The pattern of 1-aminocyclopropane-1-carboxylate oxidase induction in the tomato leaf petiole abscission zone is independent of expression of the ribonuclease-LX-encoding LeLX gene.

The abscission of tomato leaves occurs in the petiole abscission zone, and its late stage includes two spatially divided processes: cell separation and programmed cell death (PCD). Both of these processes are regulated by ethylene. The last step in ethylene biosynthesis is conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene, which is catalysed by the enzyme 1-aminocyclopropane-1-carboxylate oxidase (ACO); however, the location of ACO in the leaf petiole abscission zone is not known. The tomato gene LeLX encodes ribonuclease LX, which is a marker for PCD and is induced by ethylene during abscission, but its association with ACO has not been explored. In a tomato transgenic line 1-7 with inhibited expression of LeLX showing delayed leaf abscission, the morphology and ultrastructure of the leaf petiole abscission zone was examined. In this zone of the cv.'VF36' and of a transgenic line 1-7, spatiotemporal differences in expression of LeACO1 and LeACO4 were analysed and ACO protein was detected immunohistochemically. In comparison to wild-type plants, there were no obvious morphological and ultrastructural features in the abscission zone of plants of a transgenic line 1-7 before and after abscission induction. LeACO1 expression was low before abscission induction, and increased 24 h after induction, although with no apparent spatial pattern. In contrast, LeACO4 was expressed before abscission induction, and its transcript level declined 24 h after induction on the distal side of the abscission zone fracture. In the LeLX-inhibited transgenic line, there were no significant differences in LeACO1 and LeACO4 expression in the petiole abscission zone, in comparison to wild-type plants. In addition, the ACO protein was immunolocalised to the vascular tissues that traverse the petiole abscission zone in plants of wild type and of a transgenic line 1-7; and additionally in the plane of future abscission zone fracture of transgenic-line plants. The results suggest temporal differential expression of the LeACO genes in tomato leaf petioles and vascular localisation of ACO1 protein. Additionally, the results indicate that expression of LeACO genes is not affected by suppression of the LeLX expression.

Identification of BFN1, a bifunctional nuclease induced during leaf and stem senescence in Arabidopsis.

Nuclease I enzymes are responsible for the degradation of RNA and single-stranded DNA during several plant growth and developmental processes, including senescence. However, in the case of senescence the corresponding genes have not been reported. We describe the identification and characterization of BFN1 of Arabidopsis, and demonstrate that it is a senescence-associated nuclease I gene. BFN1 nuclease shows high similarity to the sequence of a barley nuclease induced during germination and a zinnia (Zinnia elegans) nuclease induced during xylogenesis. In transgenic plants overexpressing the BFN1 cDNA, a nuclease activity of about 38 kD was detected on both RNase and DNase activity gels. Levels of BFN1 mRNA were extremely low or undetectable in roots, leaves, and stems. In contrast, relatively high BFN1 mRNA levels were detected in flowers and during leaf and stem senescence. BFN1 nuclease activity was also induced during leaf and stem senescence. The strong response of the BFN1 gene to senescence indicated that it would be an excellent tool with which to study the mechanisms of senescence induction, as well as the role of the BFN1 enzyme in senescence using reverse genetic approaches in Arabidopsis.

Senescence-induced RNases in tomato.

A main feature of leaf senescence is the hydrolysis of macromolecules by hydrolases of various types, and redistribution of released materials. We have initiated a study for the characterization of RNases involved in nucleic acid catabolism during senescence. Using a PCR-based cloning approach we isolated from tomato two senescence-induced RNase cDNA clones. Each of these cDNAs hybridized to a senescence-induced transcript in northern analysis. One RNase cDNA was identical to the tomato LX RNase while the second corresponded to the LE RNase. Both LX and LE RNase genes had originally been demonstrated to be induced after phosphate starvation of tomato cell culture but nothing was known about their expression or function in plants. We observed that the expression of the LX and LE genes is induced in leaves during an advanced stage of senescence with the LX transcript level being much more induced than that of LE. Low-level expression of the RNase genes was observed in flowers and artificially senescing detached leaves while no expression could be detected in stems, roots, or fruits at different ripening stages. Ethylene activated the LX gene expression in detached young leaves while LE gene expression, which could be transiently induced by wounding, appeared to be activated by abscisic acid. We suggest that the LX RNase has a role in RNA catabolism in the final stage of senescence, and LE may function during wounding as a plant defense protein.

The expression of a grapefruit gene encoding an isoflavone reductase-like protein is induced in response to UV irradiation.

Exposure of harvested grapefruit to UV-C (254 nm) irradiation was previously found to induce resistance against the green mold decay caused by Penicillium digitatum. In order to gain insight into the mechanism of this UV-induced resistance we initiated a study for isolation of genes induced during this process. Using the differential display method we cloned cDNA representing an mRNA which is accumulated in grapefruit peel upon UV irradiation. Sequence analysis revealed that this cDNA represents a gene encoding for an isoflavone reductase-like protein and was termed IRL (isoflavone reductase-like). The grapefruit IRL protein sequence has high homology also to a novel family of other isoflavone reductase-like proteins present in few non-legume plants and whose function is not clear yet. The UV dose, and time following it, which lead to maximal accumulation of the IRL transcript were found to be similar to those leading to maximal induced resistance. The expression of the IRL gene was demonstrated to be induced also by wounding and pathogen infection.

Differential regulation of chloroplast gene expression in Chlamydomonas reinhardtii during photoacclimation: light stress transiently suppresses synthesis of the Rubisco LSU protein while enhancing synthesis of the PS II D1 protein.

Transfer of Chlamydomonas reinhardtii cells grown photoautotrophically in low light to higher light intensities has a dramatic transient effect on the differential expression of the two major chloroplast encoded photosynthetic proteins. Synthesis of the D1 protein of Photosystem II increases more than 10-fold during the first six hours in high light (HL), whereas synthesis of the large subunit (LSU) of Rubisco drops dramatically within 15 min and only gradually resumes at about 6 h. Synthesis of the chloroplast-encoded ATP synthase beta subunit, the nuclear-encoded Rubisco small subunit and the nuclear-encoded beta-tubulin is not noticeably affected. Up regulation of psbA mRNA translation accounts for a substantial fraction of the increased D1 synthesis, since accumulation of psbA mRNA increases 4.2- and 6.3-fold less than D1 synthesis at 6 and 18 h in HL. Down-regulation of LSU synthesis is not correlated with a reduction in the steady-state level of the rbcL transcript. Primer extension mapping of the 5' ends of the rbcL mRNAs reveals transcripts with start points located at -93 and -168 relative to the first translated ATG. Transfer of low light (LL)-grown cells to HL temporarily decreases the ratio of the -93 to -168 transcripts, but this ratio normalizes after 6 h in HL, coincident with the recovery in the synthesis of LSU. These several distinct effects of temporary light stress were correlated with a rapid, sustained increase in the reduction state of QA, a transient decline in photosynthetic efficiency, a less rapid drop in total chlorophyll content and a delay in cell division.

The carboxyl-terminal extension of the D1 protein of photosystem II is not required for optimal photosynthetic performance under CO2- and light-saturated growth conditions.

Synthesis of the photosystem II D1 protein as a precursor with a carboxyl-terminal extension occurs in almost all eukaryotic photosynthetic organisms examined so far, as well as in cyanobacteria. Processing of the D1 precursor has been recently postulated to play a regulatory role in the light-dependent migration of photosystem II units from the unstacked to the stacked thylakoids (Bowyer, J. M., Packer, J. C. L., McCormack, B. A., Whitelegge, J. P., Robinson, C., and Taylor, M. A. (1992) J. Biol. Chem. 267, 5424-5433). To test this hypothesis, site-directed mutagenesis and chloroplast transformation have been used to create a "preprocessed" mutant Chlamydomonas strain which synthesizes mature D1 protein directly. We have found that this strain is indistinguishable from wild type in terms of photosynthetic performance and cell doubling time under CO2- and light-saturated photoautotrophic growth conditions.

Co-regulation of a gene homologous to early light-induced genes in higher plants and beta-carotene biosynthesis in the alga Dunaliella bardawil.

Dunaliella bardawil, a unicellular green alga that can be induced to accumulate massive amounts of beta-carotene, is particularly suitable for studies of carotenogenesis regulation and its links to developmental and adaptive processes in the chloroplast. A cDNA clone corresponding to a transcript accumulating coordinately with carotenogenesis induction was isolated by differential hybridization of a cDNA library made from intensely illuminated cells. This transcript was also abundant in algal mutants able to accumulate beta-carotene under relatively low light intensity. DNA sequence analysis indicates that cbr (for carotene biosynthesis-related) is closely related to early light-induced genes (elip) of higher plants. Similarity also exists between repeated oligopeptide motifs in Cbr and Cab proteins of photosystems I and II. Three upstream direct repeats in cbr include an octamer and hexamer related to mammalian sterol regulatory elements. The relationship between cbr transcript and beta-carotene accumulation, the structural similarity between Cbr and Cab proteins, and the presence of potential SRE-1 elements lead us to propose that Cbr represents novel carotenoid binding proteins, whose synthesis might be coordinated with carotenogenic enzymes via an evolutionary conserved regulatory mechanism.

Photoinduction of Massive beta-Carotene Accumulation by the Alga Dunaliella bardawil: Kinetics and Dependence on Gene Activation.

The massive accumulation of beta-carotene by the halotolerant micro alga Dunaliella bardawil, in response to high light intensity and several other environmental factors, has been studied so far under different sets of fixed conditions. To determine the kinetics and characteristics of the induction of beta-carotene accumulation, cells continuously grown under white light of approximately 27 microeinsteins per square meter per second were exposed to light of approximately 1650 microeinsteins per square meter per second. The exposed cells accumulate beta-carotene in two stages: the first stage, lasting for 24 hours, starts shortly after exposure, whereas the second stage starts concomitantly with the onset of the stationary phase and persists until the cells collapse. Actinomycin D, chloramphenicol, or cycloheximide added to low-illuminated cultures abolish the subsequent induction of beta-carotene accumulation by high light intensity. These results, together with the early exponential kinetics of accumulation, point to the role of gene activation in the process. In vivo labeling of proteins and in vitro translation of poly(A)(+) mRNA revealed several pronounced differences between low-illuminated and high-illuminated cells. A strongly light-induced protein of approximately 55 kilodaltons, as well as other light-induced proteins could possibly fulfill a carotenogenic function.

Outreading promoters are located at both ends of the gamma-delta transposon.

Two plasmids were isolated containing oppositely oriented gamma-delta insertions between the wild-type transcription initiation site of the nifHDKY operon and the nifH coding sequence. The nifHDKY promoter of Klebsiella pneumoniae, similar to other nitrogen fixation (nif) promoters, normally requires the products of ntrA and nifA for activity. Mutations that allowed constitutive expression of the nifHDKY operon were searched for by transforming a plasmid, containing the regulatory region of this operon followed by an in-frame nifH'-'lacZ fusion, into a Lac- Escherichia coli strain (which contains no nifA) and screening for Lac+ derivatives. The plasmids described here were isolated from such derivatives and directed the constitutive expression of beta-galactosidase. Deletion analysis indicated that gamma-delta promoters other than those transcribing tnpA and tnpR were involved in this expression. Nuclease S1 mapping revealed outward-reading transcription initiation sites in both the gamma end and the delta end of the transposon. Most interestingly, one initiation site on each end was located in corresponding positions within the terminal inverted repeats. The sites were in the center of the longest sequence, of 12 bp, contiguously conserved between the terminal inverted repeats of gamma-delta and the related transposon Tn3. In gamma-delta and Tn3, this sequence has been recently implicated in transposase binding. These results suggest a possible interrelationship between transcription from the "end" promoters and transposition.

Stereoisomers of beta-Carotene and Phytoene in the Alga Dunaliella bardawil.

Dunaliella bardawil, a halotolerant green alga, was previously shown to accumulate high concentrations of beta-carotene when grown outdoors under defined conditions. The beta-carotene of algae cultivated under high light intensity in media containing a high salt concentration is composed of approximately 50% all-trans beta-carotene and 40% 9-cis beta-carotene. We show here that the 9-cis to all-trans ratio is proportional to the integral light intensity to which the algae are exposed during a division cycle. In cells grown under a continuous white light of 2000 microeinsteins per square meter per second, the ratio reached a value of around 1.5, while in cells grown under a light intensity of 50 microeinsteins per square meter per second, the ratio was around 0.2. As previously shown, algae treated with the herbicide norflurazon accumulate phytoene in place of beta-carotene. Electron micrographs showed that the phytoene is accumulated in many distinct globules located in the interthylakoid spaces of the chloroplast. Here too, two isomers are present, apparently all-trans and 9-cis phytoene, and their ratio is dependent upon the integral light intensity to which the algae are exposed during a division cycle. In the presence of norflurazon, Dunaliella bardawil grown under a light intensity of 2000 microeinsteins per square meter per second contained about 8% phytoene with a 9-cis to all-trans ratio of about 1.0. This ratio decreased to about 0.1 when the light intensity was reduced to 50 microeinsteins per square meter per second. These data suggest that the isomerization reaction which leads to the production of the 9-cis isomer occurs early in the path of carotene biosynthesis, at or before the formation of all-trans phytoene. The presence of the 9-cis isomer of beta-carotene and the dependence of its preponderance on light intensity seem to be a common feature of many plant parts. Thus carrots which are exposed to minimal light contain no 9-cis isomer while sun-exposed leaves, fruits, and flowers contain 20 to 50% of the 9-cis isomer.

Transcriptional analysis of promoter mutations in the Klebsiella pneumoniae nifHDKY operon.

Previously isolated promoter mutations that allow expression of the Klebsiella pneumoniae nifHDKY operon in the absence of nifA (R. Bitoun, J. Berman, A. Zilberstein, D. Holland, J.B. Cohen, D. Givol, and A. Zamir, Proc. Natl. Acad. Sci. USA, 80:5812-5816, 1983) were further characterized. pRB1 and pRB5, containing, respectively, point and duplication mutations in the nifHDKY regulatory region, were transformed into Escherichia coli and K. pneumoniae hosts with different nifA and ntrA backgrounds. nif transcription start sites were determined by nuclease S1 mapping. The results indicated that nifA-independent expression from both mutants did not require ntrA. Transcription from pBR5 started 3 base pairs (bp) upstream of the start site of nif-regulated transcription and could stem from a canonical promoter sequence generated at the junction between the two copies of the duplicated sequence. In the presence of nifA-ntrA, transcription from pRB5 started predominantly at the site characteristic of the nif-regulated promoter. The site of constitutive transcription initiation in pRB1 was located 33 bp upstream of the point mutation and 40 bp upstream of the start of nifA-ntrA-activated transcription. Low-level transcription from the upstream site was also evident, in the absence of nifA or nifA or both, with the plasmid containing the wild-type nifHDKY regulatory region. However, when nifA and ntrA were present to activate transcription from the major nif promoter, no activity was evident from the upstream site in either pRB1 or the parental plasmid. Thus, the mutation enhanced the activity of a pre-existing constitutive promoter, the activity of which was repressed on nifA-ntrA activation of the major nif promoter.