Naoyoshi Kawano

Overexpression of monodehydroascorbate reductase in transgenic tobacco confers enhanced tolerance to ozone, salt and polyethylene glycol stresses

Ascorbate (AsA) is a major antioxidant and free-radical scavenger in plants. Monodehydroascorbate reductase (MDAR; EC 1.6.5.4) is crucial for AsA regeneration and essential for maintaining a reduced pool of AsA. To examine whether an overexpressed level of MDAR could minimize the deleterious effects of environmental stresses, we developed transgenic tobacco plants overexpressing Arabidopsis thaliana MDAR gene (AtMDAR1) in the cytosol. Incorporation of the transgene in the genome of tobacco plants was confirmed by PCR and Southern-blot analysis and its expression was confirmed by Northern- and Western-blot analyses. These transgenic plants exhibited up to 2.1-fold higher MDAR activity and 2.2-fold higher level of reduced AsA compared to non-transformed control plants. The transgenic plants showed enhanced stress tolerance in term of significantly higher net photosynthesis rates under ozone, salt and polyethylene glycol (PEG) stresses and greater PSII effective quantum yield under ozone and salt stresses. Furthermore, these transgenic plants exhibited significantly lower hydrogen peroxide level when tested under salt stress. These results demonstrate that an overexpressed level of MDAR properly confers enhanced tolerance against ozone, salt and PEG stress.

Detection, isolation and characterization of a root-exuded compound, methyl 3-(4-hydroxyphenyl) propionate, responsible for biological nitrification inhibition by sorghum (Sorghum bicolor)

Nitrification results in poor nitrogen (N) recovery and negative environmental impacts in most agricultural systems. Some plant species release secondary metabolites from their roots that inhibit nitrification, a phenomenon known as biological nitrification inhibition (BNI). Here, we attempt to characterize BNI in sorghum (Sorghum bicolor). In solution culture, the effect of N nutrition and plant age was studied on BNI activity from roots. A bioluminescence assay using recombinant Nitrosomonas europaea was employed to determine the inhibitory effect of root exudates. One major active constituent was isolated by activity-guided HPLC fractionations. The structure was analysed using NMR and mass spectrometry. Properties and the 70% inhibitory concentration (IC(70)) of this compound were determined by in vitro assay. Sorghum had significant BNI capacity, releasing 20 allylthiourea units (ATU) g(-1) root DW d(-1). Release of BNI compounds increased with growth stage and concentration of supply. NH4+ -grown plants released several-fold higher BNI compounds than NO3- -grown plants. The active constituent was identified as methyl 3-(4-hydroxyphenyl) propionate. BNI compound release from roots is a physiologically active process, stimulated by the presence of NH4+. Methyl 3-(4-hydroxyphenyl) propionate is the first compound purified from the root exudates of any species; this is an important step towards better understanding BNI in sorghum.

Blocking ethylene perception enhances flooding tolerance in rice seedlings

Large areas of rainfed lowlands in South and Southeast Asia annually experience short durations of flash flooding during the rice-growing season, which dramatically affect plant survival and productivity. Submergence-intolerant cultivars usually show progressive leaf chlorosis, which could be triggered by ethylene produced during submergence. An ethylene inhibitor, 1-methyl cyclopropene (MCP), was used to evaluate the effect of ethylene on chlorophyll degradation and plant survival. Seedlings of two cultivars, FR13A (tolerant) and IR42 (intolerant) either untreated or treated with MCP for 5 h before submergence, were submerged for 6 d. Chlorophyll content and activity and gene expression of chlorophyllase, the first enzyme involved in chlorophyll degradation, were determined during submergence and recovery, and survival scores were recorded after 21 d of recovery. MCP treatment decreased chlorophyll degradation, lowered the activity and gene expression of chlorophyllase, and improved seedling survival in IR42, but with no effect on underwater relative shoot expansion. Chlorophyllase enzyme activity and gene expression were lower in FR13A, suggesting that this cultivar might have reduced ethylene level or sensitivity. Manipulation of ethylene synthesis or sensitivity through molecular approaches might therefore help improve tolerance of flash flooding in rice.

Importance of active oxygen-scavenging system in the recovery of rice seedlings after submergence

Abstract Active oxygen species (AOS) can damage cells by mutation of nucleic acids and derivatives, dysfunction of proteins, oxidation of lipids to peroxides that make membranes leaky. Excessive illumination during recovery of submerged rice seedlings may induce an oxidative stress because of abnormal amount of AOS such as hydrogen peroxide (H 2 O 2 ), superoxide and hydroxyl radicals. Though submergence-tolerant FR13A and -intolerant IR42 both have comparable H 2 O 2 production during recovery, FR13A had less lipid peroxidation and had maintained a considerably higher level of ascorbate antioxidant during recovery than IR42. Only glutathione reductase (GR) activity had significantly different levels between the two cultivars with greater level in FR13A. The high levels of ascorbate and GR activity ensure a better operation of ascorbic acid–glutathione cycle that helps detoxify H 2 O 2 more efficiently in FR13A. However, in IR42 where this cycle is expected to operate at much slower rate because of limited ascorbate and GR activity, H 2 O 2 becomes readily available for lipid peroxidation, producing more MDA. Our data reveal the involvement of active oxygen-scavenging system during recovery of submerged rice seedlings under excessive illumination.

Morphological and physiological responses of rice seedlings to complete submergence (flash flooding)

BACKGROUND AND AIMS Reducing damage to rice seedlings caused by flash flooding will improve the productivity of rainfed lowland rice in West Africa. Accordingly, the morphological and physiological responses of different forms of rice to complete submergence were examined in field and pot experiments to identify primary causes of damage. METHODS To characterize the physiological responses, seedlings from a wide genetic base including Oryza sativa, O. glaberrima and interspecific hybrids were compared using principle component analysis. KEY RESULTS Important factors linked to flash-flood tolerance included minimal shoot elongation underwater, increase in dry matter weight during submergence and post-submergence resistance to lodging. In particular, fast shoot elongation during submergence negatively affected plant growth after de-submergence. Also shoot-elongating cultivars showed a strong negative correlation between dry matter weight of the leaves that developed before submergence and leaves developing during submergence. CONCLUSIONS Enhancement of shoot elongation during submergence in water that is too deep to permit re-emergence by small seedlings represents a futile escape strategy that takes place at the expense of existing dry matter in circumstances where underwater photosynthetic carbon fixation is negligible. Consequently, it compromises survival or recovery growth once flood water levels recede and plants are re-exposed to the aerial environment. Tolerance is greater in cultivars where acceleration of elongation caused by submergence is minimal.

Comparison of adaptability to flash flood between rice cultivars differing in flash flood tolerance

Abstract The mechanism of flash flood tolerance was investigated by using a flash flood-intolerant rice cultivar (Oryza sativa), IR42 and a tolerant one, FR13A. The photosynthetic rate of the rice seedlings from both types of cultivars decreased during submergence, whereas the photosynthetic rate of FR13A remained higher than that of IR42. The decrease in photosynthesis in IR42 was partially due to a higher inhibition of the activities of ribulose-1,5-bisphosphate carboxylase, fructose-1,6-bisphosphatase, and glyceraldehyde-3-phosphate dehydrogenase, than in FR13A. After submergence receded (hereafter referred as “desubmergence”), both types of submerged rice cultivars experienced oxidative damage. However the oxidation of the lipids and proteins in FR13A was maintained at a lower level than that in IR42 after desubmergence. On the other hand, the superoxide dismutase activity in FR13A increased more than that in IR42 after desubmergence. Moreover, the content of total ascorbate (reduced and oxidized ascorbates), a main antioxidant increased in FR13A, while in IR42 the content remained low after desubmergence. It is assumed that the photosynthetic activity during submergence may be related to flash flood tolerance. Moreover, flash flood tolerance requires a rapid stimulation of antioxidant systems after desubmergence.

Relationship between shoot elongation and dry matter weight during submergence in Oryza sativa L. and O. glaberrima Steud. rice cultivars.

Abstract Rice plants are damaged by flash floods with a rapid increase in water level caused by a heavy rain. However, rice plants cope with the flash floods either by an “escape strategy” involving rapid shoot elongation or by a “quiescence strategy” involving surviving under water with minimal activity. As we found in previous experiments, Saligbeli cultivar adapted well to flash floods through rapid shoot elongation. To understand the vigorous growth process during submergence, we studied the relationship between shoot elongation and changes in dry matter weight (DMW) during submergence. O. glaberrima Steud. cv. Saligbeli and O. sativa L. cv. Ballawé and IR 49830-7-1-2-2 were used. Saligbeli and Ballawé exhibit shoot elongation, and IR 49830-7-1-2-2 exhibits flash-flood tolerance due to the presence of the Sub-1 gene. Twelve-day-old seedlings were submerged for 7 days and the plant length and DMW were measured. The plant length ratio of submerged to control plants in Saligbeli was higher than that of other cultivars during 2-6 days of submergence but IR 49830-7-1-2-2 shoot elongation was inhibited by submergence. In all three cultivars, the elongation of the developing leaf sheath conferred shoot elongation during submergence. The plant length of all submerged plants showed a strong positive correlation with DMW of the leaves developed during submergence. Submerged Saligbeli and Ballawé showed strong negative correlations between DMW of the leaves developed before and during submergence (r = –0.786 for Saligbeli and –0.772 for Ballawé , P < 0.05), suggesting that the enhancement of shoot elongation during submergence is accomplished by using dry matter of the leaves developed before submergence. However, the correlation was not observed in the submerged IR 49830-7-1-2-2. Further details from studies using isotopes are also needed to understand the plant growth during submergence.