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.

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.

Over-expression of ascorbate peroxidase in tobacco chloroplasts enhances the tolerance to salt stress and water deficit.

The role of APX (ascorbate peroxidase) in protection against oxidative stress was examined using transgenic tobacco plants. The full length cDNA, coding Arabidopsis thaliana L. APX fused downstream to the chloroplast transit sequence from A. thaliana glutathione reductase, was cloned into appropriate binary vector and mobilized into Agrobacterium tumefaciens C58C2. Leaf discs were infected with the Agrobacterium and cultured on medium supplied with kanamycin. The incorporation of the gene in tobacco genome was confirmed by Southern dot blot hybridization. Transgenic lines were generated, and the line Chl-APX5 shown to have 3.8-fold the level of APX activity in the wild-type plants. The isolated chloroplasts from this line showed higher APX activity. During early investigation, this line showed enhanced tolerance to the active oxygen-generating paraquat and sodium sulphite. The first generation of this line, also, showed enhanced tolerance to salt, PEG and water stresses, as determined by net photosynthesis. The present data indicate that overproducing the cytosolic APX in tobacco chloroplasts reduces the toxicity of H(2)O(2).

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.

Identification and cloning of a submergence-induced gene OsGGT (glycogenin glucosyltransferase) from rice (Oryza sativa L.) by suppression subtractive hybridization.

A submergence-induced gene, OsGGT, was cloned from 7-day submerged rice (Oryza sativa L. plants, FR13A (a submergence-tolerant cultivar, Indica), using suppression subtractive hybridization and both 5'- and 3'-rapid amplification of cDNA ends (RACE). The full-length OsGGT cDNA contains 1,273 bp with an open reading frame of 1,140 bp (17-1,156) that encodes 379 amino acids. Its deduced amino acid sequence is homologous with glycogenin glucosyltransferase. We found that the OsGGT gene is located in the 17,970-20,077 bp region of genome fragment AAAA01002475.1 of the Indica cultivar and in the 53,293-51,186 bp region of genome fragment AC037426.12 of chromosome 10 of the Japanica cultivar. A time-course study showed that OsGGT-gene expression increased in FR13A during submergence but decreased in IR42 (submergence-intolerant cultivar, Indica). The expression of the OsGGT gene in FR13A was induced by salicylic acid and benzyladenine. The accumulation of OsGGT mRNA in FR13A also increased in response to ethylene, gibberellin, abscisic acid, drought and salt treatment, but methyl jasmonate treatment and cold stress had no effect on expression. These results suggest that the OsGGT gene could be related to submergence stress and associated with a general defensive response to various environmental stresses.

Cloning of a putative monogalactosyldiacylglycerol synthase gene from rice (Oryza sativa L.) plants and its expression in response to submergence and other stresses.

Suppression subtractive hybridization was used to construct a subtractive cDNA library from plants of non-submerged and 7-day-submerged rice (Oryza sativa L., FR13A, a submergence-tolerant cultivar). One clone of the subtractive cDNA library, S23, was expressed abundantly during submergence. The full length of S23 was amplified using 5'- and 3'-rapid amplification of cDNA ends, and found to consist of 1,671 bp with an open reading frame of 1,077 bp (181-1257) encoding 358 amino acids. Its deduced amino acid sequence showed a high homology with monogalactosyldiacylglycerol synthase (UDPgalactose: 1,2-diacylglycerol 3-beta-D-galactosyl transferase; EC 2.4.1.46, MGDG synthase) from Arabidopsis thaliana; therefore, we named the gene OsMGD. Time-course studies showed that the expression of OsMGD in the rice cultivars FR13A and IR42 (submergence-susceptive cultivar) during submergence was gradually increased and that expression in FR13A was higher than in IR42. The expression of OsMGD in FR13A was influenced by benzyladenine and illumination. The accumulation of OsMGD mRNA in both FR13A and IR42 was also increased by ethephon, gibberellin, drought and salt treatment, but cold stress had no effect on the expression of the gene. These results suggest that the expression of OsMGD mRNA requires benzyladenine or illumination, and that the process is also mediated by ethephon and gibberellin. Salt and drought stress have an effect similar to that of submergence. Furthermore, the enhanced expression of OsMGD may relate to photosynthesis, and play an important role during submergence.

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.