These deficiencies arise due to a wide range of causes including biological factors such as decreased appetite, decreased smell, and decreased taste and behavioral factors such as difficulty cooking, difficulty obtaining ingredients, and difficulty following dietary regimens. There is a general recommendation that anyone following a plant-based diet should supplement vitamin B12 in order to prevent deficiency. Safe levels of vitamin B12 in patients on hemodialysis have yet to be determined, but regular monitoring of patients who take vitamin B12 supplements can help identify potential adverse effects. Particularly, patients on dialysis are at risk of vitamin K deficiency, which has been associated with vascular calcification, bleeding risk, and cardiovascular risk in the general population and patients on dialysis. Vitamin K is mainly present in green vegetables such as spinach, kale, Brussels sprouts, and broccoli, so dietary regimens that reduce consumption of plant foods may contribute to the development or worsening of vitamin K deficiency. It is important to know that patients on warfarin should not be deprived of vitamin K-rich vegetables. Expert guidelines recommend a stable intake of vitamin K-rich foods, which is often misinterpreted as “no K-rich food”.One of the biggest concerns when transitioning a patient on hemodialysis to a plant-based diet is the risk for hyperkalemia. It is thus recommended that patients on dialysis should be placed on a potassium-restricted diet to minimize the risk of hyperkalemia, hydroponic nft which often results in the omissions of plant-based foods.
However, many of the case reports that link hyperkalemic events to foods were the result of the consumption of a juice, sauce, or dried fruit . These foods, in particular, are all in formats that concentrate the potassium content of the food when compared to its whole-food form and facilitate a higher rate of potassium ingestion. The whole-food source is less densely composed of potassium and limits total potassium consumption. Further, fiber increases fecal bulk, facilitating excretion of potassium with bowel movements, potentially tempering a rise in serum potassium. Both juices and sauces may be prepared in forms of limited or no fiber, which may facilitate hyperkalemia. In observational studies, little to no association has been seen between dietary potassium and serum potassium among patients on dialysis. The balancewise cross-sectional study compared dietary potassium intake with serum potassium levels and found no association between the 2. Another study found that reported dietary potassium may only explain 2% of the variance in mean predialysis potassium levels.In regard to the consumption of plant foods by patients on dialysis and hyperkalemia, Saglimbene et al. conducted a prospective observational study and stratified participants into tertiles based on servings of fruit and vegetable intake per week. The tertiles corresponded to 0–5.5 , 5.6–10 , and >10 servings of fruits and vegetables per week. The predialysis potassium levels for each tertile, respectively, were 5.1, 5.0, and 5.0, showing very similar potassium levels across a wide range of plant intake. In another study by Wu et al. , potassium levels among vegetarian hemodialysis patients and nonvegetarian hemodialysis patients were not significantly different. Gonzalez-Ortiz et al. also conducted a prospective study on patients undergoing hemodialysis and found that a higher healthy plant-based diet score was not associated with the odds of hyperkalemia . The overall lack of randomized control trials in hemodialysis patients limits conclusive statements regarding potassium restriction in patients on dialysis.
As such, hyperkalemia remains a concern in the management of any patient on hemodialysis, regardless of their dietary choices. Nonetheless, studies on patients undergoing hemodialysis while eating plant-based diets have not shown increased risks of hyperkalemia. More prospective studies need to be conducted to draw more definitive conclusions on hyperkalemia and plant-based diets in hemodialysis.Another concern with a plant-based diet is the quality and quantity of protein intake. The recently released National Kidney Foundation’s Kidney Disease Outcome Quality Initiative Clinical Practice Guideline for Nutrition in CKD recommends a protein intake 1.0–1.2 g/kg of body weight per day in patients on maintenance dialysis who are metabolically stable. Patients on dialysis meet or exceed these requirements when consuming a plant-based diet without signs of under nutrition. In 2 studies, patients on dialysis consuming a vegetarian diet were shown to consume 1.2–1.25 g/kg/day. Plant and animal protein sources are composed of different proportions of amino acids and have varying availability to the human body. These differences, although biochemically relevant, have been shown to be trivial in clinical context so long as there is variety and sufficiency of foods in the plant-based diet. The idea that plant proteins need to be paired complementarily based on their amino acid profiles to prevent amino acid deficiencies is outdated as the body stores amino acids for hours to days at a time. By eating a wide variety of plants, patients on dialysis can avoid issues related to protein quantity and quality.Seeds are an undesirable feature in many fruits because they may have a hard or leathery texture, bitter taste, and in many instances accumulate harmful toxic compounds. Replacing seeds and seed cavities with edible fruit tissue is desirable . Seedlessness is especially attractive in species with many seeds per fruit such as citrus, one large seed such as mango, or large cavities filled with numerous seeds such as melon and papaya.
In tomato, seeds are in general not considered as a negative trait of fresh market fruits since they contribute in a positive way to the fruit taste. However, seedless fruits would be valuable and improve tomato processing. Seed formation is an integral component of fruit development: developing seeds promote cell expansion via synthesis of auxin and other unknown molecules . Metabolites associated with the developing embryo control the rate of cell division in surrounding fruit tissue, and seed number influences the final size and weight of fruit . Thus, seedlessness is potentially associated with agronomically undesirable changes in quality. Parthenocarpy is fruit set in the absence of fertilization. It can be induced with phytohormones, particularly auxins, and is currently used to increase fruit production under adverse conditions for fruit set and growth. Such methods are sometimes used in tomato, where they can cause malformed fruit and vegetative organs, inhibit further flowering, and usually yield poor quality fruit . Genetic strategies offer effective approaches involving specific mutations or introduction of specific genes. In tomato, pat mutations that result in parthenocarpy increase gibberellic acid in ovules during development . Parthenocarpic fruit has also been generated through ovule-specific expression of the iaaM or iaaH genes from Agrobacterium tumefacians or the rolB gene from Agrobacterium rhizogenes, hydroponic channel which affect auxin biosynthesis or response, respectively . Expression of IaaH in ovaries induced parthenocarpic fruit by hydrolysis of the auxin precursor naphthaleneacetamide in the ovary. Parthenocarpic eggplant, tobacco, and tomato fruits were also obtained by expressing iaaM under the ovule-specific promoter DefH9 . Expression of rolB under TRP-F1 induced parthenocarpy in tomato . Parthenocarpic fruits have also been obtained with the silencing of SlIAA9 and SlARF7 , and mutations in the ARF8 gene have been shown to induce parthenocarpic development in tomato . However, most of the parthenocarpic fruits were hearts haped and had a rather thick pericarp compared with wild type fruits . The challenge is to develop seedlessness by inducing parthenocarpy without needing supplemental pollination or application of plant growth regulators and without affecting fruit size and morphology. While the effect of the transgenic modifications on gross fruit morphology, productivity, and yield is known , the effects on overall gene expression and metabolism in fruit are not known. The aim of the present study was to address the following questions. Is the INNER NO OUTER promoter from Arabidopsis thaliana able to induce parthenocarpy in tomato? Can we avoid undesirable traits often associated with seedlessness such as loss of flavour or nutritional value? What changes result from seedlessness at a transcriptomic and metabolomic level? Which pathways show significant changes in seedless fruit compared with seeded fruit? The first objective of this study was to verify whether expression of iaaM and rolB driven by the INO promoter could induce parthenocarpic fruits in tomato . The second objective was to determine changes in the transcriptome and metabolite profile induced by this genetic modification, comparing them with both wild-type fruits and transgenic parthenocarpic fruits obtained through ovule-specific expression of the same genes driven by the previously described ovule-specific promoter DefH9. The third objective was to determine any differences between transgenic and wild-type fruits in soluble solids content and other important morphological parameters.Fifteen fully ripe fruits from each transgenic plant and 10 from wild-type plants were picked for analysis of morphology and soluble solids content. Each fruit was weighed and the polar and equatorial diameter, number of locules, and seed number were determined. Brix value, an index of total solids content, was determined from juice squeezed from five separate fruits on each plant with a digital refractometer.
The data were analysed with an analysis of variance univariate and ‘post hoc’ Duncan test using SPSS software.Gene expression profiles for breaker-stage fruits were generated for wild-type and parthenocarpic lines with the four transgenes. Wild-type fruits with seeds and wild-type fruits from which seeds had been removed were the controls. Three replicates were used for each treatment and control, except for DefH9-rolB, where only two replicates were available. Microarrays were used to study gene expression patterns in parthenocarpic fruit. Wild-type fruit with seeds was compared with transgenic lines INO-iaaM, DefH9-iaaM, INO-rolB, and DefH9-rolB. To find genes with seed-specific expression, the control fruit were also compared with wildtype fruit from which seeds had been manually removed. There were three biological replicates for each treatment and control except DefH9-rolB, for which only two replicates were available. The metabolites present in parthenocarpic fruit were also studied, using each transgenic line as a separate treatment. Wild-type fruit with seeds were used as the control, and wild-type fruits with seeds manually removed were not considered. There were six replicates of each treatment and control group except DefH9-rolB, of which there were only five replicates.For transgenes INO-iaaM, DefH9-iaaM, and INO-rolB, two fruits were taken from each of three different plants , for a total of six replicates per transgene. For transgene DefH9- rolB, only two different plants with very few fruits were available and two fruits were taken from one plant and three from the other, for a total of five replicates. The transgenic seedless fruits were compared with wild-type seeded controls, two fruits each from six plants. Wild-type fruit without seeds was not analysed. The replicate fruits were harvested at breaker stage, frozen in liquid nitrogen, and stored at –80 C until analysis. For each sample, 20–50 mg of pulp was ground and 1 ml of pre-chilled extraction solvent was rinsed with argon or gaseous nitrogen for 5 min and then added. After vortexing and centrifugation, the supernatant was analysed with a Pegasus III TOF mass spectrometer. The relative concentrations were determined by peak area . All peak detections were manually checked for false-positive and false-negative assignments. These mass spectra were then compared with known and commercially available mass spectral libraries. Statistical analysis was performed using pairwise comparison to determine significant differences. Metabolites that showed significant differences were grouped into functional categories.Statistical analyses of microarray data were performed using R statistical software. The RMA method was used for background subtraction and normalization to pre-process raw probe data and produce the gene expression matrix. To determine which genes were differentially expressed among different groups, one-way ANOVA was used to obtain a P-value for each gene. Then all P-values were BH adjusted for multiple hypotheses. Genes with adjusted P-values <0.05 were considered differentially expressed in different groups. The R package LMGene was used to perform one-way ANOVA and the R package mulltest was used to adjust multiple hypotheses. Annotation of some sequences was supplied by Affymetrix Inc.; additional annotations were found by BLAST comparisons with the NCBI non-redundant protein and TAIR databases. Functional classifications were based on those in the MapMan software, a user-driven tool that displays large data sets onto diagrams of metabolic pathways or other processes. . Gene set enrichment analysis was used to elucidate which functional categories of genes were more significantly associated with seeds and induced seedlessness. GSEA is a computational method that determines whether an a priori defined set of genes shows statistically significant, concordant differences between two biological states . Affymetrix tomato GeneChip targets matched Arabidopsis genes in >800 categories in the MapMan knowledge base.