Sound waves affect the total flavonoid contents in Medicago sativa, Brassica oleracea and Raphanus sativus sprouts.

BACKGROUND: Sound waves are emerging as a potential biophysical alternative to traditional methods for enhancing plant growth and phytochemical contents. However, little information is available on the improvement of the concentration of functional metabolites like flavonoids in sprouts using sound waves. In this study, different frequencies of sound waves with short and long exposure times were applied to three important varieties to improve flavonoid content. The aim of this study was to investigate the effect of sound waves on flavonoid content on the basis of biochemical and molecular characteristics. RESULTS: We examined the effects of various sound wave treatments (250 Hz to 1.5 kHz) on flavonoid production in alfalfa (Medicago sativa), broccoli (Brassica oleracea) and red young radish (Raphanus sativus). The results showed that sound wave treatments differentially altered the total flavonoid contents depending upon the growth stages, species and frequency of and exposure time to sound waves. Sound wave treatments of alfalfa (250 Hz), broccoli sprouts (800 Hz) and red young radish sprouts (1 kHz) increased the total flavonoid content by 200%, 35% and 85%, respectively, in comparison with untreated control. Molecular analysis showed that sound waves induce the expression of genes of the flavonoid biosynthesis pathway, which positively corresponds to the flavonoid content. Moreover, the sound wave treatment significantly improves the antioxidant efficiency of sprouts. CONCLUSIONS: The significant improvement of flavonoid content in sprouts with sound waves makes their use a potential and promising technology for the production of agriculture-based functional foods. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Evaluation of mature miR398 family, expression analysis and the post-transcriptional regulation evidence in gamma-irradiated and nitrogen-stressed Medicago sativa seedlings.

BACKGROUND: The miR398 (microRNA398) posttranscriptionally regulates the superoxide dismutase (Cu/Zn-SOD). MATERIALS AND METHODS: The miR398 level was quantitated in gamma-irradiated (20Gy and 200Gy) and nitrogen-stressed seedlings by stem-loop RT-PCR (Real Time-Polymerase chain reaction). The positional preference of nucleotide (nt) for miR398 families and new targets was done. The SOD enzyme was assayed in native PAGE (Polyacrylamide gel electrophoresis). RESULTS: A relative increase in miR398-3p expression in the roots, and reduction in shoots for gamma-irradiated tissue and downregulation in miR398-5p in shoots is noted. The nitrogen stress shows upregulation of miR398-3p in roots and shoots, whereas the expression of miR398-5p is upregulated in roots and downregulated in shoots. Positional preference in miR398-3p for 1-14 nt is 90% conserved unlike miR398-5p where no nucleotide positional preference is seen. Targets obtained were functionally characterized. CONCLUSIONS: The mature miR398-5p and miR398-3p levels estimated in gamma-irradiated (20 and 200Gy) and nitrogen-stressed Medicago sativa seedlings show differential levels in roots and shoots. Native PAGE of Cu/Zn-SOD suggests its negative correlation with miR398 in shoots of irradiated and nitrogen-stressed samples. The nucleotide preferences for the nucleotide for a given position and functional characterization of targets are reported herein.

Overexpression of Medicago sativa TMT elevates the α-tocopherol content in Arabidopsis seeds, alfalfa leaves, and delays dark-induced leaf senescence.

Alfalfa (Medicago sativa L.) is a major forage legume for livestock and a target for improving their dietary quality. Vitamin E is an essential vitamin that animals must obtain from their diet for proper growth and development. γ-tocopherol methyltransferase (γ-TMT), which catalyzes the conversion of δ- and γ-tocopherols (or tocotrienols) to ß- and α-tocopherols (or tocotrienols), respectively, is the final enzyme involved in the vitamin E biosynthetic pathway. The overexpression of M. sativa L.'s γ-TMT (MsTMT) increased the α-tocopherol content 10-15 fold above that of wild type Arabidopsis seeds without altering the total content of vitamin E. Additionally, in response to osmotic stress, the biomass and the expression levels of several osmotic marker genes were significantly higher in the transgenic lines compared with wild type. Overexpression of MsTMT in alfalfa led to a modest, albeit significant, increase in α-tocopherol in leaves and was also responsible for a delayed leaf senescence phenotype. Additionally, the crude protein content was increased, while the acid and neutral detergent fiber contents were unchanged in these transgenic lines. Thus, increased α-tocopherol content occurred in transgenic alfalfa without compromising the nutritional qualities. The targeted metabolic engineering of vitamin E biosynthesis through MsTMT overexpression provides a promising approach to improve the α-tocopherol content of forage crops.

The isolation and identification of a light-induced protein in alfalfa sprouts and the cloning of its specific promoter.

We used 2D-PAGE to isolate a light-induced protein (AL-A) that is expressed abundantly in light-growth alfalfa sprouts. The seven amino acids of the N-terminal region of the protein were identified, and we searched for the protein in GenBank using the BLAST program. The results of the homology analysis showed that the amino acid sequence of the isolated protein is most similar to one from a pea plastocyanin. To identify the protein, we amplified and sequenced the DNA fragment encoding AL-A from genomic alfalfa DNA. We found that the AL-A gene was highly homologous (90%) to the sequences from the pea plastocyanin via multiple alignments, and the deduced protein precursor was predicted to be chloroplast-specific via the ChloroP computer program. The protein was named alfalfa-plastocyanin (AL-P). It was characterized as being a light-inducible protein, and RT-PCR analysis showed that AL-P mRNA transcription only occurred in the leaves of the alfalfa plant and the alfalfa seedlings growth in lighted conditions. PCR was also used to amplify the DNA fragment encoding the AL-P promoter (AL-Pp) from genomic alfalfa DNA. PlantCARE analysis of the promoter sequence indicated that both a typical TATA box and a CAAT box were located in the promoter sequence, and some of the cis-elements that are responsible for light responsiveness were also identified within this promoter region. The AL-P gene promoter fused to the ß-glucuronidase (GUS) reporter gene has been examined for expression in transgenic alfalfa seedlings. Our findings have a potential application in plant genetic engineering; the AL-Pp may be used to drive the expression of heterologous genes in transgenic alfalfa plants.

The effect of microwave treatment on animal fodder.

Preliminary research has suggested that in vitro dry matter disappearance (DMD) of some poor quality animal fodder materials can be improved by microwave treatment. Laboratory scale experiments revealed that dry matter percentage of Lucerne hay increased by 1.7% as microwave treatment time increased from 0 to 80 seconds. The in vitro DMD of lucerne hay increased by 14.9% during the same microwave treatment. In addition it was also demonstrated that microwave treatment significantly increased starch digestion of oats compared to the control samples. These experiments were followed up with a larger sample experiment in which 25 kg bags of Lucerne fodder were treated for 7.5, 15, 22.5 or 30 minutes in an experimental 6 kW microwave chamber. Dry matter percentage increased by 7.2% as microwave treatment time increased from 0 to 30 minutes. Microwave treatment significantly increased DMD during an in vitro digestion study; however there were no significant differences between the various microwave treatment times. The 15 minute treatment resulted in the greatest increase in dry matter disappearance (5.9%). The crude protein retained in the digestion residues increased by 19.2% as microwave treatment increased from 0 to 30 minutes. These laboratory studies were followed up with an animal response study in which a Merino sheep group being fed the microwave treated lucerne gained 8.1% of their initial body weight by the end of the trial compared to a 0.4% increase in body weight for the control group.

Effects of gamma irradiation on ruminal DM and NDF degradation kinetics of alfalfa hay.

The effects of gamma irradiation on ruminal dry matter, Neutral Detergent Fiber (NDF) degradation of alfalfa hay were investigated. Alfalfa hay samples were irradiated by gamma irradiator at doses of 50, 100 and 150 kGy under identical conditions of temperature and humidity. Nylon bags of untreated or irradiated samples were suspended in the rumen of three Taleshi bulls for up to 96 h and resulting data were fitted to non-linear degradation model to calculate degradation parameters. Results indicated that the washout fractions of dry matter and NDF increased linearly (p < 0.001) with increasing irradiation dose. The b fraction and the degradation rate of the b fraction (c) of DM and NDF were the highest at 50 kGy dose. Effective degradability of DM and NDF increased linearly with increasing irradiation dose. Gamma irradiation at doses of 50, 100 and 150 kGy increased the effective NDF degradability of alfalfa hay at rumen outflow rate of 0.05 h(-1) by about 8, 11 and 12%, respectively. Gamma irradiation affects on the hydrogenic bonds and with theirs breakdowning causes the wander-valls power weaken, that results in the degradation of cellulose and increasing of DM and NDF degradability.

Contribution of current photosynthates to root respiration of non-nodulated Medicago sativa: effects of light and nitrogen supply.

The effects of light (PFD) and nitrogen (N) supply on root respiration of new C (currently assimilated carbon, R(new)) and old C ( R(old)) were analysed in non-nodulated Medicago sativa. Plants were pre-treated with high/low PFD and high/low N supply with a regular 16/8 h light/dark cycle. Five to eight weeks after planting current photosynthates were labelled with (13)C and their contribution to root respiration was continuously measured during a 24 h day/night cycle. PFD conditions during labelling were either those of the pre-treatments (control, 25 or 6 mol m(-2) d(-1)) or, for high PFD plants, 6 mol m(-2) d(-1) by shortening the photoperiod or reducing irradiance. The fraction of new C in the respiratory CO2 increased during the light period, but remained constant in the dark period. In control plants, R(new) contributed 40 % to the daily root respiration in high PFD/high N conditions. Continuously low PFD increased (50 %) and low N decreased (26 %) the contribution of R(new). Exposing plants from high PFD pre-treatments to a short photoperiod or to low PFD stimulated R(old), indicating mobilisation of reserve C. This stimulation was more pronounced in plants with high N supply than in those with low N supply. Comparison with other legumes suggested that R(new) in root respiration was mainly defined by the ratio between the assimilatory capacity of the shoots and the maintenance costs of roots with a short-term capacity of buffering respiratory demand by mobilisation of reserves in situations of fluctuating PFD.

Changes in growth and antioxidant status of alfalfa sprouts during sprouting as affected by gamma irradiation of seeds.

Viking 3000 alfalfa seeds irradiated with gamma rays to doses of 0, 1, 2, 3, or 4 kGy were sprouted and allowed to grow for up to 8 days at 23 degrees C. Germination, growth (yield and length), antioxidant capacity, and ascorbic acid (AA) were measured during sprouting. Results showed percent germination of the seeds and the rates of growth of the sprouts were inversely related to the radiation dose absorbed by the seeds. Both antioxidant capacity and AA content expressed on a fresh weight basis decreased during growth of the sprouts. Sprouts grown from irradiated seeds had greater antioxidant capacity and AA content on a fresh weight basis than those grown from nonirradiated seeds. However, when the nutritive values were expressed on a per gram of seed basis, irradiation had no effect on the nutritive values of sprouts.

Chemical and irradiation treatments for killing Escherichia coli O157:H7 on alfalfa, radish, and mung bean seeds.

In this study, the effectiveness of dry-heat treatment in combination with chemical treatments (electrolyzed oxidizing [EO] water, califresh-S, 200 ppm of active chlorinated water) with and without sonication in eliminating Escherichia coli O157:H7 on laboratory-inoculated alfalfa, radish, and mung bean seeds was compared with that of dry-heat treatment in combination with irradiation treatment. The treatment of mung bean seeds with EO water in combination with sonication followed by a rinse with sterile distilled water resulted in reductions of approximately 4.0 log10 CFU of E. coli O157:H7 per g. whereas reductions of ca. 1.52 and 2.64 log10 CFU/g were obtained for radish and alfalfa seeds. The maximum reduction (3.70 log10 CFU/g) for mung bean seeds was achieved by treatment with califresh-S and chlorinated water (200 ppm) in combination with sonication and a rinse. The combination of dry heat, hot EO water treatment, and sonication was able to eliminate pathogen populations on mung bean seeds but was unable to eliminate the pathogen on radish and alfalfa seeds. Other chemical treatments used were effective in greatly reducing pathogen populations on radish and alfalfa seeds without compromising the quality of the sprouts, but these treatments did not result in the elimination of pathogens from radish and alfalfa seeds. Moreover, a combination of dry-heat and irradiation treatments was effective in eliminating E. coli O157:H7 on laboratory-inoculated alfalfa, radish, and mung bean seeds. An irradiation dose of 2.0 kGy in combination with dry heat eliminated E. coli O157:H7 completely from alfalfa and mung bean seeds, whereas a 2.5-kGy dose of irradiation was required to eliminate the pathogen completely from radish seeds. Dry heat in combination with irradiation doses of up to 2.0 kGy did not unacceptably decrease the germination percentage for alfalfa seeds or the length of alfalfa sprouts but did decrease the lengths of radish and mung bean sprouts.

Quality of irradiated alfalfa sproutst.

Alfalfa (Medicago sativa L.) sprouts were irradiated with gamma rays at doses of 0, 0.85, 1.71, and 2.57 kGy at 5 degrees C. then stored at 6 degrees C for 14 days. Antioxidant power, total ascorbic acid (TAA) (ascorbic acid plus dehydroascorbic acid), carotenoid, chlorophyll, and color were measured at 1, 7, and 14 days of storage. Antioxidant power increased linearly with radiation dose at both 1 and 7 days of storage. Irradiation had minimal effect on TAA content when compared with the decrease in TAA content during storage. Carotenoid content of sprouts irradiated at 1.71 and 2.57 kGy was higher than that of control at 7 days of storage. Irradiation did not have a consistent effect on chlorophyll content or color.

Alfalfa seed germination and yield ratio and alfalfa sprout microbial keeping quality following irradiation of seeds and sprouts.

Foods can be treated with gamma radiation, a nonthermal food process, to inactivate foodborne pathogens and fungi, to kill insects on or in fruits and vegetables, and to increase shelf life. Gamma irradiation is especially well suited for these treatments because of its ability to penetrate commercial pallets of foods. Irradiated fruits, vegetables, poultry, and hamburger have been received favorably by the public and are now available in supermarkets. The use of irradiation on fresh alfalfa sprouts was studied to determine its effect on keeping quality as related to aerobic microbial load. After an irradiation dose of 2 kGy, the total aerobic count decreased from 10(5-8) to 10(3-5) CFU/g, and the total coliform counts decreased from 10(5-8) to 10(3-0) CFU/g. The results showed that the sprouts maintained their structure after irradiation, and the keeping quality was extended to 21 days, which is an increase of 10 days from the usual shelf life. The effect of various doses of irradiation on alfalfa seeds as measured by percent germination and yield ratio (wt/wt) of sprouts was determined. There was little effect on the percent germination, but as the dose increased, the yield ratio of alfalfa sprouts decreased. As the length of growing time increased, so did the yield ratio of the lower dose irradiated seeds (1 to 2 kGy). The irradiation process can be used to increase the shelf life of alfalfa sprouts, and irradiating alfalfa seeds at doses up to 2 kGy does not unacceptably decrease the yield ratio for production of alfalfa sprouts.

Nitrogen assimilation in alfalfa: isolation and characterization of an asparagine synthetase gene showing enhanced expression in root nodules and dark-adapted leaves.

Asparagine, the primary assimilation product from N2 fixation in temperate legumes and the predominant nitrogen transport product in many plant species, is synthesized via asparagine synthetase (AS; EC 6.3.5.4). Here, we report the isolation and characterization of a cDNA and a gene encoding the nodule-enhanced form of AS from alfalfa. The AS gene is comprised of 13 exons separated by 12 introns. The 5' flanking region of the AS gene confers nodule-enhanced reporter gene activity in transformed alfalfa. This region also confers enhanced reporter gene activity in dark-treated leaves. These results indicate that the 5' upstream region of the AS gene contains elements that affect expression in root nodules and leaves. Both AS mRNA and enzyme activity increased approximately 10- to 20-fold during the development of effective nodules. Ineffective nodules have strikingly reduced amounts of AS transcript. Alfalfa leaves have quite low levels of AS mRNA and protein; however, exposure to darkness resulted in a considerable increase in both. In situ hybridization with effective nodules and beta-glucuronidase staining of nodules from transgenic plants showed that AS is expressed in both infected and uninfected cells of the nodule symbiotic zone and in the nodule parenchyma. RNA gel blot analysis and in situ hybridization results are consistent with the hypothesis that initial AS expression in nodules is independent of nitrogenase activity.

Isolation of high-molecular-weight plant DNA for DNA damage quantitation: relative effects of solar 297 nm UVB and 365 nm radiation.

Quantitation of UV-induced DNA damages in nanogram quantities of non-radioactive DNA from irradiated plants by gel electrophoresis requires a prompt, efficient, high-yield method of isolating DNA yielding high-molecular-weight, enzymatically digestible DNA. To meet these criteria we devised a high-yield method for isolating from plant tissue, DNA whose single-strand molecular length is greater than about 170 kb. Leaf tissue is embedded in agarose plugs, digested with Proteinase K in the presence of detergent, and treated with phenylmethylsulfonyl fluoride (PMSF). The agarose plugs are then soaked with buffer appropriate to the desired enzyme treatment. Evaluation of the DNA on neutral and alkaline gels indicates its high molecular length and low frequency of single-strand breaks. The DNA can be digested with damage-specific and other endonucleases. The method is especially suitable for DNA damage quantitation, as tissue processing is carried out immediately after harvesting (allowing DNA lesion measurement at precisely known times after irradiation), and many samples can be easily handled at once. It should also be useful for molecular analysis of large numbers of plant samples available only in small quantities. We here use this method to quantitate DNA damage induced by 297 and 365 nm radiation, and calculate the relative damaging effects of these wavebands in today's solar spectrum.

Quantitation of pyrimidine dimers in DNA from UVB-irradiated alfalfa (Medicago sativa L.) seedlings.

Depletion of stratospheric ozone will increase the solar ultraviolet radiation in the range from 290-320 nm (UVB) that reaches the surface of the earth, placing an increased UV burden on exposed organisms. One consequence of increased UVB may be decreased productivity of crop plants. A principal lesion caused by UV in DNA is the cyclobutyl pyrimidine dimer. We have adapted a method for measuring these dimers in nanogram quantities of non-radioactive DNA for use in UV-irradiated plants. We find that biologically relevant doses of broad band UVB radiation induce easily detectable frequencies of pyrimidine dimers in the DNA of irradiated alfalfa sprout leaves and that the dose response for dimer formation is linear up to doses of at least 690 J m-2. We also find easily measurable frequencies of dimers in the leaves of seedlings grown in glass filtered sunlight but not exposed to additional UVB, suggesting that significant numbers of dimers are formed in plants exposed to normal sunlight.