Monocotyledonous leaves subjected to osmotica used for protoplast isolation accumulate a massive amount of putrescine (Put), lose chlorophyll and senesce rapidly. Treatment with spermidine (Spd) or spermine (Spm) prevents the loss of chlorophyll, indicating preservation of the thylakoid membranes at the site of the chlorophyll-protein complexes. Using several recently produced antibody probes, the effects on the stabilization of thylakoid membranes of applying either difluoromethylarginine (DFMA), a specific inhibitor of putrescine synthesis via arginine decarboxylase, or the polyamines Spd, Spm, or diaminopropane (Dap) to osmotically shocked oat leaves (Arena sativa L.) have been investigated. High protein levels were maintained in thylakoid membranes of leaf tissue incubated in the dark in the presence of 0.6 M sorbitol when pretreated with DFMA. After 48 h incubation, the level of the thylakoid protein D 1, at the core of photosystem II, was higher in the DFMA-pretreated leaves as was the stromal protein ribulose-l,5-bisphosphate carboxylase-oxygenase (Rubisco; as indicated by the level of large subunits). Applications of Spd, Spm or Dap were effective in retarding the loss of D1, D2 and cytochrome f from the thylakoid membranes as well as Rubisco large subunits and chlorophyll from the leaf tissue. The effects of polyamine applications may be mediated through Dap since most of the added Spd or Spm was converted to Dap within 6 h. The possible mechanisms of action of polyamine application s and DFMA-pretreatment on stabilizing the composition of the thylakoid membrane are also discussed.* To whom correspondence should be addressed; FAX: 44 (903) 72 67 80 Abbreviations : Cyt = cytochrorne; Dap = diaminopropane; DFMA = DL-cc-difluoromethylarginine ; LSU = large subunit (of Rubisco); Put=putrescine; Rubisco=ribulose-l,5-bisphosphate carboxylase-oxygenase; Spd = spermidine; Spin = spermine; SDS-PAGE = sodium dodecyl sulphate-polyacrylamide gel electrophoresis Key words: Polyamine -Ribulose-l,5-bisphosphate carboxylase/oxygenase (large subunit) -Thylakoid membrane (stabilization) -Thylakoid protein (D1, D2, cytochrome J)

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The abundance of transcripts of cab‐7 and cab‐3C, which code for the chlorophyll a/b binding proteins of the light‐harvesting complexes I and II, respectively, and the abundance of transcripts of Rca, which encodes Rubisco activase, were reduced in tomato plants exposed to high CO2 for up to 9d, whereas the abundance of mRNA from psa A–psa B and psb A, which encode the proteins of the core complex of PSI and the D1 protein of PSII, respectively, and the abundance of glycolate oxidase, which is involved in photorespiration, were not affected. However, the abundance of the transcript for the B subunit of ADP‐glucose pyrophosphorylase was increased after 1 d at elevated CO2. The chlorophyll a/b ratio decreased significantly over 9 d of exposure to elevated CO2. The responses of the nuclear genes to high CO2 were enhanced when leaves were detached so as to deprive them of any major sink. The responses of these transcripts to high CO2 were mimicked when sucrose or glucose was supplied to the leaf tissue, whereas acetate or sorbitol had no effect. Carbohydrate analyses of leaves grown in high CO2 or supplied with sucrose revealed that major increases occurred in the amount of glucose and fructose. Based on these and other published data, a molecular model involving the repression or activation of the transcription of nuclear genes coding for chloroplast proteins by photosynthetic end‐products is proposed to account for photosynthetic acclimation to high CO2 in tomato plants and other species.

SummaryTo test the possible interaction of polyamines in plant growth responses, transgenic tobacco plants containing the Arena sativa L. (oat) arginine decarboxylase (ADC) gene under the control of a tetracycline-inducible promoter were generated. Inducible overexpression of oat ADC in transgenic tobacco led to an accumulation of ADC mRNA, increased ADC activity and changes in polyamine levels. Transgenic lines, induced during vegetative stage, displayed different degrees of an altered phenotype, the severity of which was correlated with putrescine content. These phenotypic changes were characterized by short internodes, thin stems and leaves, leaf chlorosis and necrosis, as well as reduced root growth. This is the first report to show altered phenotypes as a consequence of polyamine changes under tetracycline-induction in in vivo conditions. Interestingly, overexpression of oat ADC in tobacco resulted in similar detrimental effects to those observed by ADC activation induced by osmotic stress in the homologous oat leaf system. In the context of the role of specific polyamines in plant growth and development, the present results indicate that activation of the ADC pathway leading to high levels of endogenous putrescine (or its catabolytes) is toxic for the vegetative growth of the plant. In contrast, no visible phenotypic effects were observed in flowering plants following tetracycline induction. Further characterization of the different transgenic lines may shed light on the action of specific polyamines in different plant developmental processes.

Tomato plants were exposed to four concentrations of CO2 (350, 700, 1050 or 1400 μmol CO2 mol−1) for 31 d. The light‐saturated rate of photosynthesis (A) of the unshaded fifth leaf was measured at either an ambient CO2 concentration of 350 μmol CO2 mol−1 [A (350)] or at the level of CO2 at which the plants were grown. The chloroplast protein composition and the level of transcripts of nuclear or plastid photosynthesis‐associated genes (PAGs), as well as the main carbohydrate content of the fifth leaf maintained horizontal and unshaded, were also measured during leaf development. At 60 and 95% leaf expansion, the A of high‐CO2‐grown plants measured at growth CO2 was higher than the A (350) of the plants grown at ambient CO2. However, in the fully mature leaves, A (growth CO2) declined linearly as growth CO2 concentration increased. The A (350) of plants exposed to elevated CO2 up to 60% leaf expanion had not acclimated to high CO2. At 95% leaf expansion, A (350) was lower in plants grown at high CO2. A versus CO2 (Ci) for mature leaves showed that A of the plants grown at high CO2 was lower over the entire range than that for plants grown at present ambient CO2 concentration. Lines fitted to the linear part of the A/Ci curves were concurrent at a Ci of 49μmol CO2 mol−1 and A=−1.21μmol CO2 m−2s−1. This Ct value is close to Λ* (46 μmol CO2 mol−1), the compensation point at 27°C calculated from the equation described in Brooks & Farquhar (1985, Planta 165, 397–406). This A is an estimate of respiration in the light (R1) and was not affected by acclimation to elevated CO2. Thylakoid proteins (photo‐system I core protein, D1 and D2 of the photosystem II core complex, cytochrome f) were all reduced by elevated CO2 only in the fully mature leaves (31d exposure), whereas the large and small subunits of Rubisco and Rubisco activase proteins had already declined after 22 d exposure. Transcript levels of the plastid‐encoded PAG (rbcL, psbA, psaA‐B) were reduced in the mature leaves by elevated CO2 when expressed on a total RNA basis, but they were not sensitive to elevated CO2 when expressed on a chloroplast 16S rRNA basis. However, rbcS, rca and cab mRNA transcripts were lower in the plants grown at high CO2 than in control plants after 22 d exposure when expressed on a nuclear rRNA basis. The loss of these nuclear PAGs was correlated with an accumulation of soluble sugars and starch.

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Plants, unlike animals, can use either ornithine decarboxylase or arginine decarboxylase (ADC) to produce the polyamine precursor putrescine. Lack of knowledge of the exact cellular and subcellular location of these enzymes has been one of the main obstacles to our understanding of the biological role of polyamines in plants. We have generated polyclonal antibodies to oat (Avena sativa L.) ADC to study the spatial distribution and subcellular localization of ADC protein in different oat tissues. By immunoblotting and immunocytochemistry, we show that ADC is organ specific. By cell fractionation and immunoblotting, we show that ADC is localized in chloroplasts associated with the thylakoid membrane. The results also show that increased levels of ADC protein are correlated with high levels of ADC activity and putrescine in osmotically stressed oat leaves. A model of compartmentalization for the arginine pathway and putrescine biosynthesis in active photosynthetic tissues has been proposed. In the context of endosymbiote-driven metabolic evolution in plants, the location of ADC in the chloroplast compartment may have major evolutionary significance, since it explains (a) why plants can use two alternative pathways for putrescine biosynthesis and (b) why animals do not possess ADC.