The light reactions of photosynthesis store energy in the form of an electrochemical gradient of protons, or proton motive force (pmf), comprised of electrical (Δψ) and osmotic (ΔpH) components. Both components can drive the synthesis of ATP at the chloroplast ATP synthase, but the ΔpH component also plays a key role in regulating photosynthesis, down-regulating the efficiency of light capture by photosynthetic antennae via the qE mechanism, and governing electron transfer at the cytochrome b6f complex. Differential partitioning of pmf into ΔpH and Δψ has been observed under environmental stresses and proposed as a mechanism for fine-tuning photosynthetic regulation, but the mechanism of this tuning is unknown. We show here that putrescine can alter the partitioning of pmf both in vivo (in Arabidopsis mutant lines and in Nicotiana wild type) and in vitro, suggesting that the endogenous titer of weak bases such as putrescine represents an unrecognized mechanism for regulating photosynthetic responses to the environment.
Citation: Ioannidis NE, Cruz JA, Kotzabasis K, Kramer DM (2012) Evidence That Putrescine Modulates the Higher Plant Photosynthetic Proton Circuit. PLoS ONE 7(1): e29864. https://doi.org/10.1371/journal.pone.0029864
Editor: Steven M. Theg, University of California – Davis, United States of America
Received: August 26, 2011; Accepted: December 5, 2011; Published: January 12, 2012
Copyright: © 2012 Ioannidis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the European Molecular Biology Organisation for post-doctoral research in the Institute of Biological Chemistry (Washington State, United States of America). DK and JC were supported by a grant from the United States Department of Energy, Chemical Sciences, Geosciences and Biosciences Division Office of Science, Basic Energy Sciences (DE-FG02-11ER16220. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The light-driven transthylakoid proton motive force (pmf) plays several essential roles in photosynthesis . Both the ΔpH (osmotic) and Δψ (electric) components of pmf contribute to ATP synthesis at the CFO-CF1 ATP synthase, in a thermodynamically equivalent fashion , but the ΔpH component of pmf is also a key signal for initiating photoprotection of the photosynthetic reaction centers through energy-dependent non-photochemical quenching (qE), a process that dissipates excess absorbed light energy as heat, thus protecting the photosynthetic apparatus from photodamage –. Acidification of the lumen also controls photosynthetic electron transfer by slowing the rate of plastoquinol oxidation at the cytochrome b6f complex , , preventing the accumulation of highly reducing species within photosystem I .
Differential partitioning of the thylakoid pmf into ΔpH and Δψ components has been observed in thylakoids  and in intact leaves  and was proposed to constitute an important fine-tuning mechanism for photosynthesis . Under optimal conditions, when down-regulation is not needed, a large fraction of pmf can be stored as Δψ, leading to moderate lumen pH and low qE, even at high pmf (and thus high rates of ATP synthesis). In contrast, under environmental stresses—e.g., high light, low CO2/O2, when photoprotection is advantageous—pmf can be predominantly stored as ΔpH, maximizing lumen acidification for a given pmf.
The mechanism by which thylakoid pmf is partitioned into Δψ and ΔpH remains unclear, but in vitro experiments and modeling have established that at least three factors are critical ,  : 1) the capacitance of the thylakoid membrane, which determines the Δψ generated for the transfer of a charge across the membrane; 2) the proton-buffering capacity of the lumen, which determines the relationship between translocated protons and changes in lumen pH; and 3) the ionic composition of the stroma and lumen, which determines the degree to which movements of counterions can dissipate the Δψ component. Of these three, ionic balance appears to be the most likely to account for the observed short-term changes in pmf partitioning in response to environmental changes, because thylakoid membrane capacitance and lumen buffering capacity are thought to change only slowly, whereas plastids can rapidly alter the ionic balance by regulating ion channels or ion pumps , .
More recently, Ioannidis et al.  proposed an alternative (but non-exclusive) hypothesis—the ‘biological weak base” (BWB) hypothesis—for Δψ/ΔpH control. This model involves biological weak bases, such as polyamines, which occur normally in chloroplasts and may act as ‘permeant buffers’, specifically dissipating the ΔpH component and thus favoring Δψ. Because the titer of these weak bases can be regulated by the organism (by synthesis, degradation, transport, covalent binding to proteins, and phenolics), this mechanism may constitute a means to adjust the ΔpH/Δψ ratio in the short (seconds) and long term (hours to days).
Putrescine (Put) is a diamine [NH2(CH2)4NH2], which, along with spermidine and spermine, constitute the major polyamines in plants. Polyamines are important or even essential for many cellular processes, such as cell growth and stress tolerance –. Although the metabolism of polyamines is well understood, their mode of action is ill defined (for reviews see refs  and ). The proposed mechanism for putrescine action is similar to that by which amines dissipate the ΔpH component of pmf in isolated thylakoids . Under physiological pH, the protonated forms of amines prevail but are in equilibrium with a small concentration of free base, which can permeate the membrane (see Figure S1). Because these forms are positively charged, they cannot readily cross the thylakoid membrane. Acidification of the lumen will displace the equilibrium toward the charged forms, in turn allowing diffusion of more free forms across the thylakoid membrane into the lumen. Net transfer of weak bases from stroma to lumen and conversion to the protonated forms dissipates (buffers) ΔpH, but builds up a gradient of charged bases. Because the process is electroneutral with respect to the thylakoid membrane, weak bases do not dissipate (or augment) the Δψ component of pmf. However, weak bases in the presence of high concentrations of counterions, which are permeable through ion channels, can dissipate both the Δψ and ΔpH components of pmf . It is important to note that the concentrations of permeable ions in chloroplasts in vivo is likely to be small , , so that weak bases should primarily affect the ΔpH component of pmf.
Results and Discussion
As a first test of the BWB hypothesis in vivo, we assessed the fractions of pmf stored as Δψ and ΔpH using in vivo spectroscopic techniques , ,  in leaves depleted of or re-infiltrated with putrescine. We found that putrescine is highly mobile and readily diffuses into (Figure S2) and out of (Figure S3) leaves through cut petioles placed in water solutions. In figure S3 we report a decrease of the endogenous titer of putrescine. It is plausible to assume that at least in part this is due to loss of putrescine from the cut petiole. This leakage of endogenous putrescine from the leaf to the water is in line with the increase of putrescine titer of the solution from 0 µM to about 3.2 µM. Using cut tobacco leaves, which are stable in such solutions for long periods (e.g., Figure S3 which shows that photosystem II is stable in tobacco leaves for 48 h), we were able to deplete and replete putrescine levels, as confirmed by HPLC analysis . Incubation for ∼15 h after leaf detachment led to a 47% decrease in putrescine titer, whereas feeding putrescine with 6 mM increased putrescine 4–5 times (Figures S2 and S3). After 16 h of incubation of the leaf petiole in a 3 mM putrescine solution, the putrescine titer was increased at the tip of the leaf about 2-fold. Figure 1a shows representative kinetic traces of the decay of the electrochromic shift (ECS) signal around 520 nm upon a light-dark transition, in control, putrescine-depleted, and re-infiltrated with putrescine samples. As described in ref. , the extent of the rapid phase reflects the total light-driven pmf, whereas the slowly recovering phase is attributed specifically to the ΔpH portion. Deconvolution, as indicated, yields an estimate of the fraction of pmf stored as ΔpH and Δψ (Figure 1a). We observed a clear increase, by ∼36%, in the ΔpH-related phase (or decrease of Δψ) upon partial depletion of putrescine, which was reversed by putrescine infiltration (Figure 1b). Complementary photosynthesis measurements were also consistent with a higher ΔpH upon depletion of putrescine. In comparison to control leaves, infiltrated with water, those with elevated putrescine showed decreased qE responses and higher rates of linear electron transfer (Figure 1c and d). The response of qE to the total light-induced pmf, estimated by ECS decay, was lower when leaf putrescine was elevated (Figure 1d), consistent with a smaller fraction of pmf stored as ΔpH (Figure 1b). Also, the sensitivity of qE to light-induced ΔpH in vivo [as estimated by analyzing the decay kinetics of the ECS ] was found to be similar between treated and control leaves (Figure S4). Noteworthy is that putrescine at higher doses decrease even more the sensitivity of qE to light induced pmf by increasing Δψ and this effect is reversed to a great extent upon transfer of the leaf petiole from the putrescine solution to distilled water (Figure S5).
Panel A shows typical deconvoluted traces obtained from intact tobacco leaves incubated with water (gray) or reinfiltrated with 3 mM putrescine (Put; black). Steady-state pmf was probed at 8 different light intensities. Leaves with elevated putrescine (open symbols) show up to 40% higher Δψ/pmf than the corresponding controls (closed symbols) (Panel B). Buffering of the thylakoid lumen by elevated putrescine levels allows more efficient electron transfer at higher light intensity in comparison to low putrescine levels (Panel C). D. Energy-dependent antenna down-regulation (qE) as a function of the ΔpH component of the light-induced pmf. The linear fit for control has a slope of 0.369 (R2 = 0.998) and for putrescine treated a slope of 0.272 (R2 = 0.992), showing that putrescine supply decreases sensitivity of qE to ECSt by about 27%. All single points are means from 4 independent experiments performed with intact tobacco leaves and bars denote standard error.
The above results and those from a time-course study (Figures 2 and S3) suggest that putrescine depletion induces an increase in the ΔpH component of thylakoid pmf, but do not determine whether this effect is direct or indirect. We thus tested for effects of mutants of Arabidopsis thaliana Columbia (Arabidopsis) deficient in putrescine synthesis. Plants completely lacking putrescine are not viable  and thus we used a mutant, adc2-2 , which under our experimental conditions accumulated putrescine to about 40% wild-type levels (∼40 vs. ∼100 nmol g FW−1). Estimates of pmf partitioning using the decay of the ECS  gave evidence that moderate decreases in putrescine in adc2-2 led to a ∼15% smaller fraction of pmf attributable to Δψ [or a larger fraction stored as ΔpH (Figure 3)]. The effect was reversed by feeding putrescine into leaf petioles, supporting a direct effect of putrescine on pmf partitioning rather than indirect biochemical or developmental effects. A larger portion of pmf stored as ΔpH in putrescine-deficient plants is also supported by a higher sensitivity of the photoprotective qE response, a process dependent on lumen acidification, to pmf as estimated by the extent of the light-induced ECS signal (Figure 3, inset). Further analysis of the ECS decay kinetics , showed that elevated putrescine had no discernable effect on the activity of the thylakoid ATP synthase (Figure S6).
Partitioning of pmf in excised tobacco leaves. Detachment of leaves and insertion of the petiole in distilled water leads to a gradual decrease of putrescine titer in the leaf, which in turn leads to a decrease in the Δψ fraction of pmf. Data correspond to the first two days after detachment (Fv/Fm decrease during this period was no more than 10%) and bars denote standard error (n = 2). Linear regression of putrescine titer versus Δψ is shown as a solid line with a slope of 0.00173 (R2 = 0.995).
The partitioning of pmf in vivo into the electric component (Δψ) for intact Arabidopsis leaves and the effect of putrescine. Low titer of putrescine (adc2-2) leads to lower Δψ/pmf in comparison to the wild type (ws). The exogenous supply of putrescine (in various doses from 1 mM up to 10 mM) leads to an increase of the fractionation of pmf to Δψ to values even higher than those of wild type. The ECS signal was measured and deconvoluted as described in Materials and Methods. Error bars denote standard error for n≥3 and the asterisk denotes statistically (t-test) significant, at the 0.05 level, difference to the adc2-2 mean values (second column). Mean values were compared only to the adc2-2 which has the lowest putrescine titer. Inset: Energy-dependent antenna down-regulation (qE) as a function of the light-induced pmf (ECSt) for putrescine-treated adc2-2 plants (closed circles) and controls in distilled water (open circles). A linear regression model was fitted to the data using log-transformed qE values. The mean ECSt response is significantly different between “putrescine fed adc2-2” and controls (P = 0.0047), across the range of the observed qE values.
The above experiments show a positive relationship between putrescine titer in leaves, altered by depletion, infiltration, or mutation, and the fraction of pmf stored as Δψ, consistent with the BWB hypothesis. At pH ∼7.5 or lower, putrescine should predominantly be in its +2 state (pK1 = 10.5, pK2 = 9.04), and its effects have been attributed to its ability to bind anions, nucleic acids, and other negatively charged molecules or domains , , . It is possible that putrescine affects pmf partitioning by scavenging anions that would otherwise permeate the thylakoid and dissipate Δψ. To test these possibilities, we assayed the effects of putrescine on Δψ/pmf using ECS assays ,  in isolated spinach thylakoids in buffer with set ionic composition. The dependence of Δψ/pmf on putrescine concentration is shown in Figure 4 with selected ECS traces shown in the inset. Putrescine is a naturally occurring solute in chloroplasts but the endogenous pool is lost during isolation procedures, together with other stromal solutes. In the presence of 0 mM KCl and only a low dose of bivalent cations (i.e., 0.15 mM MgCl2) (solution of low ionic strength), the fraction of pmf stored as Δψ was about 0.2 for control, indicating that a significant decline occurred after isolation in comparison to the in vivo conditions (Δψ/pmf∼0.5) (Figure 4). This decline at least partly reflects altered ionic composition of the suspension buffer compared to chloroplasts in vivo , but could also reflect the loss of putrescine or other mobile buffers. Adding putrescine to the suspension buffer increased Δψ/pmf of thylakoids within a few seconds, indicating that putrescine can rapidly modulate pmf partitioning. Addition of 220 µM putrescine led to an increase in Δψ/pmf to ∼0.6, and putrescine concentrations above 2 mM led to Δψ/pmf>0.9 (Figure 4). Our deconvolution suffers to some extent by drifts and noise due to in vitro conditions. Thus the absolute values of Δψ/pmf caused by putrescine could be slightly different to those of figure 4 (∼15%). However, the effect of putrescine on thylakoidal energization is initially a rapid increase of Δψ and then at a second phase a plateau.
Thylakoids isolated from spinach were pre-incubated in the dark in solution containing 0 mM, 0.02 mM, 0.22 mM, 2.2 mM, or 3.2 mM putrescine. ECS was measured during and after 16 s of actinic illumination. Selected deconvoluted traces are illustrated as an insert. Fractional storage of pmf as Δψ was estimated and plotted as a function of the putrescine concentration.
The fact that putrescine decreased ΔpH even in a solution with low concentration of scavangeable anions, supports the operation of the BWB mechanism, and is also consistent with the effects of exogenously added weak bases in photosynthetic prokaryotes  and thylakoids ,  in which amines have been shown to be concentrated by more than 100-fold on the low pH side of an energized membrane, with concomitant effects on ΔpH. We would suggest that putrescine is a good compromise between balancing ΔpH and Δψ and avoiding secondary deleterious effects. In addition, putrescine levels in the cell is so finely tuned through multiple ways (synthesis through two highly regulated pathways, conversion from Spermidine, transport from other cell compartments or neighbour cells, release from conjugate with phenolics, binding to proteins etc , ) that one can not find easily other cell metabolites that can adjust their level so rapidly and accurately to meet the ever changing demand. So although ammonia and methylamine could act in a similar way to putrescine their titer in cells is lower than that of putrescine and thus their importance in the BWB mechanism should be lower.
Assuming that putrescine is evenly distributed in leaves, we estimate its cellular concentration in tobacco leaves at about 275 µM (based on measured value of 250 nmoles Put/g fresh weight and assuming 90% of leaf mass is water; see also, ref. . When thylakoids were suspended in buffer containing this concentration of putrescine in thylakoids, we observed about 50% of pmf stored as Δψ and ΔpH (Figure 4), similar to what is observed in healthy, unstressed leaves , . However, it is important to note that the partitioning of pmf is also expected to be influenced by ionic composition, with increasing counterion concentration dissipating Δψ .
Putrescine concentrations in leaves are increased during environmental stress, in part due to up-regulation of chloroplast arginine decarboxylase (ADC, EC 220.127.116.11) –. One may thus expect to see a shift in the partitioning of pmf into Δψ under environmental stress. However, at least under drought stress in wild watermelon , short-term high light exposure in Arabidopsis , or low CO2 and O2 in tobacco , the opposite was observed, with a pronounced increase in ΔpH/pmf. A reasonable explanation is that the initial increase in ΔpH/pmf is due to stress-induced changes in ionic composition ,  and that putrescine may ameliorate these effects over the long term. At a mechanistic level, consistent with this view, Cruz et al. , showed that elevated lumen buffering capacity will increase the counterion chemical activity needed to dissipate the Δψ component of pmf, whereas Ioannidis et al.  showed that putrescine can overcome photosynthetic control and thus stimulate chemiosmotic ATP synthesis in thylakoids of higher plants .
In Arabidopsis grown under high salt stress, photosynthesis would likely need to operate under conditions where the ionic strength inside the plastid is high. In this case, pmf storage would be heavily biased toward ΔpH formation , . Consequently, energy dissipation would be more easily and strongly induced at low and moderate light intensities, severely limiting the productivity and growth of the plant, even if water and CO2 were not limiting. Thus, the accumulation of putrescine observed in plants grown under high salt stress ,  and particularly in Arabidopsis through adc2 induction  could serve to increase the BWB effect, rebalancing pmf toward Δψ and optimizing the regulation of energy transduction. In line with this view, blocking this up-regulation of putrescine during salt stress, e.g., in the adc-2-1 mutant of Arabidopsis, leads to increased sensitivity to salt stress, which is restored upon addition of putrescine , whereas over-expressing adc increased tolerance to drought .
We present evidence that putrescine plays a role in modulating pmf partitioning in vivo via the BWB mechanism, possibly operating as a part of the regulatory network of photosynthesis. Putrescine levels in the leaf are known to be regulated at several levels, including rates of synthesis, catabolism, conversion to spermidine, conjugation, intracellular or extracellular transport, gene expression, and/or allostery . Thus, putrescine homeostasis could provide the plant with an independent mechanism for adapting the qE response to pmf, optimizing the balance between energy transduction and dissipation under a variety of stress conditions.
Materials and Methods
We conducted time-resolved spectroscopic measurements for estimation of LEF, qE, ECSt, and Δψ/pmf at room temperature using wild-type (Wt) Arabidopsis thaliana (Wassilevskija ecotype; WS) plants, the low putrescine adc2-2 mutant , and wild-type Nicotiana tabacum cv Xanthi as previously described . More particularly, LEF was calculated for the following expression 0.84*PAR*(Fm′-Ft)/Fm′) where Fm′ is the maximal fluorescence value of a light adapted leaf after a saturating pulse (>7,000 µmol photons m−2s−1) and Ft is the level of fluorescence immediately before the saturating pulse. For estimates of pmf partitioning, the ECS was measured over longer dark intervals (60 sec) and deconvoluted as previously described ,  using the following equation for tobacco: ECS520 = A520 − 0.5×A535 − 0.5×A505. For more precise deconvolution of ECS signals from Arabidopsis, we derived an equation from empirically determined, relative extinction coefficients at each wavelength, based on methods described previously , : ECS520 = −(1.61*A520−0.61*A505−1.17A535).
Determination of putrescine titer
Putrescine titer in leaves was estimated after benzoylation of the amines, separation in HPLC, and quantitation of the derivatives as previously described .
In vitro experiments
Thylakoids were isolated from market spinach as previously described . Freshly isolated thylakoids (10 µg/mL Chl) were treated with various doses of the amine salt in the dark and, after equilibration, were subjected to light for 16 s. The final volume was 3 mL of a working solution containing 0.125 mM tricine, pH 7.8, 0.15 mM MgCl2, 0.3 M sucrose, 10% ficoll. Photosynthetic proton uptake was supported by 30 µM PMS. Spectroscopic measurements were performed, as above, with the sample contained in a cuvette (10-mm path length). For thylakoid samples, a simple deconvolution yielded consistent results: ECS520 = A520 − 1.2×A535.
Simplified scheme for the regulation of pmf partitioning by Putrescine.
Assay for putrescine uptake into tobacco leaves by HPLC.
Effects on Δψ/ pmf and PSII photochemical efficiency of infiltration of leaves with water.
Effect of putrescine titre on the dependence of energy-dependent antenna down-regulation (qE) on the ΔpH component of light-induced pmf .
Energy-dependent antenna down-regulation (qE) as a function of total light-induced pmf .
We thank Prof. Dr. Polychronis Kostoulas (University of Thessaly) for the linear regression model and RIKEN and Profs. Dr. Urano and Dr. Shinozaki for the generous gift of adc2-2 mutant.
Conceived and designed the experiments: NI DK. Performed the experiments: NI JC. Analyzed the data: NI JC DK. Contributed reagents/materials/analysis tools: KK DK. Wrote the paper: NI JC KK DK.
- 1. Kramer DM, Avenson TJ, Edwards GE (2004) Dynamic flexibility in the light reactions of photosynthesis governed by both electron and proton transfer reactions. Trends Plant Sci 9: 349–357.
- 2. Kramer DM, Cruz JA, Kanazawa A (2003) Balancing the central roles of the thylakoid proton gradient. Trends Plant Sci 8: 27–32.
- 3. Li X-P, Björkman O, Shih C, Grossman AR, Rosequist M, et al. (2000) A pigment-binding protein essential for regulation of photosynthetic light harvesting. Nature 403: 391–395.
- 4. Pascal AA, Liu Z, Broess K, van Oort B, van Amerongen H, et al. (2005) Molecular basis of photoprotection and control of photosynthetic light-harvesting. Nature 436: 134–137.
- 5. Ruban AV, Berera R, Ilioaia C, van Stokkum IH, Kennis JT, et al. (2007) Identification of a mechanism of photoprotective energy dissipation in higher plants. Nature 450: 575–578.
- 6. Hope AB (2000) Electron transfers amongst cytochrome f, plastocyanin and photosystem I: kinetics and mechanisms. Biochim Biophys Acta 1456: 5–26.
- 7. Takizawa K, Cruz JA, Kanazawa A, Kramer DM (2007) The thylakoid proton motive force in vivo. Quantitative, non-invasive probes, energetics, and regulatory consequences of light-induced pmf. Biochim Biophys Acta 1767: 1233–1244.
- 8. Kramer DM, Evans JR (2011) The importance of energy balance in improving photosynthetic productivity. Plant Physiol 155: 70–78.
- 9. Cruz JA, Sacksteder C, Kanazawa A, Kramer DM (2001) Contribution of electric field Δψ to steady-state transthylakoid proton motive force in vitro and in vivo. Control of pmf parsing into Δψ and ΔpH by counterion fluxes. Biochemistry 40: 1226–1237.
- 10. Avenson TJ, Cruz JA, Kramer DM (2004) Modulation of energy-dependent quenching of excitons in antennae of higher plants. Proc Natl Acad Sci USA 101: 5530–5535.
- 11. Avenson TJ, Cruz JA, Kanazawa A, Kramer DM (2005) Regulating the proton budget of higher plant photosynthesis. Proc Nat Acad Sci USA 102: 9709–9713.
- 12. Ioannidis NE, Sfichi L, Kotzabasis K (2006) Putrescine stimulates chemiosmotic ATP synthesis. Biochim Biophys Acta 1757: 821–828.
- 13. Capell T, Bassie L, Christou P (2004) Modulation of the polyamine biosynthetic pathway in transgenic rice confers tolerance to drought stress. Proc Natl Acad Sci USA 101: 9909–9914.
- 14. Kasukabe Y, He L, Nada K, Misawa S, Ihara I, et al. (2004) Overexpression of spermidine synthase enhances tolerance to multiple environmental stresses and up-regulates the expression of various stress-regulated genes in transgenic Arabidopsis thaliana. Plant Cell Physiol 45: 712–722.
- 15. Kusano T, Berberich T, Tateda C, Takahashi Y (2008) Polyamines: essential factors for growth and survival. Planta 228: 367–381.
- 16. Mehta RA, Cassol T, Li N, Ali N, Handa AK, et al. (2002) Engineered polyamine accumulation in tomato enhances phytonutrient content, juice quality, and vine life. Nat Biotechnol 20: 613–618.
- 17. Gerner EM, Meyskens LF (2004) Polyamines and cancer. Old molecules new understanding. Nat Rev Cancer 4: 781–792.
- 18. Crofts A (1967) Amine uncoupling of energy transfer in chloroplasts. I. In relation to ammonium ion uptake. J Biol Chem 242: 3352–3359.
- 19. Livingston AK, Cruz JA, Kohzuma K, Dhingra A, Kramer DM (2010) An Arabidopsis mutant with high cyclic electron flow around Photosystem I (hcef) Involving the NADPH dehydrogenase complex. Plant Cell 22: 221–233.
- 20. Kotzabasis K, Christakis-Hampsas MD, Roubelakis-Angelakis KA (1993) A narrow bore HPLC method for the identification and quantitation of free, conjugated and bound polyamines. Anal Biochem 214: 484–489.
- 21. Urano K, Tokunori H, Shinozaki K (2005) Arabidopsis ADC genes involved in polyamine biosynthesis are essential for seed development. FEBS Lett 579: 1557–1564.
- 22. Baker N, Harbinson J, Kramer DM (2007) Determining the limitations and regulation of photosynthetic energy transduction in leaves. Plant Cell Environ 30: 1107–1125.
- 23. De Robertis A, De Stefano C, Foti C, Giuffre O, Sammartano S (2001) Thermodynamic parameters for the binding of inorganic and organic anions by biogenic polyammonium cations. Talanta 54: 1135–1142.
- 24. Kramer DM, Sacksteder CA, Cruz JA (1999) How acidic is the lumen? Photosynth Res 60: 151–163.
- 25. Guarino L, Cohen SS (1979) Uptake and accumulation of putrescine and its lethality in Anacystis nidulans. Proc Natl Acad Sci USA 76: 3184–3188.
- 26. Gaensslen RE, McCarty RE (1971) Amine uptake in chloroplasts. Arch Biochem Biophys 147: 55–65.
- 27. Schuldiner S, Rotternberg H, Avron M (1972) Determination of ΔpH in chloroplasts. Fluorescent amines as a probe for the determination of ΔpH in chloroplasts. Eur J Biochem 25: 64–70.
- 28. Galston AW (2001) Plant biology—retrospect and prospect. Curr Sci 80: 150–152.
- 29. Borrell A, Culianez-Macia F, Altabella T, Besford RT, Flores D, et al. (1995) Arginine decarboxylase is localized in chloroplasts. Plant Physiol 109: 771–776.
- 30. Malmberg RL, Watson MB, Galloway GL, Yua W (1998) Molecular genetic analyses of plant polyamines. Crit Rev Plant Sci 17: 199–224.
- 31. Bortolotti C, Cordeiro A, Alcázar R, Borrell A, Culiañez-Macià FA, et al. (2004) Localization of arginine decarboxylase in tobacco plants. Physiol Plant 120: 84–92.
- 32. Kohzuma K, Cruz JA, Akashi K, Hoshiyasu S, Munekage YN, et al. (2009) The long-term responses of the photosynthetic proton circuit to drought. Plant Cell Environ 32: 209–219.
- 33. Robinson SP, Downton WJS, Millhouse JA (1983) Photosynthesis and ion content of leaves and isolated chloroplasts of salt-stressed spinach. Plant Physiol 73: 238–242.
- 34. Bagni N, Ruiz-Carrasco K, Franceschetti M, Fornalè S, Fornasiero RB, et al. (2006) Polyamine metabolism and biosynthetic gene expression in Arabidopsis thaliana under salt stress. Plant Physiol Biochem 44: 776–786.
- 35. Alcázar R, Marco F, Cuevas JC, Patron M, Ferrando A, et al. (2006) Involvement of polyamines in plant response to abiotic stress. Biotechnol Lett 28: 547–552.
- 36. Urano K, Yoshiba Y, Nanjo T, Ito T, Yamaguchi-Shinozaki K, et al. (2004) Arabidopsis stress-inducible gene for arginine decarboxylase AtADC2 is required for accumulation of putrescine in salt tolerance. Biochem Biophys Res Comm 313: 369–375.
- 37. Sacksteder CA, Kramer DM (2000) A diffused-optics flash kinetic spectrophotometer (DOFS) for measurements of absorbance changes in intact plants in the steady-state. Photosynth Res 56: 103–112.
- 38. Zhang RCJ, Kramer DM, Magallanes-Lundback ME, Dellapenna D, Sharkey TD (2009) Moderate heat stress reduces the pH component of the transthylakoid proton motive force in light-adapted, intact tobacco leaves. Plant Cell Environ 32: 1538–1547.