A. Dedonder

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.

Arabidopsis rbcS genes are differentially regulated by light.

Individual members of the Arabidopsis thaliana ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (rbcS) gene family are differentially regulated by light of different qualities. In 10-d-old etiolated seedlings, the expression of only three of the four genes is under inductive phytochrome control. rbcS mRNA levels reach a maximum (3- to 5-fold higher than the dark level) about 6 h after a red light pulse, but the rate of decay differs among the genes. Moreover, rbcS 2B requires a higher fluence for induction. At early stages of development, rbcS 1A, 2B, and 3B are highly expressed in the dark and cannot be further induced by red light, indicating a developmental component in the overall regulatory mechanism. Continuous light experiments indicate that high-irradiance responses may play a role in the induction of at least three of the four rbcS genes. Under conditions of phytochrome saturation, rbcS 1A is insensitive to blue pulses, whereas among the three B locus genes, at least rbcS 3B appears to respond to a blue-light photoreceptor. These results add to the data suggesting that individual members of rbcS gene families in higher plants may be subject to a variety of differing regulatory mechanisms.

Quantitative kinetic analysis of β-glucuronidase activities using a computer-directed microtiter plate reader

We have established a procedure for automated, kinetic analysis of β-glucuronidase (GUS) activities using a colorimetric or fluorometric microtiter plate reader connected to a computer that directs the measurements and accesses the data. Compared with end-point measurements, the procedure saves time, is more accurate, and needs 20 times less material. It allows a more precise determination of GUS activities over a range of 400,000-fold, with a limit of detection of about 0.01 units of GUS per mL in the colorimetric assay and 0.1 milliunit of GUS in the fluorometric assay. A general protocol for the determination of GUS activities in transgenic plant tissue was worked out and applied to investigate the expression of a chimeric β-glucuronidase gene in stably transformed tobacco calli.

PROBIT ANALYSIS OF LOW AND VERY‐LOW FLUENCE‐RESPONSES OF PHYTOCHROME‐CONTROLLED Kalanchoe blossfeldiana SEED GERMINATION*

Abstract— Kalanchoë blossfeldiana seeds are light‐requiring for seed germination. On water or KNO3 solution and irradiated with several daily red (R) irradiations, the seeds show a low‐fluence (LF) response which is far‐red (FR) reversible. Incubated on gibberellic acid (GA3) the seeds show a very‐low‐fluence (VLF) response which can be saturated with red as well as with far red light. As germination is a quantal response, the sub‐optimal segments of the dose‐response curves are analysed by means of probit analysis in order to calculate the seed population parameters. There is a linear relation between the probit of the germination response and the logarithm of the fluence. Moreover, the slope for the VLF as well as for the LF response is the same. The VLF requires about 8 × 104 times less fluence than the LF. VLF saturation with FR requires about 200 times more fluence than with R.

Oxidative stress in plants : EPR monitoring in DMPO-DMSO based extracts

Summary Oxygen derived radicals were measured in Brassica napus hypocotyl segments by means of EPR (electron paramagnetic resonance) after spin-trapping with 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) in a dimethylsulfoxide(DMSO)-based medium. Next to the ascorbyl radical signal, an EPR signal due to DMPO-CH3, generated by reaction of free OH radicals with DMPO, was detected. The DMPO-CH3 signals were quantified in Brassica napus hypocotyl segments at different times after wounding and in vitro culture. A maximum was detected as a function of time after wounding. When hypocotyl segments were kept in the dark, a reduction of about 70 % of both the DMPO-CH3 and ascorbyl line intensity in comparison to the light control was observed. This suggests that the production of ascorbyl and hydroxyl radicals is associated with photooxidative stress. Only small traces of DMPO-OOH (superoxide) were detected in Brassica hypocotyl segments. In contrast, in DMPO-DMSO based extracts of Arabidopsis seedlings sprayed with diquat (0.1 %), superoxide radicals were detected as adducts of DMPO. Consequently, the non-detection of DMPO-OOH in leaves of untreated Arabidopsis seedlings and the very weak signals detected in Brassica napus hypocotyls are clearly related to the absence or the very small amounts of superoxide in both tissues.

ENERGY METABOLISM OF IMBIBING KALANCHOË SEEDS. RESPONSE TO GIBBERELLIC ACID AND CONTRIBUTION OF RESPIRATION AND FERMENTATION TO ATP PRODUCTION

In Kalanchoe blossfeldiana seeds imbibed in the dark for 10 days at 20°C, very similar energy charge ( EC ) values were measured in the presence and absence of gibberellic acid (GA 3 ). This demonstrates that GA 3 does not prevent or strongly delay the induction of secondary dormancy in Kalanchoe seeds by elevating EC levels. However, GA 3 clearly increased the ATP and AMP content of the seeds. During the first hours of anoxic imbibition, the sodium-fluoride sensitive ATP production still represents 25 to 30% of the ATP amount produced in the air, indicating that in Kalanchoe seeds the fermentative pathway can partly contribute to ATP production. As the oxygen uptake by the seeds is cyanide-sensitive and a severe inhibition of the increase in ATP content by cyanide can be demonstrated during the first hours of aerobic imbibition, our results indicate the participation of the normal cytochromic pathway in ATP synthesis.

Factors Eliciting the Germination of Photoblastic Kalanchoe Seeds

In the Series “Monographies de Physiologie Vegetale” edited by professor P.E. Pilet of the University of Lausanne, Switzerland, Daniel Come (1970) wrote in the introductory remarks of his book “Les Obstacles a la Germination” : “… si l’on connait le plus souvent les causes essentielles de la non-germination d’une semence, et si l’on sait quels traitements appliquer a cette semence pour la rendre apte a germer, les raisons profondes de la non-germination sont les plus souvent inconnues ou seulement tres partiellement connues. Les recherches actuelles s’orientent progressivement vers une meilleure comprehension de ces mecanismes. L’experimentation est difficile du fait que le repos des semences ne correspond pas a un simple arret de croissance et que la germination elle-meme est encore un phenomene assez mal connu.”

Cloning and Expression Analysis of an Arabidopsis Thaliana 1-Aminocyclopropane-1-Carboxylate Synthase Gene: Pattern of Temporal and Spatial Expression

A genomic clone of one member of the Arabidopsis thaliana (L.) Heynh. 1-aminocyclopropane-l-carboxylate synthase gene family (accl) was isolated and sequenced. Genomic DNA gel blotting suggested the existence of an ACC synthase multigene family in Arabidopsis. The other members are distantly related to actg. The existence of at least another gene was confirmed by the isolation of a cDNA (acc2) from a flower-specific cDNA library. Expression of the accl gene was studied by reverse-transcription polymerase chain reaction (RT-PCR) on total RNA. The mRNA accumulated strongly in young leaves and flowers. Wounding of young leaves did not induce the accl gene. Ethylene exposure of flowering plants led to an induction of accl expression. An accl promoter-s-glucuronidase fusion was introduced in A. thaliana by the root transformation method. Temporal and spatial regulation of expression of accl were analyzed, revealing a developmental control both in shoot and root and confirming results obtained by RT-PCR analysis. A role for accl in the developmental control of ethylene synthesis is suggested.