D. Van Der Straeten

Molecular and Physiological Responses to Abscisic Acid and Salts in Roots of Salt-Sensitive and Salt-Tolerant Indica Rice Varieties

The Indica rice (Oryza sativa L.) varieties Pokkali and Nona Bokra are well-known salt tolerance donors in classical breeding. In an attempt to understand the molecular basis of their tolerance, physiological and gene expression studies were initiated. The effect of abscisic acid (ABA) on total proteins in roots from 12-d-old seedlings of Pokkali, Nona Bokra, and the salt-sensitive cultivar Taichung N1 were analyzed on two-dimensional gels. The abundance of ABA-induced proteins was highest in the most tolerant variety, Pokkali. Three ABA-responsive proteins, present at different levels in roots from tolerant and sensitive varieties, were further characterized by partial amino acid analysis. A novel histidine-rich protein and two types of late embryogenesis abundant (LEA) proteins were identified. Protein immunoblotting revealed that the levels of dehydrins and group 3 LEA proteins were significantly higher in roots from tolerant compared with sensitive varieties. Endogenous ABA levels showed a transient increase in roots exposed to osmotic shock (150 mM NaCl). Peak ABA concentrations were 30-fold higher for Nona Bokra and 6-fold higher for Pokkali compared with Taichung N1. Both the salt-induced endogenous ABA levels and a greater molecular response of root tissue to ABA were associated with the varietal differences in tolerance.

Plant enolase: gene structure, expression, and evolution.

Enolase genes were cloned from tomato and Arabidopsis. Comparison of their primary structures with other enolases revealed a remarkable degree of conservation, except for the presence of an insertion of 5 amino acids unique to plant enolases. Expression of the enolase genes was studied under various conditions. Under normal growth conditions, steady-state messenger and enzyme activity levels were significantly higher in roots than in green tissue. Large inductions of mRNA, accompanied by a moderate increase in enzyme activity, were obtained by an artificial ripening treatment in tomato fruits. However, there was little effect of anaerobiosis on the abundance of enolase messenger. In heat shock conditions, no induction of enolase mRNA was observed. We also present evidence that, at least in Arabidopsis, the hypothesis that there exists a complete set of glycolytic enzymes in the chloroplast is not valid, and we propose instead the occurrence of a substrate shuttle in Arabidopsis chloroplasts for termination of the glycolytic cycle.

The Arabidopsis 1-Aminocyclopropane-1-Carboxylate Synthase Gene 1 Is Expressed during Early Development.

The temporal and spatial expression of one member of the Arabidopsis 1-aminocyclopropane-1-carboxylate (ACC) synthase gene family (ACS1) was analyzed using a promoter-[beta]-glucuronidase fusion. The expression of ACS1 is under developmental control both in shoot and root. High expression was observed in young tissues and was switched off in mature tissues. ACS1 promoter activity was strongly correlated with lateral root formation. Dark-grown seedlings exhibited a different expression pattern from light-grown ones. The ACC content and the in vivo activity of ACC oxidase were determined. ACC content correlated with ACS1 gene activity. ACC oxidase activity was demonstrated in young Arabidopsis seedlings. Thus, the ACC formed can be converted into ethylene. In addition, ethylene production of immature leaves was fourfold higher compared to that of mature leaves. The possible involvement of ACS1 in influencing plant growth and development is discussed.

Genetic and Physiological Analysis of a New Locus in Arabidopsis That Confers Resistance to 1-Aminocyclopropane-1-Carboxylic Acid and Ethylene and Specifically Affects the Ethylene Signal Transduction Pathway

A population of M2 seedlings of Arabidopsis thaliana was screened for mutants that were insensitive to the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC). Several independent lines were obtained and proved insensitive to both ACC and ethylene. Two lines were identified as alleles of a single recessive mutation, designated ain1. Linkage analysis indicated that the ain1 gene is located on chromosome 1, adjacent to the cer5 marker and, therefore, genetically distinct from previously identified ethylene resistance loci. General phenotypic aspects of ain1 mutants were similar to wild type. For both alleles, the level of insensitivity to ethylene at the seedling stage was indistinguishable in terms of elongation growth. In contrast, the gravitropic response of ain1–1 seedlings was slower than that of wild-type and ain1–2 seedlings. At the adult stage, stress responses of mutants were similar to wild type. However, ethylene-induced leaf senescence was delayed in both mutants. In addition, we observed significant interallelic variation in ethylene production rates. Growth inhibition experiments showed that the ain1 mutation does not confer resistance to other hormones. Thus, ain1 most probably affects a step specific for the ethylene signal transduction pathway.

Early detection of nutrient and biotic stress in Phaseolus vulgaris

Prerequisites for optimal, high crop yield are disease‐free growth and an equilibrated supply of nutrients. Early signatures of stress‐altered physiology, before appearance of symptoms in the visible spectrum, allow timely treatment. Early detection of stress development was carried out on phaseolus vulgaris bean infected with the agriculturally important grey mould pathogen and under conditions of magnesium deficiency, limiting photosynthesis. During stress development, bean plants were monitored by time‐lapse imaging with thermal, video and chlorophyll fluorescence cameras, mounted on a gantry robot system. For early detection of grey mould infection, chlorophyll fluorescence imaging proved to be the most sensitive. This technique detected magnesium deficiency at least three days before visual symptoms appeared. Further development of non‐contact technology for plant health monitoring will help to achieve optimal productivity in greenhouse and field cultures. Associated establishment of a stress catalogue based on early symptoms will allow swift diagnosis.

A group of chromosomal proteins is specifically released by spermine and loses DNA-binding activity upon phosphorylation.

Biologically relevant concentrations as low as 500 [mu]M spermine led to the specific release of chromatin-associated proteins from nuclei of rice (Oryza sativa) seedlings. Using a southwestern technique, it was shown that several of these proteins bind DNA. This affinity was lost upon in organello phosphorylation by an endogenous kinase. The effect of spermine was very specific. Spermidine was far less effective and putrescine was essentially ineffective in releasing these proteins. The most abundant spermine-released protein was shown to be homologous to the maize HMG1 protein. Our results suggest that spermine induces the release of spermine-released proteins by changing DNA conformation. Binding of these proteins might be sensitive to long-range changes in chromosome structure caused by torsional stress.

Molecular and Physiological Mechanisms of Flooding Avoidance and Tolerance in Rice

Submergence is one of the major constraints in rice production. The main factor limiting rice survival during submergence is oxygen deprivation. To cope with flooding conditions, rice has developed two survival strategies: either rapid elongation of the submerged tissues to keep up with the rising water level or no elongation to save carbohydrate resources for maintenance of energy production under submerged and concomitant hypoxic conditions. The survival strategies used by rice have been studied quite extensively and the role of several phytohormones in the elongation response has been established. The mechanisms of submergence tolerance include metabolic changes, for instance, the shift to an ethanolic fermentation pathway, reduced elongation growth to save carbohydrates and energy for maintenance processes, and protective antioxidant systems. Current molecular technology can provide tools for the understanding of mechanisms developed by rice to survive submergence. In addition, cloning of genes related to submergence tolerance might open new ways to genetic improvement of this crop.

Effects of Copper and Zinc on the Ethylene Production of Arabidopsis Thaliana

The effects of toxic concentrations of the redox active metal copper and the redox inactive zinc on the ethylene production of intact, seven days old Arabidopsis thaliana (L.) Heynh. seedlings, were studied kinetically in an open flow system. Major differences in the metal stimulated increases in ethylene production were found. Using reaction rate experiments, this stimulation was shown to be entirely enzymatic in case of zinc treatment and partly enzymatic, partly non-enzymatic in case of copper addition. The existence of non-enzymatic copper stimulated ethylene production was confirmed using an oxygen-free atmosphere. By inhibiting ACC synthase activity, ACC could be indicated as the major precursor of both enzymatic and non-enzymatic copper stimulated ethylene production.

Ethylene Signaling: More Players in the Game

The Arabidopsis ethylene response pathway was established by characterization of ethylene mutants that were isolated exploiting the triple response of dark-grown seedlings. The various triple response screens were not yet exhaustive; however, a large number of ethylene-related loci have been identified, and for several of these an allelic series was isolated. To increase the chance of identifying new loci, screening for mutants at developmental stages other than the etiolated seedling stage might be a useful approach. We have isolated mutants from light-grown populations, by using the ethylene response of nutrient-deficient seedlings at two stages in development. Characterization of these mutants has resulted in the identification of new loci involved in ethylene signaling.

Isolation and characterisation of an antifolate insensitive (afi1) mutant of Arabidopsis thaliana

Antifolates can impair the synthesis and/or function of folates in living organisms. Mechanisms of resistance or tolerance to antifolates have been mainly described in plants using the drug methotrexate. In this work, the antifolate trimethoprim (TMP) was used with the aim of revealing a novel mechanism of resistance. EMS mutagenised seeds from Arabidopsis were screened to isolate individuals insensitive to TMP. Genetic analysis revealed a homozygous recessive mutation that segregates with the phenotype of tolerance to 50 μm TMP. Mapping analysis localised the mutation at the end of the short arm of chromosome 3. Preliminary characterisation demonstrated up-regulation of several genes from the folate biosynthetic pathway in the TMP insensitive mutant, and a slight increase in total folate content in the mutant as compared with the Col-0 control. Moreover, sequence analysis of the DHFR (dihydrofolate reductase) genes, which encode a known target for resistance to antifolates, did not reveal any changes. This study is the first report of a stable mutant insensitive (afi1) to the antifolate trimethoprim in plants, and suggests the existence of a novel mechanism of resistance to antifolates.