Young-Nam Hong

Photoinactivation of photosystem II complexes and photoprotection by non-functional neighbours in Capsicum annuum L. leaves

Abstract. Leaf segments from Capsicum annuum plants grown at 100 μmol photons  m−2 s−1 (low light) or 500 μmol photons  m−2 s−1 (high light) were illuminated at three irradiances and three temperatures for several hours. At various times, the remaining fraction (f) of functional photosystem II (PS II) complexes was measured by a chlorophyll fluorescence parameter (1/Fo− 1/Fm, where Fo and Fm are the fluorescence yields corresponding to open and closed PS II traps, respectively), which was in turn calibrated by the oxygen yield per saturating single-turnover flash. During illumination of leaf segments in the presence of lincomycin, an inhibitor of chloroplast-encoded protein synthesis, the decline of f from 1.0 to about 0.3 was mono-exponential. Thereafter, f declined much more slowly, the remaining fraction (≈0.2) being able to survive prolonged illumination. The results can be interpreted as being in support of the hypothesis that photoinactivated PS II complexes photoprotect functional neighbours (G. Öquist et al. 1992, Planta 186: 450–460), provided it is assumed that a photoinactivated PS II is initially only a weak quencher of excitation energy, but becomes a much stronger quencher during prolonged illumination when a substantial fraction of PS II complexes has also been photoinactivated. In the absence of lincomycin, photoinactivation and repair of PS II occur in parallel, allowing f to reach a steady-state value that is determined by the treatment irradiance, temperature and growth irradiance. The results obtained in the presence and absence of lincomycin are analysed according to a simple kinetic model which formally incorporates a conversion from weak to strong quenchers, yielding the rate coefficients of photoinactivation and of repair for various conditions, as well as gaining an insight into the influence of f on the rate coefficient of photoinactivation. They demonstrate that the method is a convenient alternative to the use of radiolabelled amino acids for quantifying photoinactivation and repair of PS II in leaves.

Photoinactivation of Photosystem II in leaves

Photoinactivation of Photosystem II (PS II), the light-induced loss of ability to evolve oxygen, inevitably occurs under any light environment in nature, counteracted by repair. Under certain conditions, the extent of photoinactivation of PS II depends on the photon exposure (light dosage, x), rather than the irradiance or duration of illumination per se, thus obeying the law of reciprocity of irradiance and duration of illumination, namely, that equal photon exposure produces an equal effect. If the probability of photoinactivation (p) of PS II is directly proportional to an increment in photon exposure (p = kΔx, where k is the probability per unit photon exposure), it can be deduced that the number of active PS II complexes decreases exponentially as a function of photon exposure: N = Noexp(−kx). Further, since a photon exposure is usually achieved by varying the illumination time (t) at constant irradiance (I), N = Noexp(−kI t), i.e., N decreases exponentially with time, with a rate coefficient of photoinactivation kI, where the product kI is obviously directly proportional to I. Given that N = Noexp(−kx), the quantum yield of photoinactivation of PS II can be defined as −dN/dx = kN, which varies with the number of active PS II complexes remaining. Typically, the quantum yield of photoinactivation of PS II is ca. 0.1μmol PS II per mol photons at low photon exposure when repair is inhibited. That is, when about 107 photons have been received by leaf tissue, one PS II complex is inactivated. Some species such as grapevine have a much lower quantum yield of photoinactivation of PS II, even at a chilling temperature. Examination of the longer-term time course of photoinactivation of PS II in capsicum leaves reveals that the decrease in N deviates from a single-exponential decay when the majority of the PS II complexes are inactivated in the absence of repair. This can be attributed to the formation of strong quenchers in severely-photoinactivated PS II complexes, able to dissipate excitation energy efficiently and to protect the remaining active neighbours against damage by light.

Expression characteristics of serine proteinase inhibitor II under variable environmental stresses in hot pepper (Capsicum annuum L.)

A cDNA clone encoding a protein (CaPI-2), which is highly homologous to proteinase inhibitor II (PI-2) from tobacco and tomato, was isolated from pericarp cDNA library of hot pepper. The 304 amino acid long primary structure contains a region of a putative signal peptide and three highly conserved repeated proteinase inhibitor domains with a trypsin- and two chymotrypsin-specific reactive sites. The mRNA encoding CaPI-2 was expressed at a steady-state level in leaves, stem, and roots. Mechanical wounding increased the CaPI-2 mRNA expression in the adjacent and remote site from wounded area. In addition to wounding, the effects of various chemical and physical stresses were investigated. Exogenous ABA, salt and electrical current was observed to increase the CaPI-2 expression, whereas acetylsalicylic acid gave an inverse effect on the wounding. In contrast, polyethylene glycol (PEG) and cold stress had no effects on the CaPI-2 expression level.

Altered physiology in trehalose-producing transgenic tobacco plants: Enhanced tolerance to drought and salinity stresses

Transgenic tobaccoNicotiana tabacum L. var. SR1) plants that over-express theEscherichia coli trehalose-6-phosphate synthase (TPS) gene(otsA) synthesized small amounts of trehalose (<400 µg g-1 leaf) while non-transformants produced no detectable trehalose. Some transgenic plants expressing a high level ofotsA exhibited stunted growth and morphologically altered leaves. We tested F22 homozygous plants devoid of phenotypic changes to determine their physiological responses to dehydration and salinity stresses. All transgenic plants maintained better leaf turgidity under a limited water supply or after treatment with polyethylene glycol (PEG). Furthermore, fresh weight was maintained at higher levels after either treatment. The initial leaf water potential was higher in transgenic plants than non-transformants, but, in both plant types, was decreased to a comparable degree following dehydration. When grown with 250 mM NaCl, transgenic plants exhibited a significant delay in leaf withering and chlorosis, as well as more efficient seed germination. Our results suggest that either trehalose or trehalose-6-phosphate can act as an osmoprotective molecule without maintaining water potential, in contrast to other osmolytes. Furthermore, both appear to protect young embryos under unfavorable water status to ensure subsequent germination.

A comparative study on the protective role of trehalose and LEA proteins against abiotic stresses in transgenic Chinese cabbage (Brassica campestris) overexpressingCaLEA orotsA

Trehalose and LEA proteins, representative low MW chemicals that are synthesized under dehydration, are known to protect plants from drought stress. To compare their effectiveness on enhancing tolerance against various abiotic stresses, we generated transgenic Chinese cabbage plants overexpressingE. ctdi trehalose-6-phosphate synthase gene (otsA) or hot pepper (Capsicum annuum) LEA protein gene(CaLEA). Both transgenic plants exhibited altered phenotype including stunted growth and aberrant root development When subjected to drought, salt or heat stress, these plants showed remarkably improved tolerance against those stresses compared with nontransformants. After dehydration treatment, leaf turgidity and fresh weight was better maintained in both transgenic plants. GaUEA-plants performed somewhat better under dehydrated condition. When treated with 250 mM NaCI, both otsA-plants and CaLEA-plants remained equally healthier than nontransformants in maintaining leaf turgidity and delaying necrosis. Furthermore, leaf Chi content and Fv/Fm was maintained considerably higher in both transgenic plants than nontransformants. After heat-treatment at 45°C, both transgenic plants appeared much less damaged in external shape and PSII function, but LEA proteins were more protective. Our results indicate that although both trehalose and LEA proteins are effective in protecting plants against various abiotic stresses, LEA proteins seem to be more promising in generating stress-tolerant transgenic plants.

Putative effects of pH in intra-chloroplast compartments on photoprotection of functional photosystem II complexes by photoinactivated neighbours and on recovery from photoinactivation in Capsicum annuum leaves

Leaf segments from Capsicum annuum L. plants grown at 100 (low light) or 500 (high light) μmol photons m-2 s-1 were illuminated in the presence of nigericin, dithiothreitol (DTT), or high [CO2] (1% in air), with or without lincomycin, an inhibitor of chloroplast-encoded protein synthesis. At various times, the remaining fraction (f ) of functional PSII complexes was measured by a dark-adapted chlorophyll fluorescence parameter (1/Fo- 1/Fm; where Fo and Fm are the fluorescence yields corresponding to open and closed PSII traps, respectively), which was calibrated by the oxygen yield per saturating single-turnover flash. The results were interpreted according to a simple kinetic model incorporating the hypothesis that photoinactivated PSII complexes photoprotect functional neighbours (Lee et al. 2001, Planta 105, 377-384), yielding the rate coefficients of photoinactivation and repair, and a parameter, a, which phenomenologically describes the effectiveness of photoprotection by photoinactivated PSII complexes. The presence of the uncoupler nigericin during illumination greatly decreased a by an order of magnitude, suggesting that a sufficiently acidic thylakoid lumen may be required for the photoprotective mechanism to operate. Both nigericin and high [CO2] decreased the rate coefficient of repair several fold, suggesting that the stromal pH was non-optimal for protein synthesis in the presence of nigericin or high [CO2]. The xanthophyll cycle, inhibited by DTT, seemed to have a minimal effect on the rate coefficients of photoinactivation and repair, and on the parameter a. The results underline the importance of optimal pH in both the stroma and lumen for photoprotection, and recovery from photoinactivation of PSII.

Increased Photoinhibition in Dehydrated Leaves of Hot Pepper (Capsicum annuum L.) Is Not Accompanied by an Incremental Loss of Functional PSII

We examined the photosynthetic responses to photoinhibition in dehydrated leaves of hot pepper (Capsicum annuum L.). Stress was induced by immersing the roots of whole plants in Hoaglands solution containing polyethylene glycol (PEG) under high light (900 μmol photons m-2 · s-1). This PEG-treatment lowered the leaf water potential and the maximal rate of photosynthetic O2 evolution (Pmax) linearly, in a time-dependent manner, to about 50% inhibition after 6 h. Pmax also decreased linearly as the period of high-light treatment lengthened. That inhibitory response was not as extreme, showing about 30% inhibition after 6 h. However, when the treatments of dehydration and high light were simultaneously administered, Pmax decreased more rapidly, in a synergistic fashion, showing about 90% inhibition within 2 h. Dehydration, in contrast to the light treatment, did not lower the maximal photochemical efficiency (Fv/Fm). Furthermore, this decline in Fv/Fm for light-treated, dehydrated leaves was almost identical to the response of photoinhibited leaves that were not dehydrated. Similar changes were observed in the number of functional PSII complexes. The decrease in Pmax and the amount of functional PSII was linearly correlated in photoinhibited leaves, but not in dehydrated leaves, regardless of light treatment. Therefore, we have demonstrated that exacerbated photoinhibition in dehydrated leaves occurs without an incremental loss of functional PSII.

GmNiR-1, a soybean nitrite reductase gene that is regulated by nitrate and light

A genomic DNA clone encoding nitrite reductase (NiR; EC 1.7.7.1), GmNiR-1, was isolated from soybean [Glycine max (L.) Merr.]. It is composed of four exons and three introns carrying an open reading frame coding for a protein of 596 amino acids. The putative MW of GmNiR-1 is 67 kDa with theoretical pI of 6.95, but the measured MW is ca 64 kDa. The difference between putative and measured MW of GmNiR-1 lies in the presence of a transit peptide. Genomic DNA blot analysis suggested that soybean NiR gene family consisted of at least three genes. In 2-week-old plants grown in the nitrate-supplemented soil, GmNiR-1 was expressed in roots and leaves, but not in hypocotyls. Transcript level of GmNiR-1 in roots was not increased by light, but was increased by nitrate even in the dark. However, light and nitrate had a synergistic effect on the increased expression of GmNiR-1. A polyclonal antibody generated against the C-terminal of GmNiR-1 hybridized to a single (62 kDa), double (62 and 64 kDa), and three bands (62, 64 and 66 kDa) in roots, hypocotyls and leaves, respectively. In etiolated seedlings, NiR proteins in roots and hypocotyls were induced by simultaneous treatment with light and nitrate, but those in cotyledons were already present substantially without induction, implying the presence of at least two kinds of NiR genes (a constitutive one and an inducible one regulated by light and nitrate) in soybean seedlings.

Regulation of ascorbate peroxidase activity in dark-grown radish cotyledons by a catalase inhibitor, 3-Amino-1,2,4-Triazole

The present study was performed to see the physiological role of cytosolic ascorbate peroxidase (APX) and its relationship to other enzymes involved in the H2O2 scavenging metabolism, and also to elucidate the regulation of APX expression in dark-grown radish (Raphanus sativus L. cv Taiwang) cotyledons. To do so, 3-amino-l,2,4-triazole (aminotriazole), a known specific inhibitor of catalase, was used to simulate a catalase-deficient phenomenon in cotyledons. Aminotriazole, in very low concetration (10-4 M), inhibited remarkably the development of catalase activity in cotyledons during dark germination. This inhibition of catalase by aminotriazole, however, did not result in any significant changes in the growth response and the H2O2 level of developing cotyledons. In addition, the development of guaiacol peroxidase (GPX) activity was also not significantly affected. Unlike GPX, cytosolic APX activity was induced rapidly and reached a 1.7-fold increase in aminotriazole treated cotyledons at day 7 after germination. However,in vitro incubation of cytosolic APX preparation from cotyledons with aminotriazole did not result in any significant change in activity. One cytosolic APX isozyme (APXa) band involved in this APX activation was predominantly intensified in a native polyacrylamide gel by activity staining assay. This means that this APXa isozyme seems to play a key role in the expression of cytosolic APX activity. On the other hand, 2-day-old control seedlings treated with exogenous 1 mM H2O2 for 1 h showed a significant increase of cytosolic APX acitivity even in the absence of aminotriazole. Also, 2 μM cycloheximide treatment substantially inhibited the increase of APX activity due to aminotriazole. Based on these results, we suggest that a radish cytosolic APX could probably be substituted for catalase in H2O2 removal and that the expression of APX seems to be regulated by a change of endogenous H2O2 level which couples to APX protein synthesis in a translation stage in cotyledons.

Photosynthetic Responses and Photoprotection in Korean Hot Pepper (Capsicum annuum L.) against High Light Stress

Photoinhibition and photoprotection of PSII in the leaves of hot pepper (Capsicum annuum L.) grown in Hoagland solution and Tap water were compared. Though changes in the rates of evolution as a function of photon fluence rate (PFR) were comparable, the rates of respiration in the dark was 3 times higher in the Hoagland solution grown leaves than in the Tap-water grown ones. Compared to Hoagland solution grown plane, PSIIs of Tap water grown pepper leaves were more susceptible to photoinhibitory light treatment. In order to inactivate functional PSII to the same extents, Hoagland solution grown plants required almost 2-fold high light treatment than those of Tap water . Interestingly, the remaining fraction of PSII in Hoagland grown pepper was able to survive under prolonged illumination in the presence of lincomycin, which probably means that the growth condition of plant seemed to have an effect on the recovery of PSII from light stress. When PSII was severly photoinactivated at a chilling temperature, recovery was observed only if the residual functional PSII were not inhibited with DCMU, Nigericin and MV during recovery. In conclusion, PSIIs grown in the Hoagland solution was more resistant to excess light than in the Tap water grown one and the recovery of PSII from photodamage was more efficient in Hoagland grown pepper leaves than Tap water grown one, which means that the increased dark respiration may play a important role in the protection of PSII from photoinhibition by helping repair photosynthetic proteins (in particular, the D1 protein of PSII) degraded by photoinhibition.