[Show abstract][Hide abstract]ABSTRACT: Background: Among cereals, rice has a genetic propensity to accumulate high levels of cadmium (Cd) in grains. Xylem-mediated root-to-shoot translocation rather than root uptake has been suggested as the main physiological factor accounting for the genotypic variation observed in Cd accumulation in shoots and grains. Several evidence indicate OsHMA2 – a putative zinc (Zn) transporter – as the main candidate protein that could be involved in mediating Cd- and Zn-xylem loading in rice. However, the specific interactions between Zn and Cd in rice often appear anomalous if compared to those observed in other staple crops, suggesting that root-to-shoot Cd translocation process could be more complex than previously thought. In this study we performed a complete set of competition experiments with Zn and Cd in order to analyze their possible interactions and reciprocal effects at the root-to-shoot translocation level.
Results: The competition analysis revealed the lack of a full reciprocity when considering the effect of Cd on Zn accumulation, and vice versa, since the accumulation of Zn in the shoots was progressively inhibited by Cd increases, whereas that of Cd was only partially impaired by Zn. Such behaviors were probably dependent on Cd-xylem loading mechanisms, as suggested by: i) the analysis of Zn and Cd content in the xylem sap performed in relation to the concentration of the two metals in the mobile fractions of the roots; ii) the analysis of the systemic movement of 107Cd in short term experiments performed using a positron-emitting tracer imaging system (PETIS).
Conclusions: Our results suggest that at least two pathways may mediate root-to-shoot Cd translocation in rice. The former could involve OsHMA2 as Zn2+/Cd2+ xylem loader, whereas the latter appears to involve a Zn-insensitive system that still needs to be identified.
[Show abstract][Hide abstract]ABSTRACT: Phosphoenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme and is utilized in the gluconeogenesis pathway in plants. Although, its catalytic and regulatory properties are quite well understood, there are uncertainties regarding its physiological role in many plants tissues such as the flesh of developing fruits. To further understand the function of PEPCK in fruits and other tissues, RNAi transgenic tomato plants in which SlPEPCK transcription was down-regulated by either CaMV 35S constitutive promoter or the fruit-specific E8 promoter were generated and characterized on the basis of their phenotypic and metabolic aspects. In the PEPCK-deficient lines, prominent growth suppression of germinated seedlings was observed and other vegetative suppression appeared during the early stage of plant growth in the 35S promoter-driven lines. In particular, root elongation was most obviously suppressed in the germinated seedlings, indicating that the gluconeogenesis pathway is involved in the root growth of seedlings. Regarding the primary metabolism in fruit, the soluble sugar content tended to decrease, whereas the malate content tended to increase in ripening fruits of the RNAi lines compared with the wild type. These results indicate that activation of the gluconeogenesis pathway from organic acids to sugars occurs during ripening but is suppressed by the knocking down of the PEPCK gene, suggesting that PEPCK participates in determining the sugar/acid ratio in ripening fruit.
Article · Sep 2015 · Plant Physiology and Biochemistry
[Show abstract][Hide abstract]ABSTRACT: After the nuclear disaster in Fukushima, radiocesium contamination became a serious scientific concern and research of its effects on plants increased. In such plant studies, high resolution images of radiocesium are required without contacting the subjects. Cherenkov light imaging of beta radionuclides has inherently high resolution and is promising for plant research. Since 137Cs and 134Cs emit beta particles, Cherenkov light imaging will be useful for the imaging of radiocesium distribution. Consequently, we developed and tested a Cherenkov light imaging system. We used a high sensitivity cooled charge coupled device (CCD) camera (Hamamatsu Photonics, ORCA2-ER) for imaging Cherenkov light from 137Cs. A bright lens (Xenon, F-number: 0.95, lens diameter: 25 mm) was mounted on the camera and placed in a black box. With a 100-μm 137Cs point source, we obtained 220-μm spatial resolution in the Cherenkov light image. With a 1-mm diameter, 320-kBq 137Cs point source, the source was distinguished within 2-s. We successfully obtained Cherenkov light images of a plant whose root was dipped in a 137Cs solution, radiocesium-containing samples as well as line and character phantom images with our imaging system. Cherenkov light imaging is promising for the high resolution imaging of radiocesium distribution without contacting the subject.
Article · Mar 2015 · Nuclear Instruments and Methods in Physics Research Section A Accelerators Spectrometers Detectors and Associated Equipment
[Show abstract][Hide abstract]ABSTRACT: To maximize fruit yield of tomatoes cultivated in a controlled, closed system such as a greenhouse or a plant factory at a limited cost, it is important to raise the translocation rate of fixed carbon to fruits by tuning the cultivation conditions. Elevation of atmospheric CO2 concentration is a good candidate; however, it is technically difficult to evaluate the effect on fruit growth by comparing different individuals in different CO2 conditions because of large inter-individual variations. In this study, we employed a positron-emitting tracer imaging system (PETIS), which is a live-imaging technology for plant studies, and a short-lived radioisotope 11C to quantitatively analyze immediate responses of carbon fixation and translocation in tomatoes in elevated CO2 conditions. We also developed a closed cultivation system to feed a test plant with CO2 at concentrations of 400, 1,500 and 3,000 ppm and a pulse of 11CO2. As a result, we obtained serial images of 11C fixation by leaves and subsequent translocation into fruits. Carbon fixation was enhanced steadily by increasing the CO2 concentration, but the amount translocated into fruits saturated at 1,500 ppm on average. The translocation rate had larger inter-individual variation and showed less consistent responses to external CO2 conditions compared with carbon fixation. Our experimental system was demonstrated to be a valuable tool for the optimization of closed cultivation systems because it can trace the responses of carbon translocation in each individual, which are otherwise usually masked by inter-individual variation.
[Show abstract][Hide abstract]ABSTRACT: Glutathione is a sulfur-containing peptide involved in various aspects of plant metabolism. Glutathione is also known to have effects on heavy metal responses in plants. In our previous work, we have found glutathione, applied to roots site-specifically, inhibited cadmium (Cd) translocation from roots to shoots and Cd accumulation in shoots in oilseed rape plants. In addition, we succeeded in visualizing inhibition of root-to-shoot translocation of Cd by using a positron-emitting tracer imaging system (PETIS). In this work, the effects of glutathione concentration in the root zone (hydroponic solution) and the glutathione treatment period on Cd partitioning in oilseed rape plants were investigated. Our experimental results demonstrated that glutathione, exceeding a certain concentration in the root zone, is needed to trigger inhibition of Cd translocation, and that treatment time from the start of glutathione application had different effects on Cd partitioning in oilseed rape plants.
[Show abstract][Hide abstract]ABSTRACT: Previously, we reported that OsNRAMP5 functions as a manganese, iron, and cadmium (Cd) transporter. The shoot Cd content in OsNRAMP5 RNAi plants was higher than that in wild-type (WT) plants, whereas the total Cd content (roots plus shoots) was lower. For efficient Cd phytoremediation, we produced OsNRAMP5 RNAi plants using the natural high Cd-accumulating cultivar Anjana Dhan (A5i). Using a positron-emitting tracer imaging system, we assessed the time-course of Cd absorption and accumulation in A5i plants. Enhanced 107Cd translocation from the roots to the shoots was observed in A5i plants. To evaluate the phytoremediation capability of A5i plants, we performed a field experiment in a Cd-contaminated paddy field. The biomass of the A5i plants was unchanged by the suppression of OsNRAMP5 expression; the A5i plants accumulated twice as much Cd in their shoots as WT plants. Thus, A5i plants could be used for rapid Cd extraction and the efficient phytoremediation of Cd from paddy fields, leading to safer food production.
[Show abstract][Hide abstract]ABSTRACT: Cadmium (Cd) accumulations in a Cd hyper-accumulator fern, Athyrium yokoscense (Ay), and tobacco, Nicotiana tabacum (Nt), were kinetically analyzed using the positron-emitting tracer imaging system under two medium conditions (Basal and No-nutrient). In Ay, maximumly 60% and 15% of the total Cd accumulated in the distal roots and the shoots under the Basal condition, respectively. Interestingly, a portion of the Cd in the distal roots returned to the medium. In comparison with Ay, a little fewer Cd accumulation in the distal roots and obviously higher Cd migration to the shoots were observed in Nt under the Basal condition (maximumly 40% and 70% of the total Cd, respectively). No-nutrient condition down regulated the Cd migration in both species, although the regulation was highly stricter in Ay than in Nt (almost no migration in Ay and around 20% migration in Nt). In addition, the present work enabled to estimate physical and physiological Cd accumulation capacities in the distal roots, and demonstrated a condition-dependent changes especially in Ay. These results clearly suggested occurrences of species-/condition-specific regulations in each observed parts. It is probable that integration of these properties govern the specific Cd tolerance/accumulation in Ay and Nt. (194 words).
[Show abstract][Hide abstract]ABSTRACT: The aims of this study are to investigate whether and how the nitrogen form (nitrate (NO3 (-)) versus ammonium (NH4 (+))) influences cadmium (Cd) uptake and translocation and subsequent Cd phytoextraction by the hyperaccumulator species Sedum plumbizincicola. Plants were grown hydroponically with N supplied as either NO3 (-) or NH4 (+). Short-term (36 h) Cd uptake and translocation were determined innovatively and quantitatively using a positron-emitting (107)Cd tracer and positron-emitting tracer imaging system. The results show that the rates of Cd uptake by roots and transport to the shoots in the NO3 (-) treatment were more rapid than in the NH4 (+) treatment. After uptake for 36 h, 5.6 (0.056 μM) and 29.0 % (0.290 μM) of total Cd in the solution was non-absorbable in the NO3 (-) and NH4 (+) treatments, respectively. The local velocity of Cd transport was approximately 1.5-fold higher in roots (3.30 cm h(-1)) and 3.7-fold higher in shoots (10.10 cm h(-1)) of NO3 (-)- than NH4 (+)-fed plants. Autoradiographic analysis of (109)Cd reveals that NO3 (-) nutrition enhanced Cd transportation from the main stem to branches and young leaves. Moreover, NO3 (-) treatment increased Cd, Ca and K concentrations but inhibited Fe and P in the xylem sap. In a 21-day hydroponic culture, shoot biomass and Cd concentration were 1.51 and 2.63 times higher in NO3 (-)- than in NH4 (+)-fed plants. We conclude that compared with NH4 (+), NO3 (-) promoted the major steps in the transport route followed by Cd from solution to shoots in S. plumbizincicola, namely its uptake by roots, xylem loading, root-to-shoot translocation in the xylem and uploading to the leaves. S. plumbizincicola prefers NO3 (-) nutrition to NH4 (+) for Cd phytoextraction.
Full-text Article · Apr 2013 · Environmental Science and Pollution Research
[Show abstract][Hide abstract]ABSTRACT: ADP-glucose pyrophosphorylase (AGPase) is a key regulatory enzyme in starch biosynthesis. In this research, 2,885 bp of the predicted promoter sequence for the AgpS1 gene encoding the AGPase small subunit was isolated from tomato. Sequence analyses revealed a number of known cis-elements related to responses to salt and dehydration stress and sugar repression; predicted TATA boxes are located at -88 to -94 bp and -114 to -120 bp. The spatial expression pattern and tissue/organ specificity of AgpS1 were analysed in during development using promoter-GUS transgenic tomato plants. Based on GUS staining, the obtained sequence was proven to be the functional promoter and directed broad expression in both sink and source tissues/organs, including seedling, stem, flower, fruit stalk, fruit and root. In source leaf and early developing fruit, GUS staining was observed in all tissues, except for epidermal tissue. In contrast, GUS staining tended to be confined to vascular tissues in seedling, stem, fruit stalk and ripening fruit. In particular, a patchy staining pattern was observed in the phloem of the stem and fruit stalk, suggesting that AgpS1 is expressed in the phloem companion cells in those organs. These results also suggest that AGPase mainly functions in the vascular tissue of those organs.
[Show abstract][Hide abstract]ABSTRACT: Seed germination is the initial step of plant development. Seed priming with salt promotes seed germination in tomato (Solanum lycopersicum L.); however, the molecular and physiological mechanisms underlying the enhancement of seed germination by priming remain to be elucidated. In this study, we examined the following in seeds both during and after priming treatment: the endogenous abscisic acid (ABA) and gibberellin (GA) concentrations; the expression of genes encoding ABA catabolic and GA biosynthesis enzymes, including 8'-hydroxylase (CYP707A), copalyl diphosphate synthase (CPS), GA 20-oxidase (GA20ox) and GA 3-oxidase (GA3ox); and endosperm cap weakening enzymes, including expansin (EXP), class I β-1,3-glucanase (GulB), endo-β-mannanase (MAN) and xyloglucan endotransglucosylase (XTH). Tomato seeds were soaked for 24 h at 25 °C in the dark in 300 mM NaCl (NaCl-priming) or distilled water (hydro-priming). For both priming treatments, the ABA content in the seeds increased during treatment but rapidly decreased after sowing. Both during and after the priming treatments, the ABA levels in the hydro-primed seeds and NaCl-primed seeds were not significantly different. The expression levels of SlGA20ox1, SlGA3ox1 and SlGA3ox2 were significantly enhanced in the NaCl-primed seeds compared to the hydro-primed seeds. The GA(4) content was quantifiable after both types of priming, indicating that GA(4) is the major bioactive GA molecule involved in tomato seed germination. The GA(4) content was significantly higher in the NaCl-primed seeds than in the hydro-primed seeds 12 h after sowing and thereafter. Additionally, the peak expression levels of SlEXP4, SlGulB, SlMAN2 and SlXTH4 occurred earlier and were significantly higher in the NaCl-primed seeds than in the hydro-primed seeds. These results suggest that the observed effect of NaCl-priming on tomato seed germination is caused by an increase of the GA(4) content via GA biosynthetic gene activation and a subsequent increase in the expression of genes related to endosperm cap weakening.
Full-text Article · Mar 2012 · Plant Physiology and Biochemistry
[Show abstract][Hide abstract]ABSTRACT: Polyamines are involved in crucial plant physiological events, but their roles in fruit development remain unclear. We generated transgenic tomato plants that show a 1.5- to 2-fold increase in polyamine content by over-expressing the spermidine synthase gene, which encodes a key enzyme for polyamine biosynthesis. Pericarp-columella and placental tissue from transgenic tomato fruits were subjected to (1)H-nuclear magnetic resonance (NMR) for untargeted metabolic profiling and high-performance liquid chromatography-diode array detection for carotenoid profiling to determine the effects of high levels of polyamine accumulation on tomato fruit metabolism. A principal component analysis of the quantitative (1)H NMR data from immature green to red ripe fruit showed a clear discrimination between developmental stages, especially during ripening. Quantification of 37 metabolites in pericarp-columella and 41 metabolites in placenta tissues revealed distinct metabolic profiles between the wild type and transgenic lines, particularly at the late ripening stages. Notably, the transgenic tomato fruits also showed an increase in carotenoid accumulation, especially in lycopene (1.3- to 2.2-fold), and increased ethylene production (1.2- to 1.6-fold) compared to wild-type fruits. Genes responsible for lycopene biosynthesis, including phytoene synthase, phytoene desaturase, and deoxy-d-xylulose 5-phosphate synthase, were significantly up-regulated in ripe transgenic fruits, whereas genes involved in lycopene degradation, including lycopene-epsilon cyclase and lycopene beta cyclase, were down-regulated in the transgenic fruits compared to the wild type. These results suggest that a high level of accumulation of polyamines in the tomato regulates the steady-state level of transcription of genes responsible for the lycopene metabolic pathway, which results in a higher accumulation of lycopene in the fruit.
Full-text Article · Feb 2011 · Journal of plant physiology
[Show abstract][Hide abstract]ABSTRACT: Tomato fruit yield per area in Japan is 88% lower than that in The Netherlands, because higher-yielding tomato cultivars are bred and cultivated in The Netherlands. In this study, we investigated differences between the Japanese common cultivar 'Reiyou' and the Dutch high-yielding cultivar 'Levanzo' in fruit yield and components contributing to an increase in fruit yield using the low-node-order pinching and high-density plant training system used in Japan. Fresh and dry fruit yield of 'Levanzo' were significantly higher than those of 'Reiyou'. Estimated leaf blade area of the whole plant and solar radiation on individual leaves were higher in the 'Levanzo' canopy than in that of 'Reiyou'. Although transpiration rate and stomatal conductance were similar in the two cultivars during the latter half of the fruit development period, the photosynthetic rate in 'Levanzo' was higher than that in 'Reiyou' during the fruit development period. The difference in photosynthetic rate was because chlorophyll a and b content had not decreased in 'Levanzo' compared with that in 'Reiyou' at 50 days after flowering, corresponding to the latter half of the fruit development period. Transcriptional levels of LeSUT1, which determines sucrose loading activity in source leaves, did not differ between the two cultivars. The higher tomato fruit yield of 'Levanzo' compared with that of 'Reiyou' was caused by a higher photosynthetic rate, increased solar radiation on individual leaves, and the large sink size due to numerous fruits, but not by sucrose loading activity.
[Show abstract][Hide abstract]ABSTRACT: Salt stress improves the quality of tomato fruits. To clarify the mechanism(s) underlying this phenomenon, we investigated metabolic alterations in tomato fruits exposed to 160 mM salt, focusing on metabolism of organic acids related to the tricarboxylic acid (TCA) cycle and gamma-aminobutyric acid (GABA). Quantitative analyses revealed that most amino acids increased in response to salt stress throughout fruit development, and the effect of the stress was greater in the pericarp than in the columella, whereas organic acids did not show a remarkable tendency to salt stress. The transcript levels of 20 genes encoding enzymes of the TCA cycle and peripheral pathways were also analyzed in salt-stressed fruit. Genes responsive to salt stress could be categorized into two types, which were expressed during early development or ripening stages. During fruit development, phosphoenolpyruvate carboxylase 2 and phosphoenolpyruvate carboxykinase displayed contrasting expression patterns between early development and ripening, suggesting a switch of carbohydrate metabolism after the turning stage. Our results revealed a new metabolic pathway for GABA during the development of tomato fruits. At the start of ripening, GABA is first converted to malate via succinate semialdehyde, and it passes into a shunt through pyruvate. Then, it flows back to the TCA cycle and is stored as citrate, which contributes as a substrate for respiration during fruit maturation.
[Show abstract][Hide abstract]ABSTRACT: Salinity stress enhances sugar accumulation in tomato (Solanum lycopersicum) fruits. To elucidate the mechanisms underlying this phenomenon, the transport of carbohydrates into tomato fruits and the
regulation of starch synthesis during fruit development in tomato plants cv. ‘Micro-Tom’ exposed to high levels of salinity
stress were examined. Growth with 160 mM NaCl doubled starch accumulation in tomato fruits compared to control plants during
the early stages of development, and soluble sugars increased as the fruit matured. Tracer analysis with 13C confirmed that elevated carbohydrate accumulation in fruits exposed to salinity stress was confined to the early development
stages and did not occur after ripening. Salinity stress also up-regulated sucrose transporter expression in source leaves
and increased activity of ADP-glucose pyrophosphorylase (AGPase) in fruits during the early development stages. The results
indicate that salinity stress enhanced carbohydrate accumulation as starch during the early development stages and it is responsible
for the increase in soluble sugars in ripe fruit. Quantitative RT-PCR analyses of salinity-stressed plants showed that the
AGPase-encoding genes, AgpL1 and AgpS1 were up-regulated in developing fruits, and AgpL1 was obviously up-regulated by sugar at the transcriptional level but not by abscisic acid and osmotic stress. These results
indicate AgpL1 and AgpS1 are involved in the promotion of starch biosynthesis under the salinity stress in ABA- and osmotic stress-independent manners.
These two genes are differentially regulated at the transcriptional level, and AgpL1 is suggested to play a regulatory role in this event.
Full-text Article · Dec 2009 · Journal of Experimental Botany
[Show abstract][Hide abstract]ABSTRACT: One direct way to identify a gene and its function is a forward genetic approach based on mutation analyses. To accumulate genetic resources for breeding and functional genomics in the tomato, we generated 6,347 lines of an M2 population with 300 Gy of gamma-ray irradiation in the inbred miniature dwarf variety 'Micro-Tom.' In total, 6,301 M2 lines were screened based on morphological alteration and brix-aberration, and 237 lines were selected as mutant candidates at the first screening. Subsequent screening of the self-fertilized M3 and M4 progeny yielded 24 lines of morphological mutants and 11 lines of aberrant brix mutants. Segregation data suggested that most of the mutant lines had single recessive mutations, with the exception of two lines. The chlorophyll mutation ratio in germinated M 2 seedlings was 0.37% and the actual mutant frequency was 0.5%. The selected mutant lines exhibited a wide range of mutations, including whole plant properties with a severe phenotype, which allowed for more efficient screening of knockout mutants. Three characterized mutants, pale leaf, pink, and short root are also described.