), MT1 and MT4 exhibited 14.4- and 15.4-fold increase respectively, in the transcript abundance, in primed seeds compared to the control. We report in our study that metallothionein and RACK1 play a vital role in the reactive oxygen species mediated signal transduction pathway to enhance the speed of germination in magnetoprimed tomato seeds. Increased enzymatic activities of catalase and ascorbate peroxidase were observed at 12 h of imbibition in the magnetoprimed seeds indicating their roles in maintaining H2O2 levels in the primed seeds. The upregulation of ABA 8'-hydroxylase and GA3 oxidase1 genes eventually, lead to the decreased abscisic acid/gibberellic acid (ABA/GA3) ratio in the primed seeds, suggesting the key role of H2O2 in enhancing the germination capacity of magnetoprimed tomato seeds.

Identification and expression profiling of miRNAs in two color variants of carrot (Daucus carota L.) using deep sequencing.

microRNAs represent small endogenous RNAs which are known to play a crucial role in various plant metabolic processes. Carrot being an important vegetable crop, represents one of the richest sources of carotenoids and anthocyanins. Most of the studies on microRNAs have been conducted in the aerial parts of the plants. However, carrot has the rare distinction of storing these compounds in roots. Therefore, carrot represents a good model system to unveil the regulatory roles of miRNAs in the underground edible part of the plant. For the first time, we report the genome wide identification and expression profiling of miRNAs in two contrasting color variants of carrot namely Orange Red and Purple Black using RNA-seq. Illumina sequencing resulted in the generation of 25.5M and 18.9M reads in Orange Red and Purple Black libraries, respectively. In total, 144 and 98 (read count >10), conserved microRNAs and 36 and 66 novel microRNAs were identified in Orange Red and Purple Black, respectively. Functional categorization and differential gene expression revealed the presence of several miRNA genes targeting various secondary metabolic pathways including carotenoid and anthocyanin biosynthetic pathways in the two libraries. 11 known and 2 novel microRNAs were further validated using Stem-Loop PCR and qRT-PCR. Also, target validation was performed for selected miRNA genes using RLM-RACE approach. The present work has laid a foundation towards understanding of various metabolic processes, particularly the color development in carrot. This information can be further employed in targeted gene expression for increasing the carotenoid and anthocyanin content in crop plants.

Five-Year Postoperative Outcomes of Bilateral Aphakia and Pseudophakia in Children up to 2 Years of Age: A Randomized Clinical Trial.

PURPOSE: Comparative evaluation of complications and visual outcomes following bilateral congenital cataract surgery in children up to 2 years of age with and without primary intraocular lens (IOL) implantation at 5 years follow-up. DESIGN: Randomized controlled clinical trial. METHODS: Sixty children (120 eyes) up to 2 years of age undergoing bilateral congenital cataract surgery were randomized to Group 1, primary aphakia (n = 30), or Group 2, primary IOL implantation (pseudophakia) (n = 30). A single surgeon performed surgeries with identical surgical technique. All patients were followed up regularly until 5 years postoperatively. At each follow-up, glaucoma, visual axis obscuration (VAO) requiring surgery, and inflammation (cell deposits, posterior synechiae) were assessed. Visual acuity was assessed until 5 years follow-up. The first operated eye was selected for statistical analysis. RESULTS: Median age of the patients at time of surgery was 5.11 months (aphakia group) and 6.01 months (pseudophakia group) (P = .56). Five years postoperatively, incidence of glaucoma was 16% and 13.8% in Groups 1 and 2 (P = .82). Incidence of posterior synechiae was significantly higher in the pseudophakia group (27.6%) compared to the aphakia group (8%) (P = .004). VAO requiring surgery was seen in 8% and 10.3% of eyes in Groups 1 and 2 (P = .76). Mean logMAR visual acuity at 5 years follow-up was 0.59 ± 0.33 and 0.5 ± 0.23 in Groups 1 and 2, respectively (P = .79). However, more eyes in the pseudophakic group started giving documentable vision earlier in their postoperative follow-ups. CONCLUSIONS: Incidence of postoperative complications was comparable between the groups, except for a higher incidence of posterior synechiae in pseudophakic eyes. Visual rehabilitation was faster in the pseudophakic group.

Evaluation of a new electronic preoperative reference marker for toric intraocular lens implantation by two different methods of analysis: Adobe Photoshop versus iTrace.

PURPOSE: The aim of this study is to compare two different methods of analysis of preoperative reference marking for toric intraocular lens (IOL) after marking with an electronic marker. SETTING/VENUE: Cataract and IOL Implantation Service, Shroff Eye Centre, New Delhi, India. PATIENTS AND METHODS: Fifty-two eyes of thirty patients planned for toric IOL implantation were included in the study. All patients had preoperative marking performed with an electronic preoperative two-step toric IOL reference marker (ASICO AE-2929). Reference marks were placed at 3-and 9-o'clock positions. Marks were analyzed with two systems. First, slit-lamp photographs taken and analyzed using Adobe Photoshop (version 7.0). Second, Tracey iTrace Visual Function Analyzer (version 5.1.1) was used for capturing corneal topograph examination and position of marks noted. Amount of alignment error was calculated. RESULTS: Mean absolute rotation error was 2.38 ± 1.78° by Photoshop and 2.87 ± 2.03° by iTrace which was not statistically significant (P = 0.215). Nearly 72.7% of eyes by Photoshop and 61.4% by iTrace had rotation error ≤3° (P = 0.359); and 90.9% of eyes by Photoshop and 81.8% by iTrace had rotation error ≤5° (P = 0.344). No significant difference in absolute amount of rotation between eyes when analyzed by either method. CONCLUSIONS: Difference in reference mark positions when analyzed by two systems suggests the presence of varying cyclotorsion at different points of time. Both analysis methods showed an approximately 3° of alignment error, which could contribute to 10% loss of astigmatic correction of toric IOL. This can be further compounded by intra-operative marking errors and final placement of IOL in the bag.

Carotenoid profiling, in silico analysis and transcript profiling of miRNAs targeting carotenoid biosynthetic pathway genes in different developmental tissues of tomato.

Carotenoid biosynthetic pathway is one of the highly significant and very well elucidated secondary metabolic pathways in plants. microRNAs are the potential regulators, widely known for playing a pivotal role in the regulation of various biological as well as metabolic processes. miRNAs may assist in the metabolic engineering of the secondary metabolites for the production of elite genotypes with increased biomass and content of various metabolites. miRNA mediated regulation of carotenoid biosynthetic genes has not been elucidated so far. To illustrate the potential regulatory role of miRNAs in carotenoid biosynthesis, transcript profiling of the known miRNAs and their possible target carotenoid genes was undertaken at eight different developmental stages of tomato, using stem-loop PCR approach combined with quantitative RT-PCR. The inter-relationship amongst carotenoid content, biosynthetic genes and miRNAs was studied in depth. Comparative expression profiles of miRNA and target genes showed variable expression in different tissues studied. The expression level of miRNAs and their target carotenoid genes displayed similar pattern in the vegetative tissues as compared to the reproductive ones, viz. fruit (different stages), indicating the possibility of regulation of carotenoid biosynthesis at various stages of fruit development. This was later confirmed by the HPLC analysis of the carotenoids. The present study has further enhanced the understanding of regulation of carotenoid biosynthetic pathway in plants. The identified miRNAs can be employed to manipulate the biosynthesis of different carotenoids, through metabolic engineering for the production of lycopene rich tomatoes.

Comparison of two different methods of preoperative marking for toric intraocular lens implantation: bubble marker versus pendulum marker.

AIM: To compare the accuracy of two different methods of preoperative marking for toric intraocular lens (IOL) implantation, bubble marker versus pendulum marker, as a means of establishing the reference point for the final alignment of the toric IOL to achieve an outcome as close as possible to emmetropia. METHODS: Toric IOLs were implanted in 180 eyes of 110 patients. One group (55 patients) had preoperative marking of both eyes done with bubble marker (ASICO AE-2791TBL) and the other group (55 patients) with pendulum marker (Rumex(®)3-193). Reference marks were placed at 3-, 6-, and 9-o'clock positions on the limbus. Slit-lamp photographs were analyzed using Adobe Photoshop (version 7.0). Amount of alignment error (in degrees) induced in each group was measured. RESULTS: Mean absolute rotation error in the preoperative marking in the horizontal axis was 2.42±1.71 in the bubble marker group and 2.83±2.31in the pendulum marker group (P=0.501). Sixty percent of the pendulum group and 70% of the bubble group had rotation error ≤3 (P=0.589), and 90% eyes of the pendulum group and 96.7% of the bubble group had rotation error ≤5 (P=0.612). CONCLUSION: Both preoperative marking techniques result in approximately 3 of alignment error. Both marking techniques are simple, predictable, reproducible and easy to perform.

Management of moderate and severe corneal astigmatism with AcrySof® toric intraocular lens implantation - Our experience.

PURPOSE: Visual performance following toric intraocular lens implantation for cataract with moderate and severe astigmatism. SETTING: Cataract services, Shroff Eye Centre, New Delhi, India. DESIGN: Case series. METHOD: This prospective study included 64 eyes of 40 patients with more than 1.50 dioptre (D) of pre-existing corneal astigmatism undergoing phacoemulsification with implantation of the AcrySof® toric IntraOcular Lens (IOL). The unaided visual acuity (UCVA), best corrected visual acuity (BCVA), residual refractive sphere and refractive cylinders were evaluated. Toric IOL axis and alignment error was measured by slit lamp method and Adobe Photoshop (version 7) method. Patient satisfaction was evaluated using a satisfaction questionnaire at 3 months. RESULTS: The mean residual refractive astigmatism was 0.57 D at the final follow-up of 3 months. Mean alignment error was 3.44 degrees (SD = 2.60) by slit lamp method and 3.88 degrees (SD = 2.86) by Photoshop method. Forty-six (71.9%) eyes showed misalignment of 5 degrees or less, and 60 (93.8%) eyes showed misalignment of 10 degrees or less. The mean log MAR UCVA at 1st post-op day was 0.172 (SD = 0.02), on 7th post-op day was 0.138 (SD = 0.11), and on 30th post-op day was 0.081 (SD = 0.11). The mean log MAR BCVA at three months was -0.04 (SD = 0.76). CONCLUSION: We believe that implantation of AcrySof® toric IOL is an effective, safe and predictable method to correct high amounts of corneal astigmatism during cataract surgery.

Development of fruit color in Solanaceae: a story of two biosynthetic pathways.

This review highlights the major differences between the regulation of two important pathways namely anthocyanin and carotenoid pathways, responsible for fruit color generation in Solanaceae mediated by transcription factors (TFs). The anthocyanin pathway is regulated by a common set of TFs (MYB, MYC and WD40) belonging to specific families of DNA-binding proteins. Their regulation is aimed at controlling the type and amount of pigments produced and the physiological conditions (like pH) at which they are finally stored. In the carotenoid pathway, the color diversity depends on the quantity of pigment produced and the point where the pathway is arrested. TFs in the latter case are accordingly found to influence the sequestration and degradation of these pigments, which determines their final concentration in the tissue. TFs (phytochrome interacting factors, MADS-BOX, HB-ZIP and B-ZIP) also regulate important rate-determining steps, which decide the direction in which the pathway proceeds and the point at which it is terminated. In the absence of a clear pattern of TF-mediated regulation, it is suggested that the carotenoid pathway is more significantly influenced by other regulatory methods which need to be explored. It is expected that common factors affecting these pathways are the ones acting much before the initiation of the biosynthesis of respective pigments.

Farnesyl pyrophosphate synthase: a key enzyme in isoprenoid biosynthetic pathway and potential molecular target for drug development.

As isoprenoid biosynthetic pathway has gained importance since last few years, key enzymes of this pathway have been characterized and their functional roles in the cell metabolism have been explored using molecular biology approaches. A key enzyme in this pathway is farnesyl pyrophosphate (EC 2.5.1.10) synthase (FPPS) which supplies precursors for the biosynthesis of essential isoprenoids like carotenoids, withanolides, ubiquinones, dolichols, sterols, among others and also helps in farnesylation and geranylation of proteins. It is a chain elongation enzyme which catalyzes head to tail condensation of two molecules of isopentenyl diphosphate with dimethylallyl diphosphate to form farnesyl pyrophosphate (FPP). Recent studies have validated FPPS as a molecular target of bisphosphonates for drug development against tumors as well as human pathogens. The present paper synthesizes the information on characterization, structural and functional relationships, evolution, localization as well as advances on FPPS enzyme as a target for drug development.

Cionni ring and in-the-bag intraocular lens implantation for subluxated lenses: a prospective case series.

PURPOSE: To report the intraoperative performance and postoperative outcomes of the Cionni-modified capsule tension ring (CTR) implantation in eyes with subluxated lenses. DESIGN: Prospective, observational case series. METHODS: This study was carried out at Iladevi Cataract & IOL Research Centre, Ahmedabad, India. The study population comprised 41 eyes with subluxated lenses that underwent lens extraction, capsular bag fixation with modified CTR, and in-the-bag single-piece AcrySof intraocular lens (IOL) implantation. Main outcome measures were intraoperative performance and postoperative best-corrected visual acuity (BCVA), IOL centration, and complications. RESULTS: The mean age was 29.46 ± 16.16 years (3-68 years). Mean extent of subluxation was 6.1 ± 1.0 clock hours. Preoperatively, vitreous was detected in the anterior chambers of 5 eyes (12.2%). Two-port anterior chamber vitrectomy was performed in 2 eyes. Mean follow-up was 45.8 ± 2.9 months. Mean preoperative BCVA was 0.66 ± 0.22 logMAR (35 eyes). Mean postoperative BCVA at final follow-up was 0.33 ± 0.21 logMAR (41 eyes) (P < .001). BCVA improved in 35 eyes (85.4%) at the last follow-up. In 3 eyes (7.3%) IOL decentration was noted and repositioning was required in 2 eyes. Posterior capsule opacification developed in 14 eyes (34.2%); 12 eyes (29.3%) required Nd:YAG capsulotomy. Other complications included cystoid macular edema in 1 eye (2.4%), posterior synechiae in 1 eye (2.4%), and retinal detachment in 1 eye (2.4%). CONCLUSION: In-the-bag implantation of a Cionni-modified CTR with IOL appears to be a safe option in eyes with subluxated cataract, ensuring a stable IOL with few complications.

Intraoperative posterior capsule flutter in posterior lentiglobus.

Posterior capsules of pediatric eyes with lens disorders have complex morphological variations with differing etiologies and visual prognoses. We describe a unique case of intraoperative posterior capsule flutter in an eye of a child undergoing cataract surgery, which we interpret as a possible expression of posterior lentiglobus.

DisCoVisc versus the soft-shell technique using Viscoat and Provisc in phacoemulsification: randomized clinical trial.

PURPOSE: To compare the effects and outcomes of DisCoVisc (hyaluronic acid 1.6%-chondroitin sulfate 4.0%) with those of the soft-shell technique using Viscoat (sodium hyaluronate 3.0%-chondroitin sulfate 4.0%) and Provisc (sodium hyaluronate 1.0%) in phacoemulsification. SETTING: Iladevi Cataract & IOL Research Centre, Ahmedabad, India. METHODS: This prospective randomized clinical trial comprised 100 eyes having phacoemulsification by the same surgeon using a standardized technique. Eyes were randomly assigned to DisCoVisc (Group 1) or Viscoat and Provisc (Group 2). Preoperative and postoperative examinations included absolute change in pachymetry, percentage difference in endothelial cell density (ECD) and coefficient of variation (CV), and anterior segment inflammation. RESULTS: The mean postoperative central corneal thickness (CCT) in Group 1 and Group 2 was 590.96+/-46.05 microm and 586.94+/-50.57 microm, respectively, at 1 day; 554.14+/-35.45 microm and 551.65+/-37.69 microm, respectively, at 7 days; and 533.74+/-29.12 microm and 536.44+/-35.59 microm, respectively, at 1 month. The between-group differences in CCT were not statistically significant. At 3 months, the mean ECD was 2427.06+/-243.26 cells/mm2 and 2475.30+/-222.83 cells/mm2, respectively, and the mean CV, 42.38+/-7.94 cells/mm2 and 41.66+/-7.71 cells/mm2, respectively. There was no significant difference in the mean ECD between preoperatively and 3 months postoperatively or in corneal thickness between preoperatively and 1, 7, and 30 days postoperatively. CONCLUSION: A single injection of DisCoVisc was effective, and its postoperative outcomes were comparable to those of combined Viscoat and Provisc.

Subtle signs of anterior vitreous face disturbance during posterior capsulorhexis in pediatric cataract surgery.

In 4 patients with congenital cataract, subtle signs of anterior vitreous face (AVF) disturbance were documented during posterior continuous curvilinear capsulorhexis. The signs were vitreous strands in the anterior chamber, vitreous strands attached to the capsule flap, and distortion of the anterior and posterior capsulorhexis margins; the latter is considered a pathognomonic sign of AVF disturbance. As a result of the early recognition, modifications to the cataract surgery technique were made.