Engineered polyamine accumulation in tomato enhances phytonutrient content,juice quality,and vine life

Polyamines, ubiquitous organic aliphatic cations, have been implicated in a myriad of physiological and developmental processes in many organisms, but their in vivo functions remain to be determined. We expressed a yeast S-adenosylmethionine decarboxylase gene (ySAMdc; Spe2) fused with a ripening-inducible E8 promoter to specifically increase levels of the polyamines spermidine and spermine in tomato fruit during ripening. Independent transgenic plants and their segregating lines were evaluated after cultivation in the greenhouse and in the field for five successive generations. The enhanced expression of the ySAMdc gene resulted in increased conversion of putrescine into higher polyamines and thus to ripening-specific accumulation of spermidine and spermine. This led to an increase in lycopene, prolonged vine life, and enhanced fruit juice quality. Lycopene levels in cultivated tomatoes are generally low, and increasing them in the fruit enhances its nutrient value. Furthermore, the rates of ethylene production in the transgenic tomato fruit were consistently higher than those in the nontransgenic control fruit. These data show that polyamine and ethylene biosynthesis pathways can act simultaneously in ripening tomato fruit. Taken together, these results provide the first direct evidence for a physiological role of polyamines and demonstrate an approach to improving nutritional quality, juice quality, and vine life of tomato fruit.     

Metabolic consequences of overproduction of phosphoenolpyruvate carboxylase in C3 plants

Phosphoenolpyruvate carboxylase (PEPC) has a variety of functions in plants, including a major anaplerotic role in replenishing the tricarboxylic acid cycle with intermediates to meet the demand of carbon skeletons for synthesis of organic acids and amino acids. Various transgenic C3 plants that overproduce PEPC have been produced and analyzed in detail. The results indicate that foreign PEPC is under the control of the regulatory mechanisms intrinsic to the host plant and down-regulated so as not to cause detrimental metabolic effects, although the anaplerotic reaction is slightly enhanced by the foreign PEPC. By use of foreign PEPCs that can avert such regulation, metabolic flow is largely directed toward synthesis of organic acids and amino acids. Observations with transgenic C3 plants also shed light on the interrelation among various metabolic pathways inside the cell.     

Response of Tomato Sugar and Acid Metabolism and Fruit Quality under Different High Temperature and Relative Humidity Conditions

The combined stress of high temperature and high relative air humidity is one of the most serious agrometeorological disasters that restricts the production capacity of protected agriculture. However, there is little information about the precise interaction between them on tomato fruit quality. The objectives of this study were to explore the effects of the combined stress of high temperature and relative humidity on the sugar and acid metabolism and fruit quality of tomato fruits, and to determine the best relative air humidity for fruit quality under high temperature environments. Four temperature treatments (32°C, 35°C, 38°C, 41°C), three relative air humidity (50%, 70%, 90%) and four duration (3, 6, 9, 12 d) orthogonal experiments were conducted, with 28°C, 50% as control. The results showed that under high temperature and relative air humidity, the activity of sucrose metabolizing enzymes in young tomato fruits changed, which reduced fruits soluble sugar content; in addition, enzyme activities involved phosphopyruvate carboxylase (PEPC), mitochondria aconitase (MDH) and citrate synthetase (CS) increased which increased the content of organic acids (especially malic acid). Eventually, vitamin C, total sugar and sugar-acid ratio decreased significantly, while the titratable acid increased, resulting in a decrease in fruit flavor quality and nutritional quality in ripe fruit. Specifically, a temperature of 32°C and a relative air humidity of 70% were the best cultivation conditions for tomato reproductive growth period under high temperature. Our results indicating that fruit quality reduced under high temperature at the flowering stage, while increasing the relative air humidity to 70% could alleviate this negative effect. Our results are benefit to better understand the interaction between microclimate parameters under specific climatic conditions in the greenhouse environment and their impact on tomato flavor quality.     

Role of phosphoenolpyruvate carboxylase in organic acid accumulation during peach fruit development

The synthesis of organic acids was studied during fruit development of two peach ( Prunus persica L. Batsch) cultivars, Fantasia and Jalousia, having fruits with high and low organic acid content, respectively. The malate content was higher in cv. Fantasia than in cv. Jalousia at the end of the first rapid growth stage (50 days after bloom [DAB]). Malate and citrate contents were higher in Fantasia than in Jalousia during the second rapid growth stage (from 100 DAB to maturity). The expression of phospho enol pyruvate carboxylase (PEPC, EC 4.1.1.31), which is involved in organic acid synthesis, was studied during peach fruit development. PEPC mRNA levels, and protein levels on a total soluble protein basis, peaked at 23 and 108 DAB in Fantasia. In Jalousia, they were very low at 23 DAB and reached levels similar to Fantasia at 108 DAB. For both cultivars, in vitro PEPC activity expressed on a dry weight basis was maximal at 24 DAB, decreased from 24 to 60 DAB, and then remained constant. The activity of peach fruit PEPC appeared extremely sensitive to malate (I_0.5 of 100 μ M for Fantasia and 65 μ M for Jalousia at pH 7.3) and low pH. PEPC may participate in the control of organic acid accumulation during fruit development in the normal-acid fruit of Fantasia. However, mechanisms other than organic acid synthesis might account for the differences in acidity between normal-acid and non-acid peach fruit.     

Tissue-specific organic acid metabolism in reproductive and non-reproductive parts of the fig fruit is partially induced by pollination

Organic acids are important components of overall fruit quality through flavor, taste, nutritional and medicinal values. Pollinated fig (Ficus carica L.) fruit quality is enhanced by increased acidity. We quantified the major organic acids and characterized the expression pattern of organic acid metabolic pathway-related genes in the reproductive part – inflorescence and non-reproductive part – receptacle of parthenocarpic and pollinated fig fruit during ripening. Essentially, pollinated fruit contains seeds in the inflorescence, as opposed to no seeds in the parthenocarpic inflorescence. The major organic acids – citrate and malate – were found in relatively high quantities in the inflorescence compared to the receptacle of both parthenocarpic and pollinated fig fruit. Notably, pollination increased citric acid content significantly in both inflorescence and receptacle. Genes related to the phosphoenolpyruvate carboxylase (PEPC) cycle, tricarboxylic acid cycle, citrate catabolism and glyoxylate cycle were identified in fig fruit. Expression levels of most of these genes were higher in inflorescences than in receptacles. In particular, FcPEPC and FcFUM (encoding fumarase) had significantly higher expression in the inflorescence of pollinated fruit. Most importantly, expression of the glyoxylate cycle genes FcMLS and FcICL (encoding malate synthase and isocitrate lyase, respectively) was induced to strikingly high levels in the inflorescence by pollination, and their expression level was highly positively correlated with the contents of all organic acids. Therefore, the glyoxylate cycle may be responsible for altering the accumulation of organic acids to upgrade the fruit taste during ripening, especially in the pollinated, seeded inflorescence.     

Seed-specific expression of a bacterial phosphoenolpyruvate carboxylase in Vicia narbonensis increases protein content and improves carbon economy

An ambitious aim in plant breeding and biotechnology is to increase the protein content of crop seeds used for food and feed. Using an approach to manipulate assimilate partitioning, we succeeded in elevating the protein content in legume seeds up to 50%. Transgenic bean plants were generated which express a Corynebacterium glutamicum phosphoenolpyruvate carboxylase (PEPC) in a seed-specific manner. The bacterial enzyme was not feedback inhibited by malate. Transgenic seeds showed a higher [14C]-CO2 uptake and about a threefold increased incorporation of labelled carbon into proteins. Changed metabolite profiles of maturing cotyledons indicated a shift of metabolic fluxes from sugars/starch into organic acids and free amino acids. These changes were consistent with an increased carbon flow through the anaplerotic pathway catalysed by PEPC. Consequently, transgenic seeds accumulated up to 20% more protein per gram seed dry weight. Additionally, seed dry weight was higher by 20%-30%. We conclude that PEPC in seeds is a promising target for molecular plant breeding.     

Overexpression of petunia chalcone isomerase in tomato results in fruit containing increased levels of flavonols

Tomatoes are an excellent source of the carotenoid lycopene, a compound that is thought to be protective against prostate cancer. They also contain small amounts of flavonoids in their peel ( approximately 5-10 mg/kg fresh weight), mainly naringenin chalcone and the flavonol rutin, a quercetin glycoside. Flavonols are very potent antioxidants, and an increasing body of epidemiological data suggests that high flavonoid intake is correlated with a decreased risk for cardiovascular disease. We have upregulated flavonol biosynthesis in the tomato in order to generate fruit with increased antioxidant capacity and a wider range of potential health benefit properties. This involved transformation of tomato with the Petunia chi-a gene encoding chalcone isomerase. Resulting transgenic tomato lines produced an increase of up to 78 fold in fruit peel flavonols, mainly due to an accumulation of rutin. No gross phenotypical differences were observed between high-flavonol transgenic and control lines. The phenotype segregated with the transgene and demonstrated a stable inheritance pattern over four subsequent generations tested thus far. Whole-fruit flavonol levels in the best of these lines are similar to those found in onions, a crop with naturally high levels of flavonol compounds. Processing of high-flavonol tomatoes demonstrated that 65% of flavonols present in the fresh fruit were retained in the processed paste, supporting their potential as raw materials for tomato-based functional food products.     

水分对有机基质栽培番茄生理特性、品质及产量的影响

以"齐粉"番茄品种为试材,研究了不同相对含水量对有机基质栽培番茄生长、生理特性、产量、品质和水分利用率的影响.结果表明:随着有机基质相对含水量的升高,番茄株高、茎粗、节间长和单株叶面积极显著增加,叶片色素含量、水势、渗透势及根系活力和果实产量显著增加,而番茄果实品质和水分利用率显著降低.从果实产量、品质和水分利用率方面综合考虑,80%基质相对含水量处理的果实产量达26 kg.m-2以上,可作为番茄有机基质栽培水分管理的量化指标;如果仅考虑果实品质,则50%基质相对含水量可以作为水分管理指标.     

Abnormal development of floral meristem triggers defective morphogenesis of generative system in transgenic tomatoes

Parthenocarpy and fruit malformations are common among independent transgenic tomato lines, expressing genes encoding different pathogenesis-related (PR) protein and antimicrobal peptides. Abnormal phenotype developed independently of the expression and type of target genes, but distinctive features during flower and fruit development were detected in each transgenic line. We analyzed the morphology, anatomy, and cytoembryology of abnormal flowers and fruits from these transgenic tomato lines and compared them with flowers and fruits of wild tomatoes, line YaLF used for transformation, and transgenic plants with normal phenotype. We confirmed that the main cause of abnormal flower and fruit development was the alterations of determinate growth of generative meristem. These alterations triggered different types of anomalous growth, affecting the number of growing ectopic shoots and formation of new flowers. Investigation of the ovule ontogenesis did not show anomalies in embryo sac development, but fertilization did not occur and embryo sac degenerated. Nevertheless, the ovule continued to differentiate due to proliferation of endothelium cells. The latter substituted embryo sac and formed pseudoembryonic tissue. This process imitated embryogenesis and stimulated ovary growth, leading to the development of parthenocarpic fruit. We demonstrated that failed fertilization occurred due to defective male gametophyte formation, which was manifested in blocked division of the nucleus in the microspore and arrest of vegetative and generative cell formation. Maturing pollen grains were overgrown microspores, not competent for fertilization but capable to induce proliferation of endothelium and development of parthenocarpic ovary. Thus, our study provided new data on the structural transformations of reproductive organs during development of parthenocarpic fruits in transgenic tomato.     

Down-regulation of TM29, a tomato SEPALLATA homolog,causes parthenocarpic fruit development and floral reversion

We have characterized the tomato (Lycopersicon esculentum Mill.) MADS box gene TM29 that shared a high amino acid sequence homology to the Arabidopsis SEP1, 2, and 3 (SEPALLATA1, 2, and 3) genes. TM29 showed similar expression profiles to SEP1, with accumulation of mRNA in the primordia of all four whorls of floral organs. In addition, TM29 mRNA was detected in inflorescence and vegetative meristems. To understand TM29 function, we produced transgenic tomato plants in which TM29 expression was down-regulated by either cosuppression or antisense techniques. These transgenic plants produced aberrant flowers with morphogenetic alterations in the organs of the inner three whorls. Petals and stamens were green rather than yellow, suggesting a partial conversion to a sepalloid identity. Stamens and ovaries were infertile, with the later developing into parthenocarpic fruit. Ectopic shoots with partially developed leaves and secondary flowers emerged from the fruit. These shoots resembled the primary transgenic flowers and continued to produce parthenocarpic fruit and additional ectopic shoots. Based on the temporal and spatial expression pattern and transgenic phenotypes, we propose that TM29 functions in floral organ development, fruit development, and maintenance of floral meristem identity in tomato.     

Identification of novel quantitative trait loci for increased lycopene content and other fruit quality traits in a tomato recombinant inbred line population

Epidemiological and clinical studies indicate that a steady dietary intake of bioavailable lycopene, a C₄₀ carotenoid and potent natural antioxidant, may be associated with a decreased incidence of prostate cancer in humans. Since fresh tomatoes and processed tomato products represent approximately 85% of the average human??s dietary lycopene intake, the identification of novel genetic factors which regulate high fruit lycopene content in tomato is imperative for the improvement of nutritional quality in this commercially valuable specialty crop. To understand the genetic control of the extraordinarily high fruit lycopene content in an accession (LA2093) of the tomato wild species Solanum pimpinellifolium, a quantitative trait locus (QTL) mapping study was conducted using a recombinant inbred line (RIL) population of a cross between LA2093 and a cultivated tomato (S. lycopersicum) breeding line, NCEBR-1. The parental lines, F1 progeny, and F7-F10 RIL populations were grown in replicated field trials in four successive years and evaluated for lycopene content as well as several other traits, including fruit fresh weight, soluble solids content, pH of puree, and plant maturity. The lycopene content of ripe fruit was estimated using three methods: high-performance liquid chromatography (HPLC), spectrophotometry, and colorimetric assays. Based on these measurements, QTL were identified and compared across generations. Among the QTL identified for lycopene, two QTL, located on chromosomes 7 and 12, had very large effects and were consistent across generations. The genomic intervals in which these two QTL reside do not correspond to known map positions of carotenoid biosynthetic genes, indicating that these QTL may represent novel alleles with potentially important implications for tomato breeding as well as increased understanding of carotenoid accumulation in tomato. Several QTL were also identified for fruit weight, soluble solids content and plant maturity. The potential implications of these results for tomato crop improvement are discussed.     

Identifying the loci responsible for natural variation in fruit size and shape in tomato

Fruit size and shape are two major factors determining yield, quality and consumer acceptability for many crops. Like most traits important to agriculture, both are quantitatively inherited. Despite their economic importance none of the genes controlling either of these traits have been cloned, and little is known about the control of the size and shape of domesticated fruit. Tomato represents a model fruit-bearing domesticated species characterized by a wide morphological diversity of fruits. The many genetic and genomic tools available for this crop can be used to unraveal the molecular bases of the developmental stages which presumably influence fruit architecture, size and shape. The goal of this review is to summarize data from the tomato QTL studies conducted over the past 15 years, which together allow the identification of the major QTLs responsible for fruit domestication in tomato. These results provide the starting point for the isolation of the genes involved in fruit-size/shape determination in tomato and potentially other fruit-bearing plants. Received: 21 January 1999 / Accepted: 12 March 1999     

Effect of suppression of ethylene biosynthesis on flavor products in tomato fruits

To elucidate the role of ethylene in the production of flavor compounds by tomato fruits, wild-type tomato (Lycopersicon esculentum L., cv. Lichun) and its transgenic antisense LeACS2 line with suppressed ethylene biosynthesis were used. The metabolism of individual sugars was ethylene-independent. However, citric acid and malic acid were under ethylene regulation. The content of these acids was higher in transgenic tomato fruits and returned to normal level after transgenic fruits were treated with ethylene. Because most of amino acids, which are important precursors of volatiles, were shown to be correlated with ethylene, we surmise that amino acid-related aroma volatiles were also affected by ethylene. Headspace analysis of volatiles showed a significant accumulation of aldehydes in wild-type tomato fruits during fruit ripening and showed a dramatic decrease in most aroma volatiles in transgenic tomato fruits as compared with wild-type fruits. The production of hexanal, hexanol, trans-2-heptenal, cis-3-hexanol, and carotenoid-related volatiles, except β-damascenone and β-ionone, was inhibited by suppression of ethylene biosynthesis. No remarkable differences were observed in the concentrations of cis-3-hexenal and trans-2-hexenal between transgenic and wild-type tomato fruits, indicating these two volatiles to be independent of ethylene. Thus, there are various regulation patterns of flavor profiles in tomato fruits by ethylene. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 1, pp. 92–101. The text was submitted by the authors in English. Both authors equally contributed to this work.