Reference gene selections for real time quantitative PCR analysis of gene expression in different oat tissues and under salt stressOriginal Papers

Z.L. DUAN, W.H. HAN, L. YAN, B. WU

Biologia plantarum 64: 838-844, 2020

Appropriate choice of reference genes for data normalization is of critical importance for accurate real time reverse transcription quantitative PCR (RT-qPCR) analysis of gene expression. Oat is an agriculturally important crop cultivated widely around the world for grain or forage, and appropriate reference genes for reliable gene expression analysis remain to be identified. In this study, we selected nine candidate reference genes based on available oat RNA-seq data. We then conducted a systematic evaluation of the relative expression stability of these genes in different tissues and under salt stress using statistical algorithms, geNorm,...

Simultaneous induction of anthocyanin and peroxidase by sucrose in hypocotyls and roots of Chinese red radish seedlingsOriginal Papers

L. WANG, X.-X. JING, P.-P. ZHAO, L.-F. WANG, Y. YIN, Y.-F. LI

Biologia plantarum 64: 828-837, 2020 | DOI: 10.32615/bp.2020.156

Anthocyanin and peroxidase (POD) are important active compounds in Chinese red radish (Raphanus sativus L.). The effects of exogenous sucrose, mixture of fructose and glucose (F/G 1:1), and mannose on anthocyanin accumulation and POD activity were investigated in hypocotyls and roots of red radish seedlings. Sucrose was most effective in inducing anthocyanin accumulation and POD activity in both organs, followed by F/G; mannose caused only a little increase of anthocyanin content and POD activity in hypocotyls, but a decrease in roots. The distribution of anthocyanin accumulation was different between hypocotyls and roots in the presence or...

Festulolium field performance under fluctuating growing conditions in LithuaniaSpecial Issue: Festulolium

V. KEMEŠYTĖ, K. JAŠKŪNĖ, G. STATKEVIČIŪTĖ

Biologia plantarum 64: 821-827, 2020

Festulolium cultivars are widely utilized in Lithuania because they are persistent under abiotic stresses and are high yielding. However, changing climate challenges the existing Festulolium cultivars to adapt to new growing conditions and still maintain the yield. In this study, we aimed at evaluating the yield stability of two Festulolium cultivars in field trials under fluctuating Lithuanian conditions. The mean total dry matter yield (DMY) of both Festulolium cultivars fluctuated greatly between the years and ANOVA analysis showed a significant effect of environment on total DMY as well as DMY of each cut, but the genotype...

Morphological diversity of seeds of Polish Festulolium cultivars depending on weather conditionsSpecial Issue: Festulolium

B. BORAWSKA-JARMUŁOWICZ, G. MASTALERCZUK, E. MAŁUSZYŃSKA, W. BORUCKI

Biologia plantarum 64: 814-820, 2020 | DOI: 10.32615/bp.2020.146

Morphological traits of seeds of Festulolium braunii (K. Richt.) A. Camus cultivars were studied in relation to weather conditions. The obtained results show that morphological traits of seeds varied among cultivars and years of cultivation. The values of seed characteristics were higher in year 2016 due to more favorable weather conditions for plant growth. It was found that the husked seeds of Festulolium cultivars were characterized by a similar length and width regardless of the year, whereas naked seeds significantly differed in these characteristics. Mean values (from the research years) of seed parameters, such as thousand seed...

Virus induced PhFTRv gene silencing results in yellow-green leaves and reduced cold tolerance in petuniaOriginal Papers

L. SANG, L. PENG, Z. QIU, F. LUO, G. CHEN, L. GAO, Y. YU, J. LIU

Biologia plantarum 64: 807-813, 2020 | DOI: 10.32615/bp.2020.151

Ferredoxin-thioredoxin reductase (FTR) is an iron-sulfur protein that supplies electrons from photochemically reduced ferredoxin (Fd) to thioredoxin (Trx) in the ferredoxin/thioredoxin system in chloroplasts. The FTR is a heterodimer with a variable subunit (FTRv) and a catalytic subunit (FTRc). The function of FTRv is not well known. In petunia (Petunia hybrida), FTRv is a single-copy gene, which is named PhFTRv. In this study, the spatio-temporal expression of PhFTRv in petunia was analyzed, and PhFTRv transcription was found to be high in leaves and stems. A tobacco rattle virus gene silencing was used in...

Transcriptome-based screening and the optimal reference genes for real-time quantitative PCR in Rehmannia chingii and R. henryi Original Papers

X. ZUO, F.-Q. WANG, X.-R. LI, M.-M. LI

Biologia plantarum 64: 798-806, 2020 | DOI: 10.32615/bp.2020.154

Real time quantitative PCR (qPCR) is a powerful tool for studying the expression of specific genes. The accuracy and reliability of qPCR analysis data require the selection of reference genes with stable expression. However, the reference genes that can be used for qPCR of Rehmannia chingii and R. henryi have not yet been identified. In this study, based on the transcriptome data of R. chingii and R. henryi, we initially selected genes with relatively stable expression in different samples. We screened six candidate reference genes in R. chingii and R. henryi and calculated their expression abundance by real...

Breeding and genetics of two new amphiploid Festulolium synthetics with improved yield and digestibilitySpecial Issue: Festulolium

J. BAERT, K. VAN LAERE, C. VAN WAES, A. GHESQUIERE, J. APER

Biologia plantarum 64: 789-797, 2020 | DOI: 10.32615/bp.2020.138

In order to introduce drought tolerance and improved cell wall digestibility from fescue in fodder ryegrasses, we developed two amphiploid Festulolium synthetics. One is a synthetic composed of three selected drought tolerant F1 hybrid genotypes from a cross between tetraploid Lolium multiflorum and hexaploid Festuca arundinacea, further on called LMFA. The other is a synthetic composed of five selected genotypes with soft leaves from a cross between tetraploid Lolium perenne and tetraploid Festuca pratensis, further on called LPFP. We produced seeds in polycrosses of two generations of both amphiploids, i.e.,...

Effects of cadmium and lead on the growth and the activity of peroxidase and superoxide dismutase of blueberry plantlets in vitro Brief Communications

J.Z. CHEN, L.P. ZHOU, C.Y. TIAN, F. YANG, G.Z. WANG

Biologia plantarum 64: 784-788, 2020 | DOI: 10.32615/bp.2020.143

To study the effects of cadmium and lead on the growth of blueberry (Vaccinium ashei Reade) plantlets in vitro and on the activity of peroxidase (POD) and superoxide dismutase (SOD) of blueberry shoots, Cd²⁺ and Pb²⁺ were added separately to a cultivation medium. The results show that 0.01 mM Cd²⁺ significantly inhibited the growth of blueberry shoots, and the height and fresh mass of the shoots were significantly lower than those of the control; 0.05 mM Cd²⁺ significantly inhibited the proliferation of the shoots; the inhibitory effect on the growth and proliferation of blueberry in vitro...

Ultraviolet-B radiation induces cell death in root tips and reprograms metabolism in ArabidopsisOriginal Papers

P. ZHANG, R. WANG, Y. WANG, J. XU

Biologia plantarum 64: 764-772, 2020 | DOI: 10.32615/bp.2020.122

Ultraviolet-B (UV-B) radiation inhibits root system growth, however, the influence of UV-B radiation on the regulation of root development remains unclear. Here, we investigated the effects of UV-B radiation on root growth and metabolism in Arabidopsis using physiological, pharmacological, and transcriptome approaches. Our previous study has demonstrated that UV-B radiation depresses auxin accumulation in roots by reducing auxin biosynthesis, transport, and response. In this study, we found that UV-B radiation inhibited primary root (PR) growth by inducing root tip cell death and thereby disrupting cell division and elongation in root tips....

Identifying citrus CBL and CIPK gene families and their expressions in response to drought and arbuscular mycorrhizal fungi colonizationOriginal Papers

B. SHU, D. CAI, F. ZHANG, D.J. ZHANG, C.Y. LIU, Q.S. WU, C. LUO

Biologia plantarum 64: 773-783, 2020 | DOI: 10.32615/bp.2020.123

The calcineurin B-like protein (CBL)-interacting protein kinase (CIPK) complex is an essential calcium sensor and contributes to biotic and abiotic stress responses. However, citrus CBL and CIPK gene family members and their underlying roles during drought and arbuscular mycorrhizal fungi (AMF) colonization remain relatively unknown. In the present study, CBLs and CIPKs were characterized in Citrus sinensis by analyzing the presence of specific domains such as the elongation factor (EF)-hand motif in CBLs, and a protein kinase and an Asn-Ala-Phe domain in CIPKs. After mining the C. sinensis genome, we identified...

Sex-specific responses of Populus deltoides to combined salinity and calcium under waterlogging conditionsOriginal Papers

L.-F. MIAO, D.-D. LI, F. YANG, Z.-H. TAN

Biologia plantarum 64: 753-763, 2020 | DOI: 10.32615/bp.2020.116

In this study, we investigated the sex-specific ion uptake and physiological and biochemical responses to combined salinity and calcium treatments of male and female Populus deltoides under waterlogging conditions. Results indicate that both P. deltoides clones were sensitive to salinity and calcium. Under well-watered conditions, salinity stress could especially increase the Na content in female P. deltoides clones, whereas salinity and calcium could increase the K and Ca content in male clones. Waterlogging could evidently stimulate Na content in leaves under salinity, especially in female P. deltoides clones. However,...

γ-Aminobutyric acid induces transcriptional changes contributing to salt tolerance in creeping bentgrassOriginal Papers

Z. LI, B.Z. CHENG, Y. PENG, Y. ZHANG

Biologia plantarum 64: 744-752, 2020 | DOI: 10.32615/bp.2020.117

γ-Aminobutyric acid (GABA) regulates plant tolerance to abiotic stresses; however, a transcriptomic change and key stress-related genes induced by GABA have not been investigated in plants during a prolonged period of salt stress. Roots of creeping bentgrass (Agrostis stolonifera) cv. Penncross were pretreated with or without 0.5 mM GABA solution for 2 days and then subjected to salt stress for 20 days (150 mM NaCl solution for 3 d, 200 mM NaCl for another 3 d, and 250 mM NaCl for 14 d) in controlled growth chambers. The application of GABA significantly increased GABA content in roots and alleviated a salt-stress induced decrease in GABA...

Effects of 5-aminolevulinic acid on osmotic adjustment and antioxidant system in mung bean under chilling stressOriginal Papers

J.J. ZHAO, N. ZHOU, N.J. FENG, D.F. ZHENG

Biologia plantarum 64: 736-743, 2020 | DOI: 10.32615/bp.2020.101

Temperature is an important factor for growth, development, productivity and geographical distribution of many plants (Nahar et al. 2015). Chilling stress is a major abiotic stress of crop production in Northeast China. Chilling stress exposure has been shown to enhance production of reactive oxygen species (ROS) and oxidative stress occurs (Nahar et al. 2015). The ROS, which include superoxide radical (O₂^*-), hydrogen peroxide (H₂O₂), hydroxyl radical (.OH), and singlet oxygen (¹O₂), cause damage to structural proteins, nucleic acids, enzymes, cell membranes, and...

Analysis of ABC1 protein family members in Lepidium apetalum seeds and the expression of LaAbc1 in seedlings in response to abiotic stressesOriginal Papers

Q.L. YANG, Z.Y. CHEN, H. LU, H.T. XIE, J.Y. LI, Y. DU, S.C. HAN, H.P. ZHAO, H.X. ZHAO

Biologia plantarum 64: 725-735, 2020 | DOI: 10.32615/bp.2020.104

To study the biological function of activity of bcl complex (ABC1) proteins in Lepidium apetalum Willd., genes encoding ABC1 family proteins were identified from the seed transcriptome. The sequence most closely related to germination at a low temperature was selected and gene expressions in response to low temperature stress further studied. The results show that 21 ABC1 genes were expressed in seeds germinating at the low temperature: 4 genes were upregulated, 6 were downregulated, and 11 were not significantly different from controls. The results of fluorescence quantification of the low-temperature stress on the seedlings of 7-d-old...

Transcriptome-sequencing analyses reveal flower color formation in Strelitzia reginae Original Papers

R.H. FAN, B. LIN, N.Y. FANG, X.X. YE, M.L. HUANG, H.Q. ZHONG

Biologia plantarum 64: 717-724, 2020 | DOI: 10.32615/bp.2020.102

Strelitzia reginae is a popular cut flower that has blue petals and orange sepals. Flower color is an important plant trait; however, little is known about its molecular mechanisms in S. reginae. In this study, cDNA libraries were constructed for blue petals and orange sepals of S. reginae. A total of 75 487 unigenes were obtained from transcriptome sequencing and de novo assembly, of which 41.86 % were annotated by public databases. Ultra-high performance liquid chromatography analysis revealed that anthocyanins were the main pigment in blue petals, and that carotenoids controled pigment formation in the orange sepals. Using a...

Rapid increases in β-1,3-glucanase and chitinase activities are markers of resistance to Microdochium nivale in grasses of the Lolium‑Festuca complexSpecial Issue: Festulolium

K. MARZEC-SCHMIDT, K. HURA, A. PŁAŻEK

Biologia plantarum 64: 710-716, 2020 | DOI: 10.32615/bp.2020.088

Microdochium nivale causes pink snow mould - a destructive disease of seedlings, stem bases, and ears of winter grasses and cereals. Glucanase and chitinase belong to pathogenesis-related proteins and exhibit a specific activity in response to pathogens. The aim of this study was to investigate changes in the activity of these enzymes in the leaves of chosen forage grasses during Microdochium nivale infection. Different cultivars of Festuca arudinacea, F. pratensis, Festulolium brauni, Lolium multiflorum, and L. perenne were prehardened at 12 °C and hardened at 2 °C and then inoculated with M. nivale...

Prolonged drought stress induced changes in yield and physiological processes of Trifolium repens and Festulolium brauniiSpecial Issue: Festulolium

M. STANIAK, J. BOJARSZCZUK, P. KRASKA, C. KWIATKOWSKI, E. HARASIM

Biologia plantarum 64: 701-709, 2020 | DOI: 10.32615/bp.2020.114

Water deficit is one of the main environmental factors, and the recognition of plant response to drought stress seems to be crucial for the yield improvement of the important crops. The aim of the study was to investigate the effect of prolonged drought stress on dry matter yield, gas exchange parameters, and relative chlorophyll content in the leaves of Festulolium braunii and Trifolium repens grown in pure stands and in mixture. In the pot experiment, different levels of soil moisture (well-watered conditions and drought stress) and of cultivation method (pure stand and mixture) have been applied. The study has shown that all measured...

Identification of potential key genes affecting soybean growth under salt stress via transcriptome studyOriginal Papers

N. LI, Z. LI, S. FAN, Y. PU, Y. GONG, R. TIAN, X. GUO, H. DING

Biologia plantarum 64: 692-700, 2020 | DOI: 10.32615/bp.2020.096

Soybean is one of the most important economic crops in the world. However, the salinization of soil results in the decrease of soybean yield as it is only a moderately salt-tolerant crop. We treated three soybean cultivars with low and high concentrations of NaCl. The differentially expressed genes between the control group and the salt treatment group were identified by mRNA sequencing and analyzed by gene ontology and Kyoto encyclopedia of genes and genomes annotations. We performed weighted gene co-expression network analysis on all samples and found genes most related to the phenotype. After verifying the results of differentially...

Spontaneous natural formation of interspecific hybrids within the Festuca-Lolium complexSpecial Issue: Festulolium

B. BOLLER, J. HARPER, E. WILLNER, J. FUCHS, M. GLOMBIK, J. MAJKA, V. MAHELKA, C. ZHAO, D. KOPECKÝ

Biologia plantarum 64: 679-691, 2020 | DOI: 10.32615/bp.2020.111

Interspecific and intergeneric hybridization within the Festuca-Lolium complex is frequently used in forage plant breeding. However, little is known about the natural occurrence and competitiveness of such hybrids. We collected naturally formed hybrids between Festuca apennina, Festuca pratensis, and Lolium perenne in different habitats of Switzerland and the British Isles and studied their origin, the ease of their spontaneous formation, and their competitiveness with parental species. A special attention was paid to the largely sterile triploid forms and their rare sexual progeny. The triploid hybrid F. apennina ×...

Cloning and characterization of a UDP-glucose dehydrogenase gene from mulberry Broussonetia kazinoki × Broussonetia papyiferaOriginal Papers

R.H. JI, Z. ZHANG, X. GUO, Y.L. BAO, W.B. ZHANG, X.F. LIN, S.L. BAI

Biologia plantarum 64: 667-678, 2020 | DOI: 10.32615/bp.2020.099

Uridine diphosphate glucose dehydrogenase (UGDH) is a key enzyme in the hemicellulose and pectin biosynthesis pathway and participates in the regulation of growth and development in plants. In this study, we isolated a BpUGDH gene from paper mulberry (Broussonetia kazinoki × Broussonetia papyifera) and analyzed its function and expression characteristics. The results show that the BpUGDH was expressed in all organs of paper mulberry with a higher expression in stems than in leaves and roots. A pBpUGDH::GUS gene construct was highly expressed in transgenic Arabidopsis thaliana seedlings, and its expression was induced...

Tensile strength of the rachilla in spikelets of Polish Festulolium braunii cultivarsSpecial Issue: Festulolium

B. GOLIŃSKA, P. GOLIŃSKI

Biologia plantarum 64: 660-666, 2020 | DOI: 10.32615/bp.2020.103

The aim of this study was to evaluate the tensile strength of the rachilla in spikelets of Polish cultivars of Festulolium braunii (K. Rich.) A. Camus during seed development and maturation. The investigations were carried out in 2009 - 2010 at the Plant Breeding, Szelejewo, Poland. The inflorescences were randomly harvested in June - July from plant collection in maintenance breeding of three Polish cultivars (Agula, Felopa, and Sulino). Using a specifically developed testing machine, tensile strength of the rachilla of individual spikelets were determined. Regardless of the F. braunii cultivar, the tensile strength of the rachilla...

Impact of nitric oxide on proline and putrescine biosynthesisin Chlamydomonas via transcriptional regulationSpecial Issue: Gasotransmitters

Z. ZALUTSKAYA, V. DERKACH, R. PUZANSKIY, E. ERMILOVA

Biologia plantarum 64: 653-659, 2020 | DOI: 10.32615/bp.2020.094

Nitric oxide plays an important role in regulating adaption of the model alga Chlamydomonas reinhardtii to various environmental stresses. One response to abiotic stresses is the accumulation of protective molecules such as proline and putrescine. The NO treatment led to a significant accumulation of proline in cells. Quantitative real-time expression analysis of proline metabolic genes in NO-treated cells showed a prolonged upregulation of the gene encoding γ-glutamyl kinase 1 (GGK1) in the glutamate biosynthetic pathway. Furthermore, truncated hemoglobin 2 (THB2)-underexpressing strains with an enhanced endogenous NO demonstrated a higher...

EditorialEditorial

J. POSPÍŠILOVÁ

Biologia plantarum 64: i, 2020

Cadmium and zinc uptake and translocation in dwarf Polish wheat seedlings as affected by calcium and potassium combinationOriginal Papers

W. SHUAI, Q. YAO, X. CHEN, Y. CHENG, X. XIAO, J. ZENG, X. FAN, H. KANG, L. SHA, H. ZHANG, Y. ZHOU, Y. WANG

Biologia plantarum 64: 642-652, 2020 | DOI: 10.32615/bp.2019.162

Combination of calcium and potassium (Ca-K) influences cadmium and zinc uptake and translocation in dwarf Polish wheat (Triticum polonicum), but its effects remain unclear. In the present study, a high concentration of Ca-K reduced uptake of Cd and Zn by roots and promoted their translocations to shoots under Cd and Zn excess. Whatever under a low or high concentration of Ca-K, Zn inhibited Cd uptake and translocation under Cd+Zn stress when compared with Cd stress alone. However, the reduced Cd content caused by Zn under the high concentration of Ca-K was significantly lower than under the low concentration of Ca-K. Under both Ca-K treatments,...

Flag leaf vein traits and their correlation with photosynthesis and grain yield in wheat genotypes of differing ploidyOriginal Papers

H.M. XU, Y.L. CHEN, Y.Y. LI

Biologia plantarum 64: 633-641, 2020 | DOI: 10.32615/bp.2020.092

Leaf venation and coupled physiological function of wild plants co-evolve during the natural selection. How artificial selection affects leaf vein traits and coordinated physiological functions of main crops are largely unknown. This study examined the changes of leaf vein traits and their correlation with gas exchange of flag leaves and yield in eight wheat genotypes of differing ploidy under the same growing conditions. The results indicate that flag leaf vein density (VLA), major-vein density (VLA_major), and minor-vein density (VLA_minor) decreased whereas the proportion of minor-vein length and interveinal distance between...

The role of gasotransmitters in movement of stomata: mechanisms of action and importance for plant immunitySpecial Issue: Gasotransmitters

S. GAHIR, P. BHARATH, A.S. RAGHAVENDRA

Biologia plantarum 64: 623-632, 2020 | DOI: 10.32615/bp.2020.071

Stomatal guard cells are specialized epidermal cells regulating gas exchange. The ability to open or close in response to external and internal cues makes stomata a dynamic and fascinating system. Stomatal closure upon infection ensures restriction of pathogen entry into the plant and forms an essential component of innate immunity. The opening or closure of stomata is dependent on the turgidity or flaccidity of guard cells, respectively, facilitated by several signaling components, including reactive oxygen species, nitric oxide (NO) and Ca²⁺. Among these, NO is the most extensively studied gasotransmitter. Its pivotal role in stomatal...

Lower photosynthetic capacity under higher spectral reflectance? The case of Actinidia polygamaOriginal Papers

Z.-X. WANG, G.-L. SHI, L. CHEN, D. SUN, P.-L. Xu, H.-Y. Qin, J. AI

Biologia plantarum 64: 616-622, 2020 | DOI: 10.32615/bp.2020.083

The variegated leaves of Actinidia polygama exhibit a striking colour change during development. However, little is known whether the photosynthetic capacity of white leaves can be maintained. Therefore, spectrum properties, leaf structure, net photosynthetic rate (P_N), and chlorophyll fluorescence in the green and white leaves were investigated. Although reflectance at 400 - 700 nm in white leaves was higher than that in green leaves, total chlorophyll content of white leaves was similar to that in green leaves. Palisade tissue cells of white leaves contained functional chloroplasts. Large intercellular spaces were observed between...

Exogenous melatonin enhances salt stress tolerance in tomato seedlingsOriginal Papers

M.A. ALTAF, R. SHAHID, M.X. REN, S. NAZ, M.M. ALTAF, A. QADIR, M. ANWAR, A. SHAKOOR, F. HAYAT

Biologia plantarum 64: 604-615, 2020 | DOI: 10.32615/bp.2020.090

Melatonin (N-acetyl-5-methoxytryptamine) is an essential molecule which regulates plant growth and development and alleviates the damaging effects of abiotic stresses. To evaluate the important functions of melatonin in response to salinity stress, the effects of exogenous melatonin on the antioxidant system and growth of tomato (Solanum lycopersicum L.) under 150 mM NaCl stress were investigated. The application of 100 μM melatonin compensated the growth inhibition caused by salt-stress. Melatonin treated seedlings had an increased fresh and dry masses of shoots and roots. The application of 1 - 200 µM melatonin notably enhanced the...

Cytogenetic insights into FestuloliumSpecial Issue: Festulolium

J. MAJKA, M. MAJKA, D. KOPECKÝ, J. DOLEŽEL

Biologia plantarum 64: 598-603, 2020 | DOI: 10.32615/bp.2020.095

Climate change calls for new methods and plant materials to breed crops adapted to new environmental conditions. Sustainable forage and amenity grass production during periods of severe drought and heat waves during summer, and unequal distribution of precipitation over the year will require drought-tolerant genotypes. However, high-yielding ryegrasses (Lolium spp.), which are the most commonly used grass species, suffer during abiotic stresses. Introgression of drought and heat tolerance from closely related fescues (Festuca spp.) offers an opportunity to develop superior hybrid cultivars to mitigate the negative impact of climate change....

The toxicity of BDE-47 to the photosystem of Lemna minor frondsOriginal Papers

N.W. QIU, W.R. ZHANG, X.H. YAN, R.J. WANG, L. TIAN, G.L. HAN, F. ZHOU

Biologia plantarum 64: 591-597, 2020 | DOI: 10.32615/bp.2020.091

To elucidate the toxicity of 2,2ʹ,4,4ʹ-tetrabromodiphenyl ether (BDE-47) on photosynthetic primary processes, in vivo and in vitro treatments of BDE-47 were performed. The 20-d treatment in vivo (5 - 20 μg dm^-3) suppressed the reproduction of duckweed (Lemna minor) and led to decline in chlorophyll (Chl) content of fronds. The most obvious features of BDE-47-treated fronds included a Chl a fluorescence rise at the J phase and a depression at the G phase, whereas significant fluorescence rises at the L, K, and J phases were found on the ΔV_t curve of thylakoid membranes treated...