Critical roles of DNA demethylation in the activation of ripening-induced genes and inhibition of ripening-repressed genes in tomato fruit.

DNA methylation is a conserved epigenetic mark important for genome integrity, development, and environmental responses in plants and mammals. Active DNA demethylation in plants is initiated by a family of 5-mC DNA glycosylases/lyases (i.e., DNA demethylases). Recent reports suggested a role of active DNA demethylation in fruit ripening in tomato. In this study, we generated loss-of-function mutant alleles of a tomato gene, SlDML2, which is a close homolog of the Arabidopsis DNA demethylase gene ROS1 In the fruits of the tomato mutants, increased DNA methylation was found in thousands of genes. These genes included not only hundreds of ripening-induced genes but also many ripening-repressed genes. Our results show that SlDML2 is critical for tomato fruit ripening and suggest that active DNA demethylation is required for both the activation of ripening-induced genes and the inhibition of ripening-repressed genes.

Critical function of DNA methyltransferase 1 in tomato development and regulation of the DNA methylome and transcriptome.

DNA methylation confers epigenetic regulation on gene expression and thereby on various biological processes. Tomato has emerged as an excellent system to study the function of DNA methylation in plant development. To date, regulation and function of DNA methylation maintenance remains unclear in tomato plants. Here, we report the critical function of tomato (Solanum lycopersicum) Methyltransferase 1 (SlMET1) in plant development and DNA methylome and transcriptome regulation. Using CRISPR-Cas9 gene editing, we generated slmet1 mutants and observed severe developmental defects with a frame-shift mutation, including small and curly leaves, defective inflorescence, and parthenocarpy. In leaf tissues, mutations in SlMET1 caused CG hypomethylation and CHH hypermethylation on a whole-genome scale, leading to a disturbed transcriptome including ectopic expression of many RIN target genes such as ACC2 in leaf tissues, which are normally expressed in fruits. Neither the CG hypomethylation nor CHH hypermethylation in the slmet1 mutants is related to tissue culture. Meanwhile, tissue culture induces non-CG hypomethylation, which occurs more frequently at gene regions than at TE regions. Our results depict SlMET1- and tissue culture-dependent tomato DNA methylomes, and that SlMET1 is required for maintaining a normal transcriptome and normal development of tomato.

Enhanced flux of substrates into polyamine biosynthesis but not ethylene in tomato fruit engineered with yeast S-adenosylmethionine decarboxylase gene.

S-adenosylmethionine (SAM), a major substrate in 1-C metabolism is a common precursor in the biosynthetic pathways of polyamines and ethylene, two important plant growth regulators, which exhibit opposing developmental effects, especially during fruit ripening. However, the flux of various substrates including SAM into the two competing pathways in plants has not yet been characterized. We used radiolabeled (14)C-Arg, (14)C-Orn, L-[U-(14)C]Met, (14)C-SAM and (14)C-Put to quantify flux through these pathways in tomato fruit and evaluate the effects of perturbing these pathways via transgenic expression of a yeast SAM decarboxylase (ySAMDC) gene using the fruit ripening-specific promoter E8. We show that polyamines in tomato fruit are synthesized both from Arg and Orn; however, the relative contribution of Orn pathway declines in the later stages of ripening. Expression of ySAMDC reversed the ripening associated decline in spermidine (Spd) and spermine (Spm) levels observed in the azygous control fruit. About 2- to 3-fold higher levels of labeled-Spd in transgenic fruit (556HO and 579HO lines) expressing ySAMDC confirmed the enzymatic function of the introduced gene. The incorporation of L-[U-(14)C]Met into Spd, Spm, ethylene and 1-aminocyclopropane-1-carboxylic acid (ACC) was used to determine Met-flux into these metabolites. The incorporation of (14)C-Met into Spd/Spm declined during ripening of the control azygous fruit but this was reversed in fruits expressing ySAMDC. However, incorporation of (14)C-Met into ethylene or ACC during ripening was not altered by the expression of ySAMDC in the fruit. Taken together these results show that: (1) There is an inverse relationship between the production of higher polyamines and ethylene during fruit ripening, (2) the inverse relationship between higher polyamines and ethylene is modulated by ySAMDC expression in that the decline in Spd/Spm during fruit ripening can be reversed without significantly altering ethylene biosynthesis, and (3) cellular flux of SAM in plants is homeostatically regulated based on its demand for competing pathways.

Role of pectin methylesterases in cellular calcium distribution and blossom-end rot development in tomato fruit.

Blossom-end rot (BER) in tomato fruit (Solanum lycopersicum) is believed to be a calcium (Ca²âº) deficiency disorder, but the mechanisms involved in its development are poorly understood. Our hypothesis is that high expression of pectin methylesterases (PMEs) increases Ca²âº bound to the cell wall, subsequently decreasing Ca²âº available for other cellular functions and thereby increasing fruit susceptibility to BER. The objectives of this study were to evaluate the effect of PME expression, and amount of esterified pectins and Ca²âº bound to the cell wall on BER development in tomato fruit. Wild-type and PME-silenced tomato plants were grown in a greenhouse. At full bloom, flowers were pollinated and Ca²âº was no longer provided to the plants to induce BER. Our results show that suppressing expression of PMEs in tomato fruit reduced the amount of Ca²âº bound to the cell wall, and also reduced fruit susceptibility to BER. Both the wild-type and PME-silenced fruit had similar total tissue, cytosolic and vacuolar Ca²âº concentrations, but wild-type fruit had lower water-soluble apoplastic Ca²âº content and higher membrane leakage, one of the first symptoms of BER. Our results suggest that apoplastic water-soluble Ca²âº concentration influences fruit susceptibility to Ca²âº deficiency disorders.

Polyamines attenuate ethylene-mediated defense responses to abrogate resistance to Botrytis cinerea in tomato.

Transgenic tomato (Solanum lycopersicum) lines overexpressing yeast spermidine synthase (ySpdSyn), an enzyme involved in polyamine (PA) biosynthesis, were developed. These transgenic lines accumulate higher levels of spermidine (Spd) than the wild-type plants and were examined for responses to the fungal necrotrophs Botrytis cinerea and Alternaria solani, bacterial pathogen Pseudomonas syringae pv tomato DC3000, and larvae of the chewing insect tobacco hornworm (Manduca sexta). The Spd-accumulating transgenic tomato lines were more susceptible to B. cinerea than the wild-type plants; however, responses to A. solani, P. syringae, or M. sexta were similar to the wild-type plants. Exogenous application of ethylene precursors, S-adenosyl-Met and 1-aminocyclopropane-1-carboxylic acid, or PA biosynthesis inhibitors reversed the response of the transgenic plants to B. cinerea. The increased susceptibility of the ySpdSyn transgenic tomato to B. cinerea was associated with down-regulation of gene transcripts involved in ethylene biosynthesis and signaling. These data suggest that PA-mediated susceptibility to B. cinerea is linked to interference with the functions of ethylene in plant defense.

Methyl jasmonate deficiency alters cellular metabolome, including the aminome of tomato (Solanum lycopersicum L.) fruit.

Exogenous treatment with jasmonates (JA) has been shown to reduce the levels of polyamines in many plants. But the role of endogenous JA on polyamine biosynthesis or other cellular metabolites has thus far remained uninvestigated. We developed transgenic tomato (Solanum lycopersicum L.) having severely reduced methyl JA levels by silencing a fruit ripening-associated lipoxygenase (LOX), SlLoxB, using a truncated LOX gene under the control of the constitutive CaMV35S promoter. The LOX suppressed and MeJA-deficient fruits had lowered polyamine levels. Thus, these transgenic fruits were used as a plant model to evaluate the effects of reduced endogenous MeJA on cellular metabolites in ripening tomato fruits using NMR spectroscopy. During on-shelf ripening, transgenic fruits were significantly reduced in the content of 19 out of 30 metabolites examined, including Ile, Val, Ala, Thr, Asn Tyr, Glu, Gln, His, Phe, Trp, GABA, citrate, succinate, myo-inositol, unidentified compound B, nucleic acid compound Nucl1, choline, and trigonelline as compared to the wild-type azygous counterparts. A significant increase in ß-glucose levels in transgenic fruits was observed at the pink stage. The transgenic fruits were equivalent to the wild type in lycopene level and chlorophyll degradation rates. Taken together, these results show that intracellular MeJA significantly regulates overall primary metabolism, especially aminome (amino acids and polyamines) of ripening fruits.

Overexpression of yeast spermidine synthase impacts ripening, senescence and decay symptoms in tomato.

Polyamines (PAs) are ubiquitous, polycationic biogenic amines that are implicated in many biological processes, including plant growth and development, but their precise roles remain to be determined. Most of the previous studies have involved three biogenic amines: putrescine (Put), spermidine (Spd) and spermine (Spm), and their derivatives. We have expressed a yeast spermidine synthase (ySpdSyn) gene under constitutive (CaMV35S) and fruit-ripening specific (E8) promoters in Solanum lycopersicum (tomato), and determined alterations in tomato vegetative and fruit physiology in transformed lines compared with the control. Constitutive expression of ySpdSyn enhanced intracellular levels of Spd in the leaf, and transiently during fruit development, whereas E8-ySpdSyn expression led to Spd accumulation early and transiently during fruit ripening. The ySpdSyn transgenic fruits had a longer shelf life, reduced shriveling and delayed decay symptom development in comparison with the wild-type (WT) fruits. An increase in shelf life of ySpdSyn transgenic fruits was not facilitated by changes in the rate of water loss or ethylene evolution. Additionally, the expression of several cell wall and membrane degradation-related genes in ySpdSyn transgenic fruits was not correlated with an extension of shelf life, indicating that the Spd-mediated increase in fruit shelf life is independent of the above factors. Crop maturity, indicated by the percentage of ripening fruits on the vine, was delayed in a CaMV35S-ySpdSyn genotype, with fruits accumulating higher levels of the antioxidant lycopene. Notably, whole-plant senescence in the transgenic plants was also delayed compared with WT plants. Together, these results provide evidence for a role of PAs, particularly Spd, in increasing fruit shelf life, probably by reducing post-harvest senescence and decay.

Differential Association of Free, Conjugated, and Bound Forms of Polyamines and Transcript Abundance of Their Biosynthetic and Catabolic Genes During Drought/Salinity Stress in Tomato (Solanum lycopersicum L.) Leaves.

Polyamines have been implicated in ameliorating the detrimental effects of drought and saline conditions on plant growth and development. The independent impact of these two abiotic stresses on polyamine (PA) biosynthesis, catabolism, and homeostasis, as well as on their transcript abundance in tomato leaves, is presented here. We show that the total levels of putrescine (PUT), spermidine (SPD), and spermine (SPM) increase up to 72 h during drought and up to 48 h during salinity stress before their precipitable drop thereafter. Thus, tomato plants maintain survivability to drought as well as salinity stress for up to 3 and 2 days, respectively. Independent multivariant analyses of drought and salinity stress kinetic data separately showed a closer association with levels of free, conjugated, and bound forms of SPD and SPM, but not with free or bound PUT. However, combined multivariant analyses showed a closer association of free SPD, conjugated SPD, and bound SPD with both stresses; SPD-bound and SPM conjugated with drought; and free SPM and conjugated PUT with salinity stress, respectively. PA biosynthesis genes, ARG1, SPDS1, and SAMDc3, segregated with drought and SPDS2 with salinity stress. PA catabolic genes CuAO4-like and PAO4 were associated with drought and salinity stresses, respectively, suggesting differential involvement of PA biosynthesis and catabolic genes in drought and salinity stresses. Pearson correlation indicated mostly positive correlations between the levels of free, conjugated, and bound forms of PUT, SPD, and SPM under drought and salinity stress. However, negative correlations were mostly seen between the levels of various forms of the PAs and their biosynthesis/catabolic genes. Levels of different PA forms had a twofold higher negative correlation during drought as compared to salinity stress (66 vs. 32) and with transcript levels of PA biosynthesis and catabolic genes. Transcripts of light-harvesting chlorophyll a/b-binding genes were generally positively associated with different forms of PAs but negatively to carbon flow genes. Most of the PA biosynthesis genes were coordinately regulated under both stresses. Collectively, these results indicate that PAs are distinctly regulated under drought and salinity stress with different but specific homologs of PA biosynthesis and catabolic genes contributing to the accumulation of free, conjugated, and bound forms of PAs.

Plant Transcriptome Reprograming and Bacterial Extracellular Metabolites Underlying Tomato Drought Resistance Triggered by a Beneficial Soil Bacteria.

Water deficit is one of the major constraints to crop production and food security worldwide. Some plant growth-promoting rhizobacteria (PGPR) strains are capable of increasing plant drought resistance. Knowledge about the mechanisms underlying bacteria-induced plant drought resistance is important for PGPR applications in agriculture. In this study, we show the drought stress-mitigating effects on tomato plants by the Bacillus megaterium strain TG1-E1, followed by the profiling of plant transcriptomic responses to TG1-E1 and the profiling of bacterial extracellular metabolites. Comparison between the transcriptomes of drought-stressed plants with and without TG1-E1 inoculation revealed bacteria-induced transcriptome reprograming, with highlights on differentially expressed genes belonging to the functional categories including transcription factors, signal transduction, and cell wall biogenesis and organization. Mass spectrometry-based analysis identified over 40 bacterial extracellular metabolites, including several important regulators or osmoprotectant precursors for increasing plant drought resistance. These results demonstrate the importance of plant transcriptional regulation and bacterial metabolites in PGPR-induced plant drought resistance.

Glutathione peroxidase regulation of reactive oxygen species level is crucial for in vitro plant differentiation.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is overexpressed in plants under abiotic and biotic stress conditions that mediate oxidative stress. To study its biological role and its ability to confer stress resistance in plants, we tried to obtain transgenic plants overexpressing citrus (Citrus sinensis) PHGPx (cit-PHGPx). All attempts to obtain regenerated plants expressing this enzyme constitutively failed. However, when the enzyme's catalytic activity was abolished by active site-directed mutagenesis, transgenic plants constitutively expressing inactive cit-PHGPx were successfully regenerated. Constitutive expression of enzymatically active cit-PHGPx could only be obtained when transformation was based on non-regenerative processes. These results indicate that overexpression of the antioxidant enzyme PHGPx interferes with shoot organogenesis and suggests the involvement of reactive oxygen species (ROS) in this process. Using transgenic tobacco (Nicotiana tabacum) leaves obtained from plants transformed with a beta-estradiol-inducible promoter, time-dependent induction of cit-PHGPx expression was employed. A pronounced inhibitory effect of cit-PHGPx on shoot formation was found to be limited to the early stage of the regeneration process. Monitoring the ROS level during regeneration revealed that upon cit-PHGPx induction, the lowest level of ROS correlated with the maximal level of shoot inhibition. Our results clearly demonstrate the essential role of ROS in the early stages of in vitro shoot organogenesis and the possible involvement of PHGPx in maintaining ROS homeostasis at this point.

Polyamines and cellular metabolism in plants: transgenic approaches reveal different responses to diamine putrescine versus higher polyamines spermidine and spermine.

Distribution of biogenic amines-the diamine putrescine (Put), triamine spermidine (Spd), and tetraamine spermine (Spm)-differs between species with Put and Spd being particularly abundant and Spm the least abundant in plant cells. These amines are important for cell viability and their intracellular levels are tightly regulated, which have made it difficult to characterize individual effects of Put, Spd and Spm on plant growth and developmental processes. The recent transgenic intervention and mutational genetics have made it possible to stably alter levels of naturally occurring polyamines and study their biological effects. We bring together an analysis of certain metabolic changes, particularly in amino acids, to infer the responsive regulation brought about by increased diamine or polyamine levels in actively growing poplar cell cultures (transformed with mouse ornithine decarboxylase gene to accumulate high Put levels) and ripening tomato pericarp (transformed with yeast S-adenosylmethionine decarboxylase gene to accumulate high Spd and Spm levels at the cost of Put). Our analysis indicates that increased Put has little effect on increasing the levels of Spd and Spm, while Spd and Spm levels are inter-dependent. Further, Put levels were positively associated with Ala (alpha and beta), Ile and GABA and negatively correlated with Gln and Glu in both actively growing poplar cell cultures and non-dividing tomato pericarp tissue. Most amino acids showed positive correlations with Spd and Spm levels in actively growing cells. Collectively these results suggest that Put is a negative regulator while Spd-Spm are positive regulators of cellular amino acid metabolism.

Genetic engineering to enhance crop-based phytonutrients (nutraceuticals) to alleviate diet-related diseases.

Nutrition studies have provided unambiguous evidence that a number of human health maladies including chronic coronary artery, hypertension, diabetes, osteoporosis, cancer and age- and lifestyle-related diseases are associated with the diet. Several favorable and a few deleterious natural dietary ingredients have been identified that predispose human populations to various genetic and epigenetic based disorders. Media dissemination of this information has greatly raised public awareness of the beneficial effects due to increased consumption of fruit, vegetables and whole grain cereals-foods rich in phytonutrients, protein and fiber. However, the presence of intrinsically low levels of the beneficial phytonutrients in the available genotypes of crop plants is not always at par with the recommended daily allowance (RDA) for different phytonutrients (nutraceuticals). Molecular engineering of crop plants has offered a number of tools to markedly enhance intracellular concentrations of some of the beneficial nutrients, levels that, in some cases, are closer to the RDA threshold. This review brings together literature on various strategies utilized for bioengineering both major and minor crops to increase the levels of desirable phytonutrients while also decreasing the concentrations of deleterious metabolites. Some of these include increases in: protein level in potato; lysine in corn and rice; methionine in alfalfa; carotenoids (beta-carotene, phytoene, lycopene, zeaxanthin and lutein) in rice, potato, canola, tomato; choline in tomato; folates in rice, corn, tomato and lettuce; vitamin C in corn and lettuce; polyphenolics such as flavonol, isoflavone, resveratrol, chlorogenic acid and other flavonoids in tomato; anthocyanin levels in tomato and potato; alpha-tocopherol in soybean, oil seed, lettuce and potato; iron and zinc in transgenic rice. Also, molecular engineering has succeeded in considerably reducing the levels of the offending protein glutelin in rice, offering proof of concept and a new beginning for the development of super-low glutelin cereals for celiac disease patients.

Polyamines and Their Biosynthesis/Catabolism Genes Are Differentially Modulated in Response to Heat Versus Cold Stress in Tomato Leaves (Solanum lycopersicum L.).

Polyamines (PAs) regulate growth in plants and modulate the whole plant life cycle. They have been associated with different abiotic and biotic stresses, but little is known about the molecular regulation involved. We quantified gene expression of PA anabolic and catabolic pathway enzymes in tomato (Solanum lycopersicum cv. Ailsa Craig) leaves under heat versus cold stress. These include arginase1 and 2, arginine decarboxylase 1 and 2, agmatine iminohydrolase/deiminase 1, N-carbamoyl putrescine amidase, two ornithine decarboxylases, three S-adenosylmethionine decarboxylases, two spermidine synthases; spermine synthase; flavin-dependent polyamine oxidases (SlPAO4-like and SlPAO2) and copper dependent amine oxidases (SlCuAO and SlCuAO-like). The spatiotemporal transcript abundances using qRT-PCR revealed presence of their transcripts in all tissues examined, with higher transcript levels observed for SAMDC1, SAMDC2 and ADC2 in most tissues. Cellular levels of free and conjugated forms of putrescine and spermidine were found to decline during heat stress while they increased in response to cold stress, revealing their differential responses. Transcript levels of ARG2, SPDS2, and PAO4-like increased in response to both heat and cold stresses. However, transcript levels of ARG1/2, AIH1, CPA, SPDS1 and CuAO4 increased in response to heat while those of ARG2, ADC1,2, ODC1, SAMDC1,2,3, PAO2 and CuPAO4-like increased in response to cold stress, respectively. Transcripts of ADC1,2, ODC1,2, and SPMS declined in response to heat stress while ODC2 transcripts declined under cold stress. These results show differential expression of PA metabolism genes under heat and cold stresses with more impairment clearly seen under heat stress. We interpret these results to indicate a more pronounced role of PAs in cold stress acclimation compared to that under heat stress in tomato leaves.

Engineered Ripening-Specific Accumulation of Polyamines Spermidine and Spermine in Tomato Fruit Upregulates Clustered C/D Box snoRNA Gene Transcripts in Concert with Ribosomal RNA Biogenesis in the Red Ripe Fruit.

Ripening of tomato fruit leads, in general, to a sequential decrease in the endogenous levels of polyamines spermidine (SPD) and spermine (SPM), while the trend for the diamine putrescine (PUT) levels is generally an initial decrease, followed by a substantial increase, and thereafter reaching high levels at the red ripe fruit stage. However, genetic engineering fruit-specific expression of heterologous yeast S-adenosylmethionine (SAM) decarboxylase in tomato has been found to result in a high accumulation of SPD and SPM at the cost of PUT. This system enabled a genetic approach to determine the impact of increased endogenous levels of biogenic amines SPD and SPM in tomato (579HO transgenic line) and on the biogenesis, transcription, processing, and stability of ribosomal RNA (rRNA) genes in tomato fruit as compared with the non-transgenic 556AZ line. One major biogenetic process regulating transcription and processing of pre-mRNA complexes in the nucleus involves small nucleolar RNAs (snoRNAs). To determine the effect of high levels of SPD and SPM on these latter processes, we cloned, sequenced, and identified a box C/D snoRNA cluster in tomato, namely, SlSnoR12, SlU24a, Slz44a, and Slz132b. Similar to this snoRNA cluster housed on chromosome (Chr.) 6, two other noncoding C/D box genes, SlsnoR12.2 and SlU24b, with a 94% identity to those on Chr. 6 were found located on Chr. 3. We also found that other snoRNAs divisible into snoRNA subclusters A and B, separated by a uridine rich spacer, were decorated with other C/D box snoRNAs, namely, J10.3, Z131a/b, J10.1, and Z44a, followed by z132a, J11.3, z132b, U24, Z20, U24a, and J11. Several of these, for example, SlZ44a, Slz132b, and SlU24a share conserved sequences similar to those in Arabidopsis and rice. RNAseq analysis of high SPD/SPM transgenic tomatoes (579HO line) showed significant enrichment of RNA polymerases, ribosomal, and translational protein genes at the breaker+8 ripening stage as compared with the 556AZ control. Thus, these results indicate that SPD/SPM regulates snoRNA and rRNA expression directly or indirectly, in turn, affecting protein synthesis, metabolism, and other cellular activities in a positive manner.

Transcript Abundance Patterns of 9- and 13-Lipoxygenase Subfamily Gene Members in Response to Abiotic Stresses (Heat, Cold, Drought or Salt) in Tomato (Solanum lycopersicum L.) Highlights Member-Specific Dynamics Relevant to Each Stress.

Lipoxygenases (LOXs; EC 1.13.11.12) catalyze the oxygenation of fatty acids to produce oxylipins including the jasmonate family of plant hormones. The involvement of jasmonates in plant growth and development and during abiotic stress has been documented, however, the response and regulation of each member of the LOX gene family under various abiotic stresses is yet to be fully deciphered. Previously, we identified fourteen members of the tomato LOX gene family, which were divisible into nine genes representing the 9-LOX family members and five others representing the 13-LOX family members based on the carbon oxidation position specificity of polyunsaturated fatty acids. Here, we have determined the transcript abundance patterns of all the 14 LOX genes in response to four independent abiotic stresses, namely, heat, cold, drought and salt. Our results show that each of these stresses leads to a time-dependent, variable or indifferent response of specific and different set(s) of LOX gene members of both subfamilies, differentiating functional relevance of the 14 LOX genes analyzed. Out of the 14 gene members, three LOX genes were expressed constitutively or were non-responsive to either heat (SlLOX9), cold (SlLOX9) or salt (SlLOX4) stress. An in-silico LOX gene promoter search for stress-responsive elements revealed that only some but not all of the LOX genes indeed are decorated with specific and known stress responsive cis-acting elements. Thus, these data implicate some other, yet to be discovered, cis-acting elements present in the LOX gene family members, which seemingly regulate tomato responses to defined abiotic stresses presented here.

Nexus Between Spermidine and Floral Organ Identity and Fruit/Seed Set in Tomato.

Polyamines (PAs) constituting putrescine (Put), spermidine (Spd), and spermine (Spm) are ubiquitous in all organisms and play essential roles in the growth and developmental processes in living organisms, including plants. Evidences obtained through genetic, biochemical, and transgenic approaches suggest a tight homeostasis for cellular PA levels. Altered cellular PA homeostasis is associated with abnormal phenotypes. However, the mechanisms involved for these abnormalities are not yet fully understood, nor is it known whether cellular ratios of different polyamines play any role(s) in specific plant processes. We expressed a yeast spermidine synthase gene (ySpdSyn) under a constitutive promoter CaMV35S in tomato and studied the different phenotypes that developed. The constitutive expression of ySpdSyn resulted in variable flower phenotypes in independent transgenic lines, some of which lacked fruit and seed set. Quantification of PA levels in the developing flowers showed that the transgenic plants without fruit and seed set had significantly reduced Spd levels as well as low Spd/Put ratio compared to the transgenic lines with normal fruit and seed set. Transcript levels of SlDELLA, GA-20oxidase-1, and GA-3oxidase-2, which impact gibberellin (GA) metabolism and signaling, were significantly reduced in bud tissue of transgenic lines that lacked fruit and seed set. These findings indicate that PAs, particularly Spd, impact floral organ identity and fruit set in tomato involving GA metabolism and signaling. Furthermore, we suggest that a nexus exists between PA ratios and developmental programs in plants.

Fruit Architecture in Polyamine-Rich Tomato Germplasm Is Determined via a Medley of Cell Cycle, Cell Expansion, and Fruit Shape Genes.

Shape and size are important features of fruits. Studies using tomatoes expressing yeast Spermidine Synthase under either a constitutive or a fruit-ripening promoter showed obovoid fruit phenotype compared to spherical fruit in controls, suggesting that polyamines (PAs) have a role in fruit shape. The obovoid fruit pericarp exhibited decreased cell layers and pericarp thickness compared to wild-type fruit. Transgenic floral buds and ovaries accumulated higher levels of free PAs, with the bound form of PAs being predominant. Transcripts of the fruit shape genes, SUN1 and OVATE, and those of CDKB2, CYCB2, KRP1 and WEE1 genes increased significantly in the transgenic ovaries 2 and 5 days after pollination (DAP). The levels of cell expansion genes CCS52A/B increased at 10 and 20 DAP in the transgenic fruits and exhibited negative correlation with free or bound forms of PAs. In addition, the cell layers and pericarp thickness of the transgenic fruits were inversely associated with free or bound PAs in 10 and 20 DAP transgenic ovaries. Collectively, these results provide evidence for a linkage between PA homeostasis and expression patterns of fruit shape, cell division, and cell expansion genes during early fruit development, and suggest role(s) of PAs in tomato fruit architecture.

A field-grown transgenic tomato line expressing higher levels of polyamines reveals legume cover crop mulch-specific perturbations in fruit phenotype at the levels of metabolite profiles, gene expression, and agronomic characteristics.

Genetic modification of crop plants to introduce desirable traits such as nutritional enhancement, disease and pest resistance, and enhanced crop productivity is increasingly seen as a promising technology for sustainable agriculture and boosting food production in the world. Independently, cultural practices that utilize alternative agriculture strategies including organic cultivation subscribe to sustainable agriculture by limiting chemical usage and reduced tillage. How the two together affect fruit metabolism or plant growth in the field or whether they are compatible has not yet been tested. Fruit-specific yeast S-adenosylmethionine decarboxylase (ySAMdc) line 579HO, and a control line 556AZ were grown in leguminous hairy vetch (Vicia villosa Roth) (HV) mulch and conventional black polyethylene (BP) mulch, and their fruit analysed. Significant genotypexmulch-dependent interactions on fruit phenotype were exemplified by differential profiles of 20 fruit metabolites such as amino acids, sugars, and organic acids. Expression patterns of the ySAMdc transgene, and tomato SAMdc, E8, PEPC, and ICDHc genes were compared between the two lines as a function of growth on either BP or HV mulch. HV mulch significantly stimulated the accumulation of asparagine, glutamate, glutamine, choline, and citrate concomitant with a decrease in glucose in the 556AZ fruits during ripening as compared to BP. It enables a metabolic system in tomato somewhat akin to the one in higher polyamine-accumulating transgenic fruit that have higher phytonutrient content. Finally, synergism was found between HV mulch and transgenic tomato in up-regulating N:C indicator genes PEPC and ICDHc in the fruit.

Polyamines: Bio-Molecules with Diverse Functions in Plant and Human Health and Disease.

Biogenic amines-polyamines (PAs), particularly putrescine, spermidine and spermine are ubiquitous in all living cells. Their indispensable roles in many biochemical and physiological processes are becoming commonly known, including promoters of plant life and differential roles in human health and disease. PAs positively impact cellular functions in plants-exemplified by increasing longevity, reviving physiological memory, enhancing carbon and nitrogen resource allocation/signaling, as well as in plant development and responses to extreme environments. Thus, one or more PAs are commonly found in genomic and metabolomics studies using plants, particulary during different abiotic stresses. In humans, a general decline in PA levels with aging occurs parallel with some human health disorders. Also, high PA dose is detrimental to patients suffering from cancer, aging, innate immunity and cognitive impairment during Alzheimer and Parkinson diseases. A dichotomy exists in that while PAs may increase longevity and reduce some age-associated cardiovascular diseases, in disease conditions involving higher cellular proliferation, their intake has negative consequences. Thus, it is essential that PA levels be rigorously quantified in edible plant sources as well as in dietary meats. Such a database can be a guide for medical experts in order to recommend which foods/meats a patient may consume and which ones to avoid. Accordingly, designing both high and low polyamine diets for human consumption are in vogue, particularly in medical conditions where PA intake may be detrimental, for instance, cancer patients. In this review, literature data has been collated for the levels of the three main PAs, putrescine, spermidine and spermine, in different edible sources-vegetables, fruits, cereals, nuts, meat, sea food, cheese, milk, and eggs. Based on our analysis of vast literature, the effects of PAs in human/animal health fall into two broad, Yang and Yin, categories: beneficial for the physiological processes in healthy cells and detrimental under pathological conditions.

Functional analysis of a tomato (Solanum lycopersicum L.) rhamnogalacturonan lyase promoter.

The enzyme rhamnogalacturonan lyase (RGL) cleaves α-1,4 glycosidic bonds located between rhamnose and galacturonic acid residues in the main chain of rhamnogalacturonan-I (RG-I), a component of the plant cell wall polymer pectin. Although the mode of action of RGL is well known, its physiological functions associated with fruit biology are less understood. Here, we generated transgenic tomato plants expressing the ß-glucuronidase (GUS) reporter gene under the control of a -504 bp or a -776 bp fragment of the promoter of a tomato RGL gene, Solyc11g011300. GUS enzymatic activity and the expression levels of GUS and Solyc11g011300 were measured in a range of organs and fruit developmental stages. GUS staining was undetectable in leaves and roots, but high GUS enzymatic activity was detected in flowers and red ripe (RR) fruits. Maximal expression levels of Solyc11g011300 were detected at the RR developmental stage. GUS activity was 5-fold higher in flowers expressing GUS driven by the -504 bp RGL promoter fragment (RGFL3::GUS) than in the isogenic line, and 1.7-fold higher when GUS gene was driven by the -776 bp RGL promoter fragment (RGLF2::GUS) or the constitutive CaMV35S promoter. Quantitative real-time polymerase chain reaction analysis showed that the highest expression of GUS was in fruits at 40 days after anthesis, for both promoter fragments. The promoter of Solyc11g011300 is predicted to contain cis-acting elements, and to be active in pollen grains, pollen tubes, flowers and during tomato fruit ripening, suggesting that the Solyc11g011300 promoter is transcriptionally active and organ-specific.