Explore the therapeutic potential and bioevaluation of giloy leaves (Tinospora cordifolia) powder for the management of type 2 diabetes.

Giloy (Tinospora cordifolia) is an important Ayurvedic medication. Numerous illnesses, including general senility, fever, diabetes, dyspepsia, urinary infections, jaundice and skin conditions are treated with it. The biological description and chemical components of cordifolia are critically reviewed in this essay, with a focus on its ayurvedic properties and pharmaceutical applications. The goal of the current study was to investigate the chemical, phytochemical and mineral profile and anti-diabetic potential of giloy leaves powder. The results showed that the moisture content was 6.2%, ash content was 13.12%, crude protein was 17.27% and fiber was 5.5%. While in mineral analysis, sodium was 22.12±1.78, magnesium was 15.78±1.70, calcium was 9.78±1.27, potassium was 32.24±1.40, iron was 8.37±1.078 and zinc was 4.87±0.89. Furthermore, total phenolic content was 156.78±1.18 and total flavonoid content was 45.78±0.57. Afterwards, the anti-diabetic potential was analyzed by givingthe giloy leaves powder to human experimental group G1 and G2 at adose of 400mg/kg and 800mg/kg, respectively. The effect of giloy leaves powder on diabetes patients' blood sugar levels was monitored every seventh day for 2 months and HbA1c tests were done initially and after 2 months. Random blood sugar and HbA1c were significant in ANOVA.

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.

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.

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.

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.

Pathogenesis-Related Protein 1b1 (PR1b1) Is a Major Tomato Fruit Protein Responsive to Chilling Temperature and Upregulated in High Polyamine Transgenic Genotypes.

Plants execute an array of mechanisms in response to stress which include upregulation of defense-related proteins and changes in specific metabolites. Polyamines - putrescine (Put), spermidine (Spd), and spermine (Spm) - are metabolites commonly found associated with abiotic stresses such as chilling stress. We have generated two transgenic tomato lines (556HO and 579HO) that express yeast S-adenosylmethionine decarboxylase and specifically accumulate Spd and Spm in fruits in comparison to fruits from control (556AZ) plants (Mehta et al., 2002). Tomato fruits undergo chilling injury at temperatures below 13°C. The high Spd and Spm tomato together with the control azygous line were utilized to address role(s) of polyamines in chilling-injury signaling. Exposure to chilling temperature (2°C) led to several-fold increase in the Put content in all the lines. Upon re-warming of the fruits at 20°C, the levels of Spd and Spm increased further in the fruit of the two transgenic lines, the higher levels remaining stable for 15 days after re-warming as compared to the fruit from the control line. Profiling their steady state proteins before and after re-warming highlighted a protein of ∼14 kD. Using proteomics approach, protein sequencing and immunoblotting, the ∼14-kD protein was identified as the pathogenesis related protein 1b1 (PR1b1). The PR1b1 protein accumulated transiently in the control fruit whose level was barely detectable at d 15 post-warming while in the fruit from both the 556HO and 579HO transgenic lines PR1b1 abundance increased and remained stable till d 15 post warming. PR1b1 gene transcripts were found low in the control fruit with a visible accumulation only on d 15 post warming; however, in both the transgenic lines it accumulated and increased soon after rewarming being several-fold higher on day 2 while in 556HO line this increase continued until d 6 than the control fruit. The chilling-induced increase in PR1b1 protein seems independent of ethylene and methyl jasmonate signaling but may be linked to salicylic acid. We propose that polyamine-mediated sustained accumulation of PR1b1 protein in post-warmed chilled tomato fruit is a pre-emptive cold stress response and possibly a defense response mechanism related to Cold Stress-Induced Disease Resistance (SIDR) phenomenon.

Fruit metabolite networks in engineered and non-engineered tomato genotypes reveal fluidity in a hormone and agroecosystem specific manner.

INTRODUCTION: Metabolomics provides a view of endogenous metabolic patterns not only during plant growth, development and senescence but also in response to genetic events, environment and disease. The effects of the field environment on plant hormone-specific metabolite profiles are largely unknown. Few studies have analyzed useful phenotypes generated by introducing single or multiple gene events alongside the non-engineered wild type control at field scale to determine the robustness of the genetic trait and its modulation in the metabolome as a function of specific agroecosystem environments. OBJECTIVES: We evaluated the influence of genetic background (high polyamine lines; low methyl jasmonate line; low ethylene line; and isogenic genotypes carrying double transgenic events) and environments (hairy vetch, rye, plastic black mulch and bare soil mulching systems) on the metabolomic profile of isogenic reverse genetic mutations and selected mulch based cropping systems in tomato fruit. Net photosynthesis and fruit yield were also determined. METHODS: NMR spectroscopy was used for quantifying metabolites that are central to primary metabolism. We analyzed both the first moment (means) of metabolic response to genotypes and agroecosystems by traditional univariate/multivariate methods, and the second moment (covariances) of responses by creating networks that depicted changes in correlations of paired metabolites. This particular approach is novel and was necessary because our experimental material yielded highly variable metabolic responses that could not be easily understood using the traditional analytical approaches for first moment statistics. RESULTS: High endogenous spermidine and spermine content exhibited strong effects on amino acids, Krebs cycle intermediates and energy molecules (ADP + ATP) in ripening fruits of plants grown under different agroecosystem environments. The metabolic response to high polyamine genotypes was similar to the response to hairy vetch cover crop mulch; supported by the pattern of changes in correlation between metabolites. Changes in primary metabolites of genotypes mutated for the deficiency of ethylene or methyl jasmonate were unique under all growth conditions and opposite of high polyamine genotype results. The high polyamine trait was found to dominate the low ethylene and low jasmonate mutations under field conditions. For several metabolites low ethylene and low methyl jasmonate genotypes had an inverse relationship. Collectively, these results affirm that interactions between metabolite pathways and growth environments are affected by genotype, and influence the metabolite quality of a crop. CONCLUSION: This study portrays how metabolite relationships change, both in mean and in correlation, under different genotypic and environmental conditions. Although these networks are surprisingly dynamic, we also find examples of selectively conserved associations.

Metabolite profiling and expression analysis of flavonoid, vitamin C and tocopherol biosynthesis genes in the antioxidant-rich sea buckthorn (Hippophae rhamnoides L.).

In this study, phenolic compounds were analyzed in developing berries of four Canadian grown sea buckthorn (Hippophae rhamnoides L.) cultivars ('RC-4', 'E6590', 'Chuyskaya' and 'Golden Rain') and in leaves of two of these cultivars. Among phenolic acids, p-coumaric acid was the highest in berries, while gallic acid was predominant in leaves. In the flavonoid class of compounds, myricetin/rutin, kaempferol, quercetin and isorhamnetin were detected in berries and leaves. Berries of the 'RC-4' cultivar had approximately ⩾ 2-fold higher levels of myricetin and quercetin at 17.5mg and 17.2 mg/100 g FW, respectively, than the other cultivars. The flavonoid content in leaves was considerably more than in berries with rutin and quercetin levels up to 135 mg and 105 mg/100 g FW, respectively. Orthologs of 15 flavonoid biosynthesis pathway genes were identified within the transcriptome of sea buckthorn mature seeds. Semi-quantitative RT-PCR analysis of these genes in developing berries indicated relatively higher expression of genes such as CHS, F3'H, DFR and LDOX in the 'RC-4' cultivar than in the 'Chuyskaya' cultivar. Vitamin C levels in ripened berries of the Canadian cultivars were on the high end of the concentration range reported for most other sea buckthorn cultivars. Orthologs of genes involved in vitamins C and E biosynthesis were also identified, expanding the genomic resources for this nutritionally important plant.

Seed dormancy is modulated in recently evolved chlorsulfuron-resistant Turkish biotypes of wild mustard (Sinapis arvensis).

Biotypes of the broad-leaved wild mustard (Sinapis arvensis L.) found in wheat fields of Aegean and Marmara region of Turkey were characterized and shown to have developed resistance to sulfonylurea (chlorsulfuron), an inhibitor of acetolactate synthase (ALS). DNA sequence analysis of the ALS genes from two such resistant ("R") biotypes, KNF-R1 and KNF-R2, revealed point mutations, CCT (Pro 197) to TCT (Ser 197) in KNF-R1 and CCT (Pro 197) to ACT (Thr 197) in KNF-R2; these substitutions are consistent with the presence of chlorsulfuron-insensitive ALS enzyme activity in the "R" S. arvensis biotypes. An additional phenotype of chlorsulfuron resistance in the Turkish S. arvensis "R" biotypes was revealed in the form of an altered seed dormancy behavior over 4-48 months of dry storage (after-ripening) compared to the susceptible ("S") biotypes. Seeds of the "S" biotypes dry stored for 4 months had a higher initial germination, which sharply decreased with storage time, while the seeds of the "R" biotypes had lower germination after 4-months storage, rising sharply and peaking thereafter by 24 months' of dry storage. The "R" biotype seeds continued to maintain a higher germination percentage even after 48 months of after-ripening. The seed weight of "R" and "S" biotypes after-ripened for 4 months was similar but those after-ripened for 48 months differed, "R" seeds were significantly heavier than those of the "S" seeds. Differential seed germinability between "S" and "R" biotypes was found not a case of differential viability, temperature regimen or non-response to pro-germination hormone GA3. These studies are of relevance to ecological fitness of herbicide-resistant biotypes in terms of seed viability and germination.

Development of psychiatric risk evaluation checklist and routine for nurses in a general hospital: ethnographic qualitative study / Desenvolvimento de check-list e rotina de avaliação de risco psiquiátrico para enfermeiras num hospital geral: pesquisa qualitativa etnográfica

CONTEXT AND OBJECTIVE:There is high prevalence of mental and behavioral disorders in general hospitals, thus triggering psychiatric risk situations. This study aimed to develop a psychiatric risk assessment checklist and routine for nurses, the Psychiatric Risk Evaluation Check-List (PRE-CL), as an alternative model for early identification and management of these situations in general hospitals.DESIGN AND SETTING:Ethnographic qualitative study in a tertiary-level private hospital.METHOD:Three hundred general-unit nurses participated in the study. Reports were gathered through open groups conducted by a trained nurse, at shift changes for two months. The questions used were: "Would you consider it helpful to discuss daily practice situations with a psychiatrist? Which situations?" The data were qualitatively analyzed through an ethnographic approach.RESULTS:The nurses considered it useful to discuss daily practice situations relating to mental and behavioral disorders with a psychiatrist. Their reports were used to develop PRE-CL, within the patient overall risk assessment routine for all inpatients within 24 hours after admission and every 48 hours thereafter. Whenever one item was present, the psychosomatic medicine team was notified. They went to the unit, gathered data from the nurses, patient files and, if necessary, attending doctors, and decided on the risk management: guidance, safety measures or mental health consultation.CONCLUSION:It is possible to develop a model for detecting and intervening in psychiatric and behavioral disorders at general hospitals based on nursing team observations, through a checklist that takes these observations into account and a routine inserted into daily practice.

CONTEXTO E OBJETIVO:Existe alta prevalência de transtornos mentais e comportamentais em hospitais gerais, propiciando situações de risco psiquiátrico. Este estudo objetivou desenvolver uma rotina e um check-list para enfermeiras, a Avaliação de Risco Psiquiátrico (ARP-CL), como modelo alternativo de identificação e manejo precoce destas situações no hospital geral.TIPO DE ESTUDO E LOCAL:Pesquisa qualitativa etnográfica, em hospital particular terciário.MÉTODO:Trezentas enfermeiras de unidades gerais participaram do estudo. Os relatos foram coletados em grupos abertos, conduzidos por enfermeira treinada, durante passagens de plantão, por dois meses, através das questões: "Você consideraria útil discutir com um psiquiatra situações da sua prática diária? Quais situações?" Os dados foram analisados qualitativamente através do método etnográfico.RESULTADOS:Enfermeiras consideraram útil poder discutir rotineiramente com um psiquiatra situações relacionadas a transtornos mentais e de comportamento da sua prática diária. Seus relatos foram utilizados no desenvolvimento da ARP-CL, na rotina da avaliação de risco global do paciente, para todos os internados nas primeiras 24 horas e posteriormente a cada 48 horas. Quando um item era presente, a equipe de medicina psicossomática era notificada, indo à ala e coletando dados com a enfermagem, no prontuário do paciente, ou com o médico assistente, se necessário, decidindo conduta no risco: orientação, medidas de segurança ou consulta em saúde mental.CONCLUSÃO:É possível desenvolver um modelo de detecção e intervenção precoces para transtornos psiquiátricos e de comportamento num hospital geral baseado na observação de enfermeiras, através de check-list que leve em conta essas observações e de uma rotina inserida na prática diária.

Development of psychiatric risk evaluation checklist and routine for nurses in a general hospital: ethnographic qualitative study.

CONTEXT AND OBJECTIVE: There is high prevalence of mental and behavioral disorders in general hospitals, thus triggering psychiatric risk situations. This study aimed to develop a psychiatric risk assessment checklist and routine for nurses, the Psychiatric Risk Evaluation Check-List (PRE-CL), as an alternative model for early identification and management of these situations in general hospitals. DESIGN AND SETTING: Ethnographic qualitative study in a tertiary-level private hospital. METHOD: Three hundred general-unit nurses participated in the study. Reports were gathered through open groups conducted by a trained nurse, at shift changes for two months. The questions used were: "Would you consider it helpful to discuss daily practice situations with a psychiatrist? Which situations?" The data were qualitatively analyzed through an ethnographic approach. RESULTS: The nurses considered it useful to discuss daily practice situations relating to mental and behavioral disorders with a psychiatrist. Their reports were used to develop PRE-CL, within the patient overall risk assessment routine for all inpatients within 24 hours after admission and every 48 hours thereafter. Whenever one item was present, the psychosomatic medicine team was notified. They went to the unit, gathered data from the nurses, patient files and, if necessary, attending doctors, and decided on the risk management: guidance, safety measures or mental health consultation. CONCLUSION: It is possible to develop a model for detecting and intervening in psychiatric and behavioral disorders at general hospitals based on nursing team observations, through a checklist that takes these observations into account and a routine inserted into daily practice.

Genetic introgression of ethylene-suppressed transgenic tomatoes with higher-polyamines trait overcomes many unintended effects due to reduced ethylene on the primary metabolome.

Ethylene regulates a myriad physiological and biochemical processes in ripening fruits and is accepted as the ripening hormone for the climacteric fruits. However, its effects on metabolome and resulting fruit quality are not yet fully understood, particularly when some of the ripening-associated biochemical changes are independent of ethylene action. We have generated a homozygous transgenic tomato genotype (2AS-AS) that exhibits reduced ethylene production as a result of impaired expression of 1-aminocyclopropane-1-carboxylate synthase 2 gene by its antisense RNA and had a longer shelf life. Double transgenic hybrid (2AS-AS × 579HO) developed through a genetic cross between 2AS-AS and 579HO (Mehta et al., 2002) lines resulted in significantly higher ethylene production than either the WT or 2AS-AS fruit. To determine the effects of reduced ethylene and introgression of higher polyamines' trait, the metabolic profiles of ripening fruits from WT (556AZ), 2AS-AS, and 2AS-AS × 579HO lines were determined using (1)H-NMR spectroscopy. The levels of Glu, Asp, AMP, Adenosine, Nucl1, and Nucl2 increased during ripening of the WT fruit. The increases in Glu, Asp, and AMP levels were attenuated in 2AS-AS fruit but recovered in the double hybrid with higher ethylene and polyamine levels. The ripening-associated decreases in Ala, Tyr, Val, Ile, Phe, malate, and myo-inositol levels in the 2AS-AS line were not reversed in the double hybrid line suggesting a developmental/ripening regulated accumulation of these metabolites independent of ethylene. Significant increases in the levels of fumarate, formate, choline, Nucl1, and Nucl2 at most stages of ripening fruit were found in the double transgenic line due to introgression with higher-polyamines trait. Taken together these results show that the ripening-associated metabolic changes are both ethylene dependent and independent, and that the fruit metabolome is under the control of multiple regulators, including ethylene and polyamines.

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.

Fatty acid composition of developing sea buckthorn (Hippophae rhamnoides L.) berry and the transcriptome of the mature seed.

BACKGROUND: Sea buckthorn (Hippophae rhamnoides L.) is a hardy, fruit-producing plant known historically for its medicinal and nutraceutical properties. The most recognized product of sea buckthorn is its fruit oil, composed of seed oil that is rich in essential fatty acids, linoleic (18:2 ω-6) and α-linolenic (18:3 ω-3) acids, and pulp oil that contains high levels of monounsaturated palmitoleic acid (16:1 ω-7). Sea buckthorn is fast gaining popularity as a source of functional food and nutraceuticals, but currently has few genomic resources; therefore, we explored the fatty acid composition of Canadian-grown cultivars (ssp. mongolica) and the sea buckthorn seed transcriptome using the 454 GS FLX sequencing technology. RESULTS: GC-MS profiling of fatty acids in seeds and pulp of berries indicated that the seed oil contained linoleic and α-linolenic acids at 33-36% and 30-36%, respectively, while the pulp oil contained palmitoleic acid at 32-42%. 454 sequencing of sea buckthorn cDNA collections from mature seeds yielded 500,392 sequence reads, which identified 89,141 putative unigenes represented by 37,482 contigs and 51,659 singletons. Functional annotation by Gene Ontology and computational prediction of metabolic pathways indicated that primary metabolism (protein>nucleic acid>carbohydrate>lipid) and fatty acid and lipid biosynthesis pathways were highly represented categories. Sea buckthorn sequences related to fatty acid biosynthesis genes in Arabidopsis were identified, and a subset of these was examined for transcript expression at four developing stages of the berry. CONCLUSION: This study provides the first comprehensive genomic resources represented by expressed sequences for sea buckthorn, and demonstrates that the seed oil of Canadian-grown sea buckthorn cultivars contains high levels of linoleic acid and α-linolenic acid in a close to 1:1 ratio, which is beneficial for human health. These data provide the foundation for further studies on sea buckthorn oil, the enzymes involved in its biosynthesis, and the genes involved in the general hardiness of sea buckthorn against environmental conditions.

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.

Tomato response to legume cover crop and nitrogen: differing enhancement patterns of fruit yield, photosynthesis and gene expression.

Excessive use of nitrogen (N) in crop production has impacted ecosystems by contaminating soil and water. Management of N in agriculture is therefore of global concern. Sustainable agriculture systems that use leguminous cover crops such as hairy vetch (Vicia villosa Roth) to fix N and enrich soil organic matter by fixing carbon provide an alternative strategy. N signalling pathways were found associated with delayed leaf senescence and disease tolerance of hairy vetch-grown tomatoes. To test whether N in hairy vetch is the only contributing factor leading to these phenotypes, we designed a pot experiment in the field to analyse growth and gene expression in tomatoes, one set with soil overwintered without a cover crop (bare soil) and the other with soil overwintered with a hairy vetch cover crop including the vetch residue on the soil surface. Additionally, supplementary N fertiliser was also provided to aid distinguishing tomato responses to vetch from those to inorganic N. Tomato fruit yield, plant biomass and photosynthesis were higher in plants grown in vetch than bare soil. Tomato growth and photosynthesis metrics exhibited a parabolic response to inorganic N in bare soil, suggesting the potential for N toxicity in pots with the highest N rate. Vetch-grown tomato plants mitigated these effects and maintained elevated photosynthetic rates at high inorganic N levels. Vetch-grown plants also mitigated a decline in expression of several genes regulating nitrogen and carbon metabolism and upregulated the defence-related gene, osmotin, relative to plants grown in bare soil. Thus, some of the positive responses of tomatoes to a hairy vetch cover crop observed in the field seem mediated by physiological cues other than the additional N provided by the vetch cover crop.

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.

Overaccumulation of higher polyamines in ripening transgenic tomato fruit revives metabolic memory, upregulates anabolism-related genes, and positively impacts nutritional quality.

Vegetables and fruits are essential components of the human diet as they are sources of vitamins, minerals, and fiber and provide antioxidants that prevent chronic diseases. Our goal is to improve durable nutritional quality of tomato fruit. We developed transgenic tomatoes expressing yeast S-adenosylmethionine decarboxylase (ySAMdc) gene driven by a fruit-specific E8 promoter to investigate the role of polyamines in fruit metabolism. Stable integration of E8-ySAMdc chimeric gene in tomato genome led to ripening-specific accumulation of polyamines, spermidine (Spd) and spermine (Spm), which in turn affected higher accumulation of glutamine, asparagine, and organic acids in the red fruit with significant decrease in the contents of valine, aspartate, sucrose, and glucose. The metabolite profiling analysis suggests that Spd/Spm are perceived as "signaling" organic-N metabolites by the fruit cells, resulting in the stimulation of carbon sequestration; enhanced synthesis of biomolecules; increased acid to sugar ratio, a good attribute for the fruit flavor; and in the accumulation of another "vital amine," choline, which is an essential micronutrient for brain development. A limited transcriptome analysis of the transgenic fruit that accumulate higher polyamines revealed a large number of differentially expressed genes, about 55% of which represented discrete functional categories, and the remaining 45% were novel, unknown, or unclassified: amino acid biosynthesis, carotenoid biosynthesis, cell wall metabolism, chaperone family, flavonoid biosynthesis, fruit ripening, isoprenoid biosynthesis, polyamine biosynthesis, signal transduction, stress/defense-related, transcription, translation, and vacuolar function. There was a good correspondence between some gene transcripts and their protein products, but not in the case of the tonoplast intrinsic protein, which showed post-transcriptional regulation. Higher metabolic activity of the transgenic fruit is reflected in higher respiratory activity, and upregulation of chaperones and mitochondrial cytochrome oxidase transcripts compared to the control. These transgenic plants are a new resource to understand the role of Spd/Spm in fruit biology. Transcriptome analysis and metabolic profiles of Spd/Spm accumulating, transgenic fruit suggest the presence of an intricate regulation and interconnection between certain metabolic pathways that are revived when Spd and Spm likely reach a certain threshold. Thus, polyamines act as antiapoptotic regulatory molecules and are able to revive metabolic memory in the tomato fruit.

Metabolic control of seedling development by invertases.

Invertases are important enzymes in higher plants, which are involved in regulating developmental processes and responses to external factors. In a functional approach the role of invertases was investigated using transgenic plants ectopically expressing inhibitor proteins to decrease invertase activity. For generating specific effects, these inhibitor proteins were expressed in Arabidopsis under the control of synthetic promoters consisting of tetramers of pathogen-inducible elements, which were reported to yield low constitutive expression. Unexpectedly, seedling growth of putative transgenic plants was arrested at the four-leaf stage. Analysis of ß-glucuronidase activity of corresponding reporter gene lines showed a correlation of the growth arrest with high activity of these promoters in seedlings grown under tissue culture conditions. The negative effect of invertase inhibition on seedling growth was substantiated by transgenic tobacco plants expressing an invertase inhibitor under control of a tetracycline inducible promoter. Ectopic induction of the invertase inhibitor during early seedling development resulted in a reduced fresh weight of seedlings. The importance of invertase in seedling development is further supported by results of expression profiling of invertases in Arabidopsis, which was confirmed by expression analyses. The mRNA for the vacuolar invertases Atßfruct3 and Atßfruct4 and cell wall invertase AtcwINV1 are specifically and strongly expressed during seedling development. These complementing results show that invertase activity is required for normal seedling development.

Extracellular invertase is an essential component of cytokinin-mediated delay of senescence.

Leaf senescence is the final stage of leaf development in which the nutrients invested in the leaf are remobilized to other parts of the plant. Whereas senescence is accompanied by a decline in leaf cytokinin content, exogenous application of cytokinins or an increase of the endogenous concentration delays senescence and causes nutrient mobilization. The finding that extracellular invertase and hexose transporters, as the functionally linked enzymes of an apolasmic phloem unloading pathway, are coinduced by cytokinins suggested that delay of senescence is mediated via an effect on source-sink relations. This hypothesis was further substantiated in this study by the finding that delay of senescence in transgenic tobacco (Nicotiana tabacum) plants with autoregulated cytokinin production correlates with an elevated extracellular invertase activity. The finding that the expression of an extracellular invertase under control of the senescence-induced SAG12 promoter results in a delay of senescence demonstrates that effect of cytokinins may be substituted by these metabolic enzymes. The observation that an increase in extracellular invertase is sufficient to delay leaf senescence was further verified by a complementing functional approach. Localized induction of an extracellular invertase under control of a chemically inducible promoter resulted in ectopic delay of senescence, resembling the naturally occurring green islands in autumn leaves. To establish a causal relationship between cytokinins and extracellular invertase for the delay of senescence, transgenic plants were generated that allowed inhibition of extracellular invertase in the presence of cytokinins. For this purpose, an invertase inhibitor was expressed under control of a cytokinin-inducible promoter. It has been shown that senescence is not any more delayed by cytokinin when the expression of the invertase inhibitor is elevated. This finding demonstrates that extracellular invertase is required for the delay of senescence by cytokinins and that it is a key element of the underlying molecular mechanism.