Nobuyuki Fukuoka

The occurrence of internal browning induced by high soil temperature treatment and its physiological function in Raphanus root

Abstract Internal browning (IB), consisting of a brown coloration at the central stele of the root of radish ( Raphanus sativus ), is a physiological disorder induced by high temperature conditions. In this study, different durations of high soil temperature treatment were compared on the basis of their influence on both the occurrence of IB and its related enzyme fluctuations. In affected roots, the degree of IB was positively connected with a variation in the enzyme activities of polyphenol biosynthesis and the ascorbate–glutathione cycle. Accordingly, the heat stress led to significant increases of glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), l -phenylalanine ammonia-lyase (PAL), as well as polyphenol oxidase (PPO) activities and IB formation, in the susceptible cultivar Fukutenka, but not in the resistant cultivar Minowase. Although no correlation was found between catalase (CAT) activity and IB occurrence, a pronounced increase in ascorbate peroxidase (APX) and decreases of dehydroascorbate reductase (DHAR) and glutathione reductase (GSHR) activities were detected in roots suffering from IB. These observations strongly suggest that detoxification of H 2 O 2 by polyphenol biosynthesis was operating, and that the pentose phosphate pathway was activated in order to supply the NADPH needed for its biosynthesis.

Effects of soil temperature on hollowness in Japanese radish (Raphanus sativus L. cv. 'Gensuke')

Abstract To examine the effect of high soil temperature on the development of hollowing in the root of radish, seeds were sown in the field on 27 May, 10 July and 16 August 1992. Plants of the July sowing were subjected to soil temperatures above 32 °C during the middle of the growth period. Root weight was reduced and hollowness occurred in roots from the July sowing. Roots of the July sowing had the greatest concentration of vessels in the central region. Lignin formed in walls surrounding the central cavity only in roots of the July sowing. In a second experiment, seeds were sown on 30 April 1991, in plastic pots with heating cables to increase soil temperature. Heating from the 16th day after sowing (DAS) to the 30th DAS and from the 31st to the 45th DAS slowed root growth, and produced some hollow cavities, caused more vessels to form in the central area, and promoted lignification near the central cavity. In contrast, heating from the 10th to the 15th DAS or from the 46th to the 60th DAS produced roots with no hollow cavities, and resulted in the production of a smaller number of vessels in the central region of the root. The heating during these periods had no effect on lignin formation. In conclusion, a large hollow cavity develops in the central region of the root as a result of active lignin formation in the surrounding cells induced by heating in the middle of the growth period.

Suppressive effects of CPPU on lignification of xylem parenchymatous cells and on hollowing in the root of Japanese radish (Raphanus sativus L.)

Abstract To see the effects of CPPU on the development of hollowing and lignin formation in the roots of Japanese radish ( Raphanus sativus L.) ‘Wakayama’, ‘Yamada’ and ‘Osakashijunichi’, solutions of 5 mg l −1 of 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU) were sprayed on the leaves of plants every 3 days from the 11th to the 40th days after sowing (DAS). The size of the hollow cavity in the roots of ‘Wakayama’, ‘Yamada’ and ‘Osakashijunichi’ became significantly smaller with CPPU treatment, in contrast to that of the control. Vessels in the roots of all cultivars became more sparsely arranged with CPPU treatment, compared with vessels in the root of the control. In addition, the central region of the root of all cultivars treated with CPPU was filled with parenchymatous cells and these cells showed little staining reaction to phloroglucinol-HCl reagent. In contrast, in the controls, there was clear evidence of the presence of lignin in parenchymatous cells facing the intercellular space.

Role of endogenous cytokinin in the development of hollowing in the root of Japanese radish (Raphanus sativus L.)

Abstract The size of the hollow cavity increased with time in plants of the early sown plot (ESP), whereas its size remained almost unchanged throughout the growth period in plants of the late sown plot (LSP). The daily maximum soil temperatures in the ESP were 6–12 °C higher than those in the LSP. A much higher level of cytokinin was detected in the ammonia eluate fraction than in the acidic ethyl acetate fraction and none was detected in the effluent and washings. Cytokinin levels in the ammonia eluate fraction and the acidic ethyl acetate fraction were low in roots from the ESP in comparison with those from the LSP. A much larger size of hollow cavity was observed at any days after sowing (DAS) in control roots, that were exposed to temperatures of 30 °C and above for 30 days, than in roots grown at lower soil temperatures between 25 °C and 30 °C throughout the growth period. Cytokinin levels in control roots grown at high soil temperature were consistently lower than those in the roots grown at low soil temperature. The production of endogenous cytokinin in the roots of cvs. Sobuto and Fukumi, cultivars that were prone to hollowness, was reduced at higher soil temperatures, while in cultivars that were resistant to hollowness cytokinin production was higher. The role of endogenous cytokinin in the development of hollowing is discussed.

Enzyme activity changes in relation to internal browning of Raphanus roots sown early and late

Summary Early sown radish seedlings were subjected to heat stress from the middle to late growth stage. Roots exhibiting internal browning (IB) were more prevalent in the susceptible cultivars whose seeds were sown early in the summer compared with those which were sown late. However, IB occurrence in the resistant ones was not nearly so marked regardless of the sowing date. In the IB susceptible cultivars, higher levels of L-phenylalanine ammonia-lyase (PAL) and polyphenol oxidase (PPO) activities were detected in roots from the early sown plot (ESP), but these enyzme inductions did not occur to a significant extent in the resistant ones. Roots suffering internal browning were also detected to have pronounced increases in ascorbate peroxidase (APX) activity, but decreases in both dehydroascorbate reductase (DHAR) and glutathione reductase (GHAR) activities. These observations strongly suggest that a decline in H2O2 decomposing capacity of the ascorbate-glutathione cycle by heat stress is responsible for the activation of H2O2 detoxyifying pathway derived from PPO.

Relationship between the development of hollowing and the separation of vessel sectors in the central region of the root of Japanese radish (Raphanus sativus L.)

Abstract The present investigations were carried out to clarify the relationship between the separation of two vessel sectors, especially in the later growth period, and the development of hollowing in the root and the effect of varying the cultural conditions. Roots with hollow cavities were more prevalent in plants whose seeds were sown early in the summer compared with those sown later. The gap between the bifurcated vessel sectors became larger in roots sown earlier. A larger number of hollow roots was observed in the sparsely planted plot than in the high planting density plot, and the gap between the bifurcated vessel sectors became larger in roots at low planting density. Late defoliation, after rapid thickening growth, produced a large number of hollow roots, while early defoliation prevented the root from hollowing due to slower thickening growth. The gap between the bifurcated vessel sectors became wider the later the time of defoliation. A larger number of hollow roots occurred in proportion to increasing amounts of basal fertilizer and larger amounts of basal fertilizer application resulted in a wider space between the two vessel sectors. These facts strongly indicate that the development of hollowing may pass through two indispensable steps. First is the obstruction of cell formation inside the intercellular air space during the middle of the growth period, caused by high soil temperatures. Second is the rapid divergence of bifurcated vessel sectors during the later growth period as a result of enhanced thickening growth. If the first condition is present, the second is the determining factor for the development of hollowing.

Relationship between the concentration of pectin-like substances and the severity of internal browning in radish (Raphanus sativus L.) roots induced by high soil temperature

Summary In white radish (Raphanus sativus L.) roots, internal browning (IB), a physiological disorder characterised by a brown discolouration in the central stele, is frequently associated with summer-sown crops. In this study, nine cultivars of white radish were studied to clarify the association between pectin-like substances and the severity of IB. Radish cultivars could be classified into three types according to the severity of the IB disorder: resistant, moderately susceptible, or susceptible. An increase in the severity of the disorder was accompanied by a parallel increase in polyphenol oxidase activity.The mean cell length in each IB-resistant cultivar was < 135 µm, while the mean cell length in each moderately susceptible or susceptible cultivar, with one exception, was 1.2- to 1.8-times larger. Although there was no association between the severity of IB and the concentration of water-soluble pectin or pectic acid, the IB-resistant cultivars contained a higher concentration of protopectin than the moderately susceptible or susceptible cultivars. These results suggest that IB in radish roots is closely associated with the concentration of protopectin, which varies according to cell size.

Changes in polyphenol biosynthesis induced in Gynura bicolor DC. leaves by infrared irradiation

Summary The aim of the present study was to determine the effects of infrared irradiation (IRI) on polyphenol biosynthesis and anti-oxidant activity in Gynura bicolor DC. leaves. To achieve this, infrared light lamps were placed 75 cm above the top of the canopy, and the plants were covered with a black nylon mesh to exclude the influence of natural light. Higher activities of ascorbate peroxidase (APX), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PDH), L-phenylalanine ammonia-lyase (PAL), and polyphenol oxidase (PPO) were detected in leaves when plants were subjected to IRI. Although IRI resulted in a significant decrease in anthocyanin content, HPLC analysis of the phenolic components showed a higher content of chlorogenic acid that was accompanied by a higher 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) radical-scavenging activity in IRI-treated leaves. These observations suggest that IRI causes oxidative stress and stimulates the activities of enzymes that regulate non-flavonoid polyphenol biosynthesis in G. bicolor leaves.

Histochemical observations and gene expression changes related to internal browning in tuberous roots of sweet potato (Ipomea batatas)

The mechanism underlying internal browning (IB), or brown discoloration, of the central region of tuberous roots of sweet potato (Ipomoea batatas) was examined. IB disorder begins in roots from approx. 90 days after transplanting, and the severity increases significantly with time. IB damage initially occurs in cells around the secondary vascular tissue, and the area per cell occupied by starch grains in this region was larger than in the unaffected region. High levels of reducing sugars, polyphenol oxidase (PPO) activities, chlorogenic acid, and hydrogen peroxide (H2O2) were detected in cells from the IB damaged regions. The content of sugar and polyphenols was higher in disks (transverse sections) with larger amounts of damaged tissues than in disks of sound root. The transcript levels of acid invertase (IbAIV) tended to be higher with greater IB severity, whereas fluctuation patterns of ADP-glucose pyrophosphorylase (IbAGPase), granule bound starch synthase (IbGBSS), and starch branching enzyme 1 (IbSBE1) were lower with higher IB severity. These observations suggest that the incidence of IB disorder in sweet potato is largely dependent on the excessive generation of reactive oxygen species (ROS) in cells around the secondary vascular tissues due to the abundant accumulation of sugar and/or starch grains during the root maturation period.

Easy Method for the Approximate Quantitation of 4-Methylthio-3-Butenyl Isothiocyanate of Daikon (Raphanus sativus L.)

Daikon (Japanese white radish, Raphanus sativus L.) is widely consumed across Japan. The characteristic pungent flavor of daikon is derived from 4-methylthio-3-butenyl isothiocyanate. Herein, we propose an easy method for the approximate quantitation of this compound. This method does not require an expensive analytical apparatus such as a high-performance liquid chromatograph but only an ultra violet-visible spectrophotometer to measure the absorbance of a daikon n-hexane extract, at 231 nm, against a standard. The estimation of the concentration of 4-methylthio-3-butenyl isothiocyanate by this method can sort the pungency of daikon, because this isothiocyanate is a principal isothiocyanate of daikon.