Antimicrobial peptides (AMPs) are multifunctional effector molecules that often combine direct antimicrobial activities with signaling or immunomodulatory functions. The skin secretions of anurans contain a variety of such bioactive peptides. The identification of AMPs from frog species often requires sacrificing several specimens to obtain small quantities of crude peptides, followed by activity based fractionation to identify the active principles.We report an efficient alternative approach to selectively amplify AMP-coding transcripts from very small amounts of tissue samples, based on RNA extraction and cDNA synthesis, followed by PCR amplification and high-throughput sequencing of size-selected amplicons. This protocol exploits the highly conserved signal peptide region of the AMP precursors from Ranidae, Hylidae and Bombinatoridae for the design of family-specific, forward degenerate primers, coupled with a reverse primer targeting the mRNA poly-A tail.Analysis of the assembled sequencing output allowed to identify more than a hundred full-length mature peptides, mostly from Ranidae species, including several novel potential AMPs for functional characterization. This (i) confirms the effectiveness of the experimental approach and indicates points for protocol optimization to account for particular cases, and (ii) encourages the application of the same methodology to other multigenic AMP families, also from other genera, sharing common features as in anuran AMPs.The online version of this article (10.1186/s12864-018-5225-5) contains supplementary material, which is available to authorized users. Rana palustris) with rare exceptions such as in Rana sylvatica, in which only one antimicrobial peptide has been isolated to date [AMPs are endogenous antibiotics present in all organisms, with a direct antimicrobial activity towards pathogens, often also showing immunomodulatory properties, and a regulated gene expression to facilitate and modulate immune responses , 2. The  to date . It is w to date –9, whichIdentifying novel anuran peptides normally requires handling several individuals, which are either sacrificed or held in captivity and treated with electric shocks/norepinephrine to obtain small amounts of crude peptide. This is followed by several rounds of purification using different precipitation and chromatographic techniques combined with activity testing of fractions to identify the active principles . This apWe have developed a potentially faster, less invasive and more efficient approach based on the selective amplification and subsequent sequencing of transcripts encoding for antimicrobial peptides, starting from very small amounts of tissue. This method however requires accurate primer design to capture the diversity of AMPs. In general, anuran antimicrobial peptide precursors consist of a highly conserved signal sequence, a negatively charged propeptide and a hypervariable cationic mature region –16. Datalater® buffer  and stored at − 20 °C according to the manufacturers’ instructions. Total RNA was extracted using the TRIzol protocol  from ~ 50 mg of this tissue, resuspended in RNAase free water, quantified with NanoDrop 2000 , quality checked using denaturing 1.5% agarose gel electrophoresis, and stored at − 80 °C until further use  . The sam) were also synthesized with a 5’-CAGGACCAGGGTACGGTG-3′ tail required for multiplex sequencing through attachment of the barcodes in a secondary outer amplification. All primers were synthesized by BMR Genomics   contained a poly-dT stretch to match the poly-A tail of the mRNA and was used for cDNA synthesis. It also contained a 5′ tail sequence trP1) which was required for the cDNA amplification protocol and subsequent parallel sequencing. All forward primers  together with 0.5 μl of 10 μM dNTP solution, 0.5 μl of 10 μM specific forward primer solution, 0.5 μl of 10 μM trP1 primer solution and 1 μl of cDNA template in a total volume of 20 μl. The PCR started with an initial denaturation at 95 °C for 2 min, followed by 10 cycles including 10 s at 95 °C, annealing at 45–50 °C for 20 s (ramping 0.5 °C/cycle) and 20 s elongation at 72 °C. For the next 25 cycles the annealing temperature was set to 52 °C for 20 s ending with 5 min final elongation at 72 °C .First strand cDNA was synthesized from 1 μg of total RNA using the qScript™ Flex cDNA Synthesis Kit  according to the manufacturers’ instructions. The reverse transcription reaction was performed at 37 °C for 1 h. The mixture for cDNA amplification contained 0.2 μl of DNA polymerase (5 U/μl), 2.5 μl of 25 mM MgClOuter PCR was performed to attach the barcodes (a sample-specific 10 nucleotide sequence used for de-multiplexing) on the 5’ end of the amplicon, which were followed by sequencing adapters. The mixture for outer PCR contained 0.2 μl of DNA polymerase (5 U/μl), 2.5 μl of 10× buffer A  together with 1 μl of 20× EvaGreen™ , 0.5 μl of 10 μM dNTP solution, ~ 20 ng of nucleic acid from the primary PCR and 3 μl of 10 μM primer solution in a total volume of 20 μl. This secondary PCR run was performed for 8 cycles including denaturation at 95 °C for 10 s, annealing at 60 °C for 10 s and 40 s elongation at 65 °C with 3 min final elongation at 72 °C. The quality of the amplification products was visualized by electrophoresis on 1.5% agarose gel after each amplification run. Based on this analysis, some amplicons were discarded due either to an unsuccessful PCR or out of range size.The size range and quantity of nucleic acid in each individual library were assessed using a DNA 1000 kit on an Agilent Bioanalyzer 2100   and the library prepared using Ion PGM Hi-Q View OT2 Kit  according to the manufacturers’ instructions. The library was loaded on an Ion 314 chip , and sequenced for 800 cycles using an Ion PGM Hi-Q View Sequencing Kit .Raw sequencing data from PGM runs were imported into CLC Genomics Workbench 10.1.1  to perform trimming. Briefly, low quality (trimming limit = 0.05) and ambiguous nucleotides, adapters and short residual reads (< 100 nucleotides) were removed. Filtered reads were then assembled into contigs with an overlap-layout-consensus (OLC) approach. The original reads were re-mapped on the assembled contigs to allow visual inspection of the correctness of the assembly of each contig. Sequences supported by less than 3 reads were discarded without further analysis and in some cases contigs displaying a high amount of polymorphism were re-assembled with more stringent parameters to obtain all the possible sequence variants. Transcripts were blasted (using BLASTx) against a custom sequence database containing all known Class-1 (Ranidae and Hylidae) or Class-3 (Bombinatoridae) nucleotide sequences to detect all positive hits based on E-value threshold of 0.05. The longest ORFs for each of the resulting contigs were translated into amino acid sequences using the ExPASy translate tool  and grouEscherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, obtained from the American Type Culture Collection , using the serial two-fold microdilution method as described previously [Two peptides with amidated C-terminus were obtained from GenicBio Ltd.  at > 98% purity as confirmed by RP-HPLC and MS. They were dissolved in doubly distilled water and stock concentration determined as described previously , 28. Mineviously . Cytotoxeviously .Hyla arborea  was much lower , as evaluated by Agilent Bioanalyzer 2100. This was suggested by the DNA fragment size restrictions (< 450) of the Ion Torrent™ PGM sequencer that was used.Selective amplification with AMP specific primers of cDNA libraries obtained from seven anuran species , followed by long-read high-throughput sequencing, resulted in 120.371 reads. Out of the assembled contigs, 5–15% of the sequences from the Ranidae family corresponded to AMPs, depending on the species. The success rate for Hyla arborea, from the Hylidae family  and none from Bombina variegata (Bombinatoridae) (see Table Rana or Pelophylax genus (Ranidae), whereas limited data with satisfactory phylogenetic relations was available for Hylidae. The high number of AMP transcripts available in the public databases for the two Ranidae genera enabled to construct three primers suitable for efficiently amplifying the different expressed AMP transcripts in the five target species, improving the chances of correct annealing and amplification in PCR. As the nucleotide sequence data available for other genera increases in the databases, it should become possible to refine primer design and provide suitable primer options also for other more distantly related families.Selective amplicon sequencing resulted in the simultaneous identification of antimicrobial peptide encoding transcripts from 5 out of 7 different anuran species tested. All the species with a positive result pertain to the Ranidae family, with a single sequence obtained from Bombina species were excluded from sequencing for this reason, as their average size was > 500 nucleotides. Using the new isothermal amplification for Ion Torrent, or a different sequencing platform, may partly counteract this issue.Another important parameter is transcript size. All known Class-1 Ranidae peptides are shorter than 100 AA long (mostly 70–80). Furthermore, our transcriptome screening revealed that the 3’ UTR region of the encoding transcripts was generally shorter than 200 nucleotides. The Ion Torrent™ PGM  sequencing kit used is suitable for sequencing DNA strands up to about 450 base pairs. The design of degenerate primers based on the Class-1 signal peptide region should therefore have generated amplicons with a size range compatible with this sequencing method. This was confirmed using an Agilent Bioanalyzer 2100 prior to sequencing, which revealed a library size of between 300 and 400 bp for all the Ranidae species , the explanation could thus be a combination of i) the poor representation of transcriptomic datasets from Hyla spp. in the SRA database; ii) the size range of the library, which was close to the maximum capabilities of Ion Torrent sequencing  a different AMP gene organization for this family. With respect to the first consideration, 4 out of 5 transcriptomes used for primer design pertained to a species of a different subfamily (Pelodryadinae) then Hyla (Hylinae) and the only transcriptome available for Hyla arborea was not obtained from AMP-producing tissues. It thus seems likely the designed forward primer did not include all the polymorphisms present in AMPs from the Hyla genus, resulting in the amplification of a single but highly represented sequence . Concerning the second consideration, it is likely that AMP amplicons had been removed during the E-gel purification procedure, due to excessive size. The third consideration could be relevant if, unlike Ranidae family peptides, Hyla Class-1 AMPs do not have a multi-gene organization, even though this seems to be disproved by previous reports [Considering that only one Class-1 AMP encoding transcript was obtained from  reports . Based oB. bombina) or very close (B. variegata) to maximal input length capabilities of the sequencing technologies used. While the former library was discarded altogether, the latter one was subjected to sequencing but did not produce any positive matches. Despite a similar apparent size range between the libraries obtained from B. variegata and some of the longer ones obtained from Ranidae species, the concentration of the former was approximately 7 times lower . Although the chromatogram was not clean, suggesting that multiple products of the same size were amplified, the consensus sequence clearly confirmed the amplification of a Class-3 AMP precursor. Therefore, while our strategy was not suitable with Bombinatoridae for Ion Torrent PGM, other massive parallel sequencing platforms allowing higher read lengths (such as SMRT PacBio or Oxford Nanopore) could enable its application also in this anuran family.Unsuccessful results with Bombinatoridae are most certainly linked to the longer AMP precursor and, consequently, longer length of the encoding mRNA molecules. During the initial phases of the experimental design, we tried to overcome this issue by designing more internal primers based on the propeptide rather than on the signal peptide region, thereby reducing the size of the expected amplicons. However, the assessment of the library size range indicated that amplicons were above  confirm the effectiveness of this experimental design, as long as the degenerate primers are properly designed, and the amplicon size is tailored to the sequencing platform used. Geographical location would have been very important if the selected species were endemic to Croatia. However, in this case all the species targeted display a relatively broad and partially overlapping area of distribution across Europe and are, in some cases, evolutionarily closely related (e.g. the latest common ancestor of  Miocene ). ConseqH. arborea indicates that the panel of species analyzed can be quite wide, but this requires a particular effort in collecting as many sequences as possible from species phylogenetically closely related to the target species in the analysis panel to optimize primer design, thereby maximizing the chances of annealing during selective amplification. In this respect, PCR experimental conditions, and the annealing temperature in particular, need to be carefully selected to allow pairing with templates that are not perfectly matching, and allow capturing of as many sequence variants as possible. The lack of success in obtaining Bombinatoridae AMPs instead pinpoints the importance of tailoring the sequencing platform to the expected amplicon length, or alternatively, to identify useable sequences and design primers as close as possible to 3′ end of the mRNAs in order to reduce the amplicon size.The single result obtained from (see Table 4). This confirms the reliability of our approach to identify novel, functional antimicrobial peptide sequences (manuscript in preparation).Although the novel identified peptides display physico-chemical features compatible with antimicrobial activity  tail. These characteristics are well known in many different animal AMPs –41, toxiAdditional file 1:Complete list of frog species obtained from Croatian wild. (DOCX 13 kb)Additional file 2:List of transcriptome data downloaded from SRA database. (DOCX 14 kb)Additional file 3:List of signal peptides used for transcriptome screening. (XLSX 46 kb)Additional file 4:Clusters of nucleotide alignments used for forward primer design. (DOCX 7338 kb)Additional file 5:Size range of each individual library obtained prior to pooling. (DOCX 14 kb)Additional file 6:Classification of identified peptides. (DOCX 1600 kb)Additional file 7:BLASTp output for novel identified putative AMP sequences. (XLSX 47 kb)Additional file 8:Success rate of amplicon synthesis in Ranidae species based on used primer. (DOCX 13 kb)


Genotyping for ACE I/D polymorphism was performed using polymerase chain reaction. VO2max was determined by an incremental test to volitional exhaustion on a motorized treadmill.The insertion (I) rather than deletion (D) of human angiotensin converting enzyme gene (ACE) is associated with lower circulating ACE activity and with endurance performance among Caucasians. The frequency of the ACE gene I/D allele in Iranian sample seems to be more similar to the Caucasians. To assess the possible relationship between I/D polymorphism of ACE gene with athletic status and selected cardiovascular indices and VO2max, resting heart rate, systolic and diastolic blood pressure. There was no interaction effect of training statue x ACE genotype for each of the examined indices.I/D genotype was neither associated with elite athlete status nor with VO2max in either trained or untrained Iranian participating in soccer. The absence of an association between either I/D genotype and elite Iranian athlete status and better cardiovascular function also suggests that the ACE gene does not contribute significantly to the phenomenal success of Iranian soccer players.ACE gene variation was not a determinant of cardiovascular function and VO Progressing endurance, having a lower resting heart rate and a stronger heart volume are all valid physiological indicators of the cardiovascular system’s ability to adjust to physical overload in well-trained athletes ; indeed,2max is about 50% [2max.The individual variability of physical performance characteristics and the potential to become an elite athlete have a powerful genetic base. The genetic effect of VObout 50% ; but, thbout 50% . The I about 50% , high pebout 50% , therefo2max [2max for the DD genotype (57 ml.kg-1.min− 1) versus the ID or II (50 ml.kg− 1 min− 1) genotype was demonstrated in Chinese males [2max [While ACE is involved in the metabolism of substances that impact on vascular remodeling, it may regard in the cardiorespiratory fitness of individuals and for the differences among individuals in response to physical training . High fr2max . In contse males . Other ses [2max  or elitees [2max .Besides, cardiac response to certain physiological stimuli is affected by racial differences. It seems that certain ethnicities demonstrate a stronger association between ACE activity or I/D polymorphism and athletic results . Saadat In a population association study, a homogeneous cohort of subjects from the same sporting discipline is needed when investigating whether a genetic marker (the ACE I/D polymorphism) occurs more frequently in cases (elite athletes) than in controls . Therefo2max and other cardiovascular determinants had been measured was assessed post-hoc for its influence.Soccer players (white men of Iranian descent) were recruited through an open invitation from a large soccer club. In total, 57 subjects gave written informed consent to participate after the study had been explained in details. Physical activity levels, medical health and quality of life of the subjects was assessed by a validated questionnaire  soccer players, competitors in the first national league who had at least a history of 5 years of regular training , recorded a level of intense physical activity above 8 h per week (in the questionnaire). Untrained subjects (n = 28) were active members of the community (soccer) who were not involved in a regular training program. In this study, the untrained group with a level of intense physical activity below 2 h per week and a range of VO2max of (29.2–49.7) is in consistence with the study of Valdivieso (2017) who considered healthy subjects which documented a level of intense physical activity below 6 h per week or demonstrated a VO2max below 50 ml·Kg− 1·min− 1 as not being trained, i.e., being untrained [For the analysis, subjects were categorized based on their training status and ACE I/D genotype as assessed by questionnaire, functional exploration and genotyping. Volunteers included highly trained male , mass and skinfold measurements were taken according to standard procedures used by ISAK . Skinfol2max during a graded incremental exercise test on a treadmill , using Bruce protocol in line with the ACSM guidelines [2 of less than 2 ml · Kg− 1 · min− 1 with increasing running speed (VO2 plateau); a final heart rate (bpm) defined as a heart rate within 10 beats.min− 1 of the sex and age predicted maximum heart rate (HRmax = 220- age). Heart rate was measured and recorded by a HR monitor . The final result of the Bruce test is exercise time (ET) which is necessary for determining the value of maximal oxygen consumption (VO2max).Aerobic fitness level of each soccer player was determined by measuring VOidelines . The tesVenous blood from all participants in this study, obtained by venipuncture of the antecubital vein in a sitting position were collected in EDTA tubes and stored at − 20 °C. Genomic DNA was extracted from whole blood leukocytes using the salting-out method as previously described by Miller et al. . We usedP < 0.05. Data were analyzed using SPSS version 16.0 .Data are presented as mean ± standard deviation (SD). The significance of differences in allele frequencies was estimated using the χ2 test. Two-way analysis of variance (factors: training state and ACE I/D polymorphism) was used to assess mean differences. Subjects were tested at both loci for Hardy Weinberg Equilibrium (HWE) by Chi-square tests with one degree of freedom. Statistical significance was set at n = 18), ID , II . No differences were found in the ACE genotype frequencies between trained and untrained males. Also, the allele frequency was statistically similar  higher in untrained and VO2max in trained group , ET and RHR were significantly (up Table .Table 3C2max is 49.6 ml.kg− 1.min− 1 (range 34.2–68.1 ml.kg− 1.min− 1) in DD genotype group, 46.2 ml.kg− 1.min− 1 (range 29.2–66.2 ml.kg− 1.min− 1) in ID group and 48.7 ml.kg− 1.min− 1 (range 31.5–61.8 ml.kg− 1.min− 1) in II group.The median of VO2max, resting SBP and DBP, fat mass and RHR (Table P < 0.09) and DD (P < 0.08) genotypes  is in agreement with previous studies from Iran , 26. SaaShahmoradi et al. (2014) grouped Iranian elite athletes in three categories of mixed, endurance and power sports. It was demonstrated that frequency of the I allele (53.13%) was higher than the D allele in mixed-oriented athletes. Besides, I allele in athletes was significantly higher than controls . Accordi2max which is considered the gold standard for cardiorespiratory evaluation. Improvements in VO2max have been associated with improved soccer performance during competition  [Cardiovascular performance plays an important role in athletic success . Bradykihe ball) . Physicahe ball) .In our study, we did not find any association between the ACE gene I/D polymorphism and cardiovascular determinants in univariate analysis. These results are in accordance with the results from other studies, where no association was found between ACE gene variation and endurance performance , 31. It 2max . Our trained group with a VO2max of (56.0 ± 6.9 ml · Kg− 1 · min− 1) supports the above statement. But the untrained group with a VO2max of (39.0 ± 5.8 ml · Kg− 1 · min− 1) is far from the above mentioned data and is closer to the healthy untrained men at this age [Wells et al. (2012)  indicatethis age .2max. But even in a study by Rankinen et al. (2000) on 192 elite endurance athletes with VO2max values over 83 ml.kg− 1.min− 1 (highest decile), they did not find any association between ACE genotype and VO2max [Perhaps it would have been better if we used sports that their athletes had higher levels of VOd VO2max . These f2max levels. Williams et al. (2017) [2max. They reviewed 35 studies that reported 97 genes associated with improvements in VO2max. It has been estimated that VO2max trainability has a significant heritable component of around 50% [2max.In addition to the ACE variants, many other genetic variants may be responsible for interindividual variability of VO. (2017)  conducteound 50% . It is c2max.Despite inconsistency among past reports, an association between the ACE I/D polymorphism and circulating ACE levels has been almost accepted. The I allele has been associated with endurance phenotypes and the D allele with power. A greater maximal oxygen extraction (a-vO2 difference) in ACE II versus ID and DD genotypes was reported . Also, mOn the other hand, there are some other studies that contradict these findings and suggest an association of D allele/DD genotype with endurance athletes’ phenotypes , 19, 39.2max response.Traits such as response to exercise do not only depend on the genetic code, but also on epigenetic signals. Epigenetic modulation of gene activity occurs in response to non-genetic factors such as physical activity , body weEthnic as well as geographic differences might be responsible for the observed disagreements . The eviGene-specific patterns of LD make it difficult to provide general guidelines for selecting the most useful polymorphisms in association studies. So, these kinds of studies should concentrate on the overall sequence variation of functionally important regions of candidate genes and not only on a few polymorphisms. Evaluating the pattern of LD in the human genome is important to identify tagging SNPs. these SNPs are often correlated due to linkage disequilibrium (LD). But in the present study the LD structure among SNPs was not examined.Many physical and physiological traits have a multifactorial etiology resulting from the interplay of genetic factors (G) and environmental exposures (E). Differential classification may increase the power to detect true interactions in a genetic based study. An implication of our null finding is that, in real-life situations that is in studies by which more genetic markers will have detectable non-null effects for a given sample size, the number of markers identified will be good deal more than those obtained from G-E interaction methods. One must then investigate which markers are implicated in G-E interaction.Several limitations of this study exist. We did not consider tests for additive interaction. There are many possible reasons why gene studies lack power to detect G-E interactions, including small sample size or more complex multimarker interactions.Using convenient single-nucleotide polymorphism simulation routines that generate true LD structure would make our study more realistic, changing it from a fixed null scenario to a reasonable genetic interaction.In addition to the endurance performance traits, the data on the effects of the ACE I/D polymorphism on various cardiovascular phenotypes in general are incoherent . PreviouThis investigation was performed under several constraints. Lack of serum ACE activity measurement and also the small sample size are main limitations of this study. When small sample size is used, the risk is high that observations will be due to chance and the estimates derived may be biased. So the data should be interpreted cautiously due to the small sample size. Therefore, there is an urgent need for more investigations on markedly larger sample size to understand the function of the ACE gene and its possible effects on cardiac function and athletic performance.2max) in Iranian men. The absence of an association between either I/D genotype and elite Iranian athlete status also suggests that the ACE gene does not contribute significantly to the phenomenal success of Iranian soccer players. Nevertheless, demographic and ethnic profile should be taken into account in large samples to confirm these results.In conclusion, although there are conflicting results about the effects of ACE I/D polymorphism on cardiovascular variables, the current study does not support the hypothesis that ACE I/D genotype is either a determinant of cardiovascular function or aerobic performance (VO


In two poplar species with contrasting growth performance, sugar metabolism in xylem and cambial regions can explain their different grades of tolerance to drought and their recovery rates following water deficits. Populus deltoides ‘Dvina’ and Populus alba ‘Marte’, demonstrated contrasting growth performance and water–carbon balance strategies; a mechanistic understanding of the water deficit response was provided by these poplar species. ‘Marte’ was found to be more anisohydric than ‘Dvina’. This characteristic was associated with the capacity to reallocate carbohydrates during water deficits. In contrast, ‘Dvina’ displayed more conservative water management; carbohydrates were preferably stored or used for cellulose production rather than to achieve an osmotic balance between the phloem and the xylem. Data confirmed that the more ‘risk-taking’ characteristic of ‘Marte’ allowed a rapid recovery following water deficit and was connected to a different carbohydrate metabolism.Drought dramatically affects wood production by adversely impacting cambial cells and their derivatives. Photosynthesis and assimilate transport are also affected by drought conditions. Two poplar genotypes,  Drought dramatically affects the production and yield of plants worldwide, and it is expected to affect plants more frequently due to ongoing global climatic changes . MoreoveThe cambial region of a plant includes the cambium, which is composed of meristematic cells called ‘initials’, and mother cells of the xylem and phloem that undergo expansion and differentiation processes . The ratPopulus, different species show contrasting water-use efficiencies and adaptive strategies under drought conditions  and α- and β-amylases (Amy and Bam). Starch synthases were found to elicit a drought response in poplar species  poplar species showing rapid recovery following water deficit exhibit the greatest plasticity in the sucrose/starch pathway; (ii) carbohydrate metabolism of the cambial region is associated with different genotypic responses under water deficit conditions; and (iii) genes related to the cell cycle and sucrose/starch pathway elucidate different degrees of poplar tolerance to water deficit.Populus deltoides Marsh ‘Dvina’ and Populus alba L. ‘Marte’ clones were grown in pots with a combination of peat, sand, and perlite  at a nursery of CNR-IVALSA in Florence (43°49'06''N 11°12'08''E), as described by t0), three plants of each genotype were sampled, and the remaining plants were subjected to different watering regimes (n=4) during 21 d of treatment. Soil moisture was kept at field capacity [water content was 30% (w/w) of total soil weight] in well-watered (WW) plants. Watering was suspended for 8 d (tmax) and then resumed up to day 13 (trec) in water-limited (WL) plants. http://www.lamma.rete.toscana.it; the weather station was located at the experimental site).One-year-old plants of Substrate volumetric water content was measured daily in WW and WL plants by ‘HydroSense’ probes , which were based on time-domain reflectometry. The relative extractable water in the substrate (REW) was calculated as follows:a is the water content of the actual substrate in the root zone; SWCwp is the water content of the substrate at the wilting point (9% w/w); and SWCfc is the water content of the substrate at field capacity (30% w/w).Here SWCpd and Ψwmd, MPa) was determined using two fully expanded leaves from each plant (the leaf plastochron index was between 5 and 7) and a pressure chamber . Leaf water content (LWC) was determined using three fully expanded leaves that were excised before dawn (LWCpd) and at midday (LWCmd). Fresh leaves were excised and weighed (FW) and then dried at 80 °C for 48 h, after which dry weight (DW) was determined.Leaf water potential at pre-dawn and midday  was also calculated.The difference between pre-dawn and midday LWC , transpiration rate , and net CO2 assimilation rate  were measured using intact, fully expanded leaves (leaf plastochron index was either 6 or 7) between 13.00 h and 14.00 h with a portable open photosynthesis system . This system operated at a flow rate of 5.7 ml s–1 and an ambient CO2 pressure of 33 ± 1 Pa. Photosynthetic flux density was >1200 µmol m–2 s–1. Instantaneous water-use efficiency  was determined as instantaneous leaf transpiration efficiency, Amax/E. Specific leaf area  was calculated at tmax.Leaf stomatal conductance (×10–6 K–1), which was pressed against the bark. The transducer was mounted on a polytetrafluoroethylene frame attached to the stem by three titanium rods at 10–15 cm above the ground. The frame was anchored to a polytetrafluoroethylene holder attached on two sides of the pot. As the stem expanded or contracted, the rod transmitted the signal to the transducer. The sensor output, V1/Vx ratio, was converted into a numerical value (length of sensor in millimetres) using a linear calibration regression equation . A total of 16 plants (four plants for each genotype and treatment) were monitored with dendrometers, installed at a quarter of the height of the stem from the collar. Aluminium foil was used to shield all the dendrometers from direct sunlight and weather damage. Raw data were recorded every 15 min, and averages were calculated hourly.Variations in stem radius were monitored with automatic point dendrometers that were constructed for use with small stems. The dendrometers consisted of a linear variable transducer  used to measure linear displacement of a stainless-steel sensing rod  and the current stem radius (SRt) when SRt<SRmax. Furthermore, GRO (µm) was calculated as the difference between SRt and the highest previous value of SRmax when SRt>SRmax.Growth-induced irreversible stem expansion (GRO) and tree water deficit-induced reversible stem shrinkage (TWD) were determined following the procedure reported by t, µm h–1) were calculated as follows:The rates of stem growth over time (ΔSR/ΔJXB online).Instantaneous stem water deficit, ΔW (µm) was extracted by de-trending the original growth data collected from the dendrometers with the help of piecewise linear regression (see f) and then fresh volume (Vf) by water displacement (d) was determined after 96 h at 103 °C. Relative water content  was determined for the bark and the xylem using the following formula:At each sampling time, stem portions (50 mm) were excised 20 cm from the collar. The pith was discarded immediately after separating the bark from the xylem. Samples were weighed within 5 min after harvest to determine fresh mass (Md) was estimated by dividing Md by 1.53; the density value was assumed for dry cell wall material  was determined according to f):w is the density of liquid water (kg m–3).Here, ρf is the amount of free water available to support the hydraulic network.The difference α–αThe stems were divided into logs of 10–15 cm; these were immediately frozen in liquid nitrogen and then subjected to freeze-drying under vacuum. To obtain samples from the cambial region, the bark was detached from the logs, and differentiating phloem and xylem were gently scraped from the inner side of the bark and the outermost side of the xylem. The xylem was converted to a fine powder using an Ultra Centrifugal Mill ZM 200 . The complete procedure has been reported in –1. Soluble sugars were identified with a refractive index detector ; carbohydrate standards were used to corroborate the identified sugars . Finally, sorbitol was used to normalize sugar amounts  of individual sugars to Ψπ; RDW is the relative dry weight at saturation (kg m–3); C is the molar concentration of solutes (mol kg-1); and the RT value at 25 °C is –0.002479 m3 MPa–1 mol–1.Here, Ψπg for 5 min, and supernatants were analysed using a freezing point osmometer . The results were expressed in mOsm g–1 DW.At each sampling, osmotically active solutes were determined following the procedure described in a study conducted by At each sampling, stem discs of 10–20 mm thickness were cut 30 cm from the collar. They were then placed in ethanol (96%) and stored at 4 °C. Next, disc sections were fixed with ice on a Peltier plate. Finally, these sections were cut into transverse or radial sections of 8–12 μm thickness with a microtome. The sections were stained with Lugol’s solution (Sigma-Aldrich) and observed with a Nikon Eclipse E800 light microscope .CycB1.3-4 and CycB2.1; amylase Amy1.1-2 and Bam5; starch synthase SS2 and SS4; sucrose synthase Sus2 and Sus3; and sugar transporter Suc2.1, SUT2.a-b, and SUT4. Primers for the housekeeping genes Act-2 and Ef-1 were obtained according to CycB1.3-4 and CycB2.1 genes were obtained from P. trichocarpa genes were retrieved from the PopGenIE database  of all cDNA samples. For each gene and each poplar genotype, a five-point dilution set of two standard samples was amplified in duplicate reactions, and reaction efficiency was verified. Then, RT–PCRs were carried out in duplicate reactions for each sample; outcomes were discarded if the difference in the Cq numbers was >0.5 for the replicates. For each gene, a no-template control was amplified in each run. For all primers, RT–PCRs were conducted with an annealing temperature of 60 °C using the iTaq™ Universal SYBR® Green Supermix kit . In a final volume of 10 μl, the reaction was performed with 10 μM of each primer and 1 μl of a 1:5 dilution of template cDNA. The RT–PCR protocol was as follows: 95 °C for 5 s and 60 °C for 30 s; a total of 40 amplification cycles were performed. At the end of the amplification cycles, the melting curve was calculated for each amplification.According to a procedure described by n=4). Then, RT–PCR analysis was performed on three biological replicates (n=3). Data were checked for normal distribution (D’Agostino–Pearson’s K2 test). The effects of the water regime and genotype were assessed with two-way ANOVA. Post-hoc analysis was conducted using Fisher’s LSD test. Furthermore, t-test analysis was performed at sampling time t0 (n=3). Data that did not have a normal distribution and percentages were transformed before ANOVA. Correlation was evaluated by performing Pearson’s test. All analyses were executed using NCSS Data Analysis software. All graphs were plotted using Prism 5 software .All analyses were performed on four biological replicates . Compared with ‘Dvina’, ‘Marte’ had a higher RWC in the bark  of the xylem were significantly higher in ‘Dvina’ than in ‘Marte’. However, the amount of free water available to support the hydraulic network (α–αf) did not differ significantly in the two genotypes . Despite the similar water deficit, following 8 d of suspension of irrigation (tmax), several traits were affected to different extents, depending on the genotype. Stem elongation (data not shown) was negatively affected in ‘Dvina’ (P=0.004), but not in ‘Marte’ (P=0.220). In contrast, ΔSR/Δt  was recorded in WL plants compared with WW plants; however, no significant differences were observed in ‘Dvina’ plants. In addition, ‘Dvina’ WL plants maintained a significantly higher leaf ΔΨwpd-md than ‘Marte’ . Both genotypes showed a decrease in RWCs of the bark and xylem . In contrast, the sucrose content decreased significantly in the cambial region of ‘Marte’ . Compared with WW plants, no variations were observed in the soluble sugars and starch of ‘Dvina’ WL plants.Water deficits induced contrasting effects on genotype in regard to soluble sugar and starch contents within the xylem and cambial region . Starch trec), both genotypes demonstrated a complete restoration of primary and secondary growth, ΔSR/Δt, ΔW, bark RWC . The only exception was the RWC of the xylem . With regard to carbohydrates , confirming that storage was not influenced by water deficit but was driven mainly by cambium phenology  in the xylem. The high ‘risk-taking’ behaviour of ‘Marte’ under water deficit was coupled with similar carbohydrate variations, which were also observed in other woody plants during embolism response (A higher transcription of the t=0), the transcription levels of CycB1.3-4 and CycB2.1 genes were at the detection limit.B-type cyclins regulated a crucial checkpoint for mitosis . Under WIn conclusion, several physiological features were associated with different genotype-dependent responses. These features were related to the capacity to reallocate carbohydrates during water deficit. Moreover, the importance of cambial region activity under water deficit was clearly demonstrated and related to xylem traits. ‘Marte’ showed an active response and a rapid recovery following water deficit, which was related to greater plasticity in sugar/starch conversion. In contrast, less anisohydric behaviour was demonstrated by ‘Dvina’ during water deficit. This might be related to the inability to use carbohydrates in the osmotic processes. These carbohydrates were preferentially stored or used for cellulose production.To elucidate plant responses to water deficits, it is necessary to understand how plants’ water status and leaf gas exchange can be integrated with the transportation of photoassimilates within the phloem and xylem. Moreover, basic information is required about daily carbon balances and the limits of plants to shift energy among competing sinks under water-limited conditions.JXB online.Supplementary data are available at Table S1. List of primers used in this research.sat in the cambial region and the xylem.Table S2. Glucose and fructose contents, and glucose and fructose ΨπFig. S1. Calculation of the instantaneous stem water deficit.Fig. S2. Starch localization using Lugol’s solution.Fig. S3. Osmotically active molecules in the cambial region and xylem.SS2 and SS4 genes.Fig. S4. Accumulation of mRNAs SUT2 a-b, Suc2.1.a-b, and SUT4 genes.Fig. S5. Accumulation of mRNAs of Supplementary MaterialClick here for additional data file.


Colloids are mainly divided into two types defined by size. Micron-scale colloids are widely used as model systems to study phase transitions, while nanoparticles have physicochemical properties unique to their size. Here we study a promising yet underexplored third type: anisometric colloids, which integrate micrometer and nanometer dimensions into the same particle. We show that our prototypical system of anisometric silver plates with a high polydispersity assemble, unexpectedly, into an ordered, three-dimensional lattice. Real-time imaging and interaction modeling elucidate the crucial role of anisometry, which directs hierarchical assembly into secondary building blocks—columns—which are sufficiently monodisperse for further ordering. Ionic strength and plate tip morphology control the shape of the columns, and therefore the final lattice structures . Our joint experiment–modeling study demonstrates potentials of encoding unconventional assembly in anisometric colloids, which can likely introduce properties and phase behaviors inaccessible to micron- or nanometer-scale colloids. Anisometric colloids—which have both nanometer and micrometer dimensions—combine features of their two constituent length scales. Here, using optical microscopy and modeling, the authors find that highly polydisperse anisometric colloids self-assemble into 3D, periodic lattices in a manner hardly inaccessible to nano- or micron-scale colloids. For example, bundling of anisometric tropocollagen helices into the fibrous protein collagen allows multiple building blocks to work in concert under loads, giving collagen its exceptional elastic energy absorption4. Nanoscale DNA strands can be engineered to braid into micron-scale filaments to build programmed architectures6. Similarly shaped synthetic nanowires can serve as ideal energy transport materials to direct longitudinal conduction of quantum particles such as electrons, phonons, and photons9. However, despite extensive work on micron-scale colloids16 and nanoparticles21, anisometric colloids as a promising and emergent particle type are still greatly underexplored. Most work has focused on one-dimensional anisometric building blocks 6. Recent work by our group and others explored shapes like anisometric metallic plates3, which were shown to be great candidates for self-assembly studies. On the one hand, a nanoscale thickness  allows the plates to diffuse freely in solution, which enables their assembly in three dimensions (3D). In comparison, previous work on micron-scale colloids has required either a precise match with solvent density22 or an external field23 to build 3D structures against gravity. These experimental requirements pose limitations which are in principle irrelevant here 26, or electron microscopy with dried samples29. In comparison, although nanoparticles can assemble into 3D structures31, understanding and predictive engineering of their assembly pathways have been challenging due to the technical difficulty in direct imaging of such processes in solution33. However, despite the great advantages from anisometric plates, the observed assembly into one-dimensional lamellar superlattices from these plates is the same behavior seen in nano-sized metallic plates35; the potential of anisometry to enrich phase behavior was not fully harnessed.Anisometry is a potent strategy for biological and artificial building blocks to integrate the merits of both micron- and nanometer-scale dimensions36, which can further assemble into ordered 3D lattices due to a size-focusing effect (an effective narrowing of the building block size distribution). The apparent shape of columns can be predictively manipulated by ionic strength or the extent of plate tip truncation, which leads to distinct final lattice structures. Such unexpected 3D lattices have not been observed in previous systems of metallic nanoplates35. Our study demonstrates the unique secondary building block engineering opportunity enabled by anisometry as well as the greatly enriched phase behaviors of anisometric colloids, which opens doors to incorporating their unique mechanical, optical, and electronic properties into 3D assemblies18.Here we show that the anisometry of silver plates , when utilized meticulously, opens the design space of colloidal self-assembly to a regime not easily accessible before. Anisometry not only brings the technical ease for studying self-assembly, it also renders strongly directional interactions that favor hierarchical self-assembly along one plate axis at a time. In this way, the initial products in our system, hereafter referred to as “columns”, embody the concept of secondary building blocks in molecular crystals37. Tip truncation results from the formation of alternating sides during the kinetic expansion of the basal plane, as explained by Lofton and Sigmund38 in the silver halide model. The first plate type we studied has a long side length L of 1.49 ± 0.24 µm, a short side length L′ of 0.79 ± 0.27 µm, and a plate thickness tplate of 35 ± 5 nm  between adjacent plates overcomes screened electrostatic repulsion (Eel), and because the basal plane has a ~63 times larger interacting area than the plate side, face-to-face stacking into columns is favored over lateral assembly. Specifically, based on the pairwise interaction (Etot = EvdW + Eel) we computed following a discretized model41, the face-to-face stacking as observed in experiments has an Etot minimum of ‒6.1 kBT . The key difference is that, due to their larger basal plane area, the columns in this work experience enhanced van der Waals attraction with the substrate and larger gravitational force, so the columns prefer to stand vertically on the substrate , leading to the formation of standing column secondary building blocks by face-to-face plate stacking Fig. . The colkBT Fig. , while tL, 34% based on L′) because the growth of micron-sized particles from nano-sized seeds is highly susceptible to local fluctuations in solution, as reported previously37. This polydispersity is usually considered too high for forming an ordered crystal 42. However, the secondary building blocks of columns average their sizes due to plate stacking, thereby rendering them greatly reduced polydispersity (8%) to assemble into an ordered lattice , indicating that the columns in the lattice are closely packed. The plates in a nearest pair of columns are expected to stay “enveloped” by the circular rims, experiencing little physical contact as they rotate. Indeed, the orientations of two such plates span a range over 120° over time as shown in Fig. 45 of the plate orientations  sampled for n continuous frames, defined as f being the frame number; n = 300), confirming a weak angular correlation between plates in adjacent columns.A close investigation into the relative rotations of plates in the columns further reveals how the effective shape of columns is reduced into monodisperse, radially symmetric circular columns. As shown in Fig. vj for each column j  reaches to 6 of a perfect hexagonal lattice  of the column lattice is similar to that of an ideal hexagonal lattice with persisting peaks  Fig. , indicatm as the ratio between the short (L′) and the long (L) side lengths of a plate, to quantify the extent of tip truncation. As shown in both the schematics and SEM images , we calculated the interactions Etot between a stacked plate pair with varying relative in-plane orientations Δθ at their energy minimum spacings, as a function of ionic strength  ≈ E is the Etot difference for two plates at different relative orientations, see Fig. Etot calculated at a constant ionic strength and a constant basal plane area, but at varying m, we find a more hexagonal shape (a larger m) renders a flatter interaction profile over different Δθ values , on the other hand, have a steeper potential well disfavoring relative rotations between plates. This interaction profile, on the column level, maintains the generic three-fold symmetry of triangular plates instead of a smeared-out circular shape. This detail of tip truncation was not recognized in the previous literature2, due to the lack of importance in inter-column interactions in those systems.Having elucidated that the circular shape of columns leads to hexagonal lattices, we next demonstrated that the effective shape of columns can be controlled by changing the ionic strength or the extent of plate tip truncation, potentially leading to different lattice structures. Here we define a parameter ges Fig. , an m vamentclasspt{minimaues Fig. . The pla= 0.17, Fig. kBT. The disfavoring of plate misalignment in the columns leads to a triangular column projection, which encodes an assembly symmetry distinct from the isotropic, circular ones studied above. At low column concentration, the triangular columns assemble side-by-side into small clusters via individual plate or column addition , we observed a transition from the space-filling honeycomb lattice composed of triangular columns to a loosely packed structure which exhibits hexagonal packing due to increased electrostatic repulsion and lowered penalty for relative plate rotation  and electrostatic repulsion57. The formation of covalent organic frameworks is similar to the formation of our honeycomb lattice from triangular plates, upon a balance of interparticle interactions. The real-time dynamics observed here can potentially provide insight into phenomena taking place in those molecular systems.In our study of anisometric colloidal self-assembly, two otherwise separated length scales merge to induce 3D hierarchical self-assembly. Fundamentally the anisometry encodes highly directional interactions and induces the formation of secondary building blocks, which enable the selection of final structures from polymorphs over an otherwise complicated free energy landscape. Beyond the conventional small molecular ligands used here, macromolecular shelling2COONa)2·2H2O, Sigma-Aldrich), sodium borohydride , silver nitrate , l-ascorbic acid , citric acid monohydrate 2-C(OH)(CO2H)·H2O; Fisher Scientific), sodium chloride , hydrogen peroxide , and 2-(2-[2-(11-mercapto-undecyloxy)-ethoxy]-ethoxy)-ethoxy-ethoxy-ethoxy-ethoxy-acetic acid 11(OC2H4)6OCH2COOH; Prochimia Surfaces) were purchased and used without further purification. All glassware was treated with aqua regia (prepared by mixing HCl and HNO3 with a volume ratio of 3:1), thoroughly rinsed with water, and dried immediately before use. Nanopure water (with a resistivity of 18.2 MΩ cm at 25 °C) purified by a Milli-Q Advantage A10 system was used for all washing and solution preparation.Sodium citrate tribasic dihydrate (CH37. Silver seeds were prepared by adding 25 mL AgNO3 (0.1 mM), 600 μL sodium citrate (75 mM), and 60 μL hydrogen peroxide (30 wt %) in sequence to a 125 mL Erlenmeyer flask stirring at 300 r.p.m. at room temperature. Next, the stirring speed was increased to 1050 r.p.m. before rapid injection of 250 μL freshly prepared aqueous NaBH4 solution (0.1 M). This NaBH4 solution was prepared by adding solid NaBH4 to water, which had been precooled in an ice bath for ~10 min and then vortexing immediately. The obtained light yellow solution turned blue about 4–10 min after the injection of the NaBH4 solution and was kept stirring for another 5 min after this color change. Then the mixture was centrifuged (Eppendorf Centrifuge 5804) at 10,500 r.p.m. for 8 min and most of the supernatant was carefully removed, leaving ~50 μL sediment. This sediment was redispersed in 10 mL 0.94 mM sodium citrate solution as the seed solution. A UV–Vis spectrum of the as-prepared seeds was measured using a Scinco S-4100 PDA spectrophotometer. In different batches of seed preparation, the extinction peak positions were at 700–800 nm. Based on the UV–Vis spectrum, we diluted the seed solution using 0.94 mM sodium citrate solution to a concentration with an extinction intensity of 0.012 or 0.0024 at the maximum peak position; this diluted solution was later used as our seed solution stock to grow silver plates.The silver plates of controlled extent of truncation were synthesized following a slightly modified seeded-growth methodm) are listed in Supplementary Table l-ascorbic acid (0.1 M) was added to 20 mL of the seed solution stock kept in a 40 mL glass vial  as growth solution I. At the same time, a separate growth solution II was prepared by mixing 20 mL AgNO3 (1.0 mM), 125 μL citric acid (0.1 M), and 100 μL sodium citrate (1.5 mM). Growth solution II was added to growth solution I dropwise through a syringe pump  at a rate of 0.2 mL min–1 to start the plate growth. During the growth process, the growth solution was shaken vigorously at room temperature at a speed of 700 r.p.m. for the first cycle and 1000 r.p.m. for the later cycles . After every 10 min of injection, which is one growth cycle, two-thirds of the growth solution in the vial was removed, and the remaining solution was used for the next growth cycle to grow larger plates. The solution removed after growth cycle 5 was collected and stored in 8 mL glass vials  for further thiol modification. For plates with –1, with 5 min injection as one growth cycle; and (iii) plates after growth cycle 3 were collected and used for thiol modification.All the parameters used in silver plate growth (different size and different extent of truncation mentclass2pt{minim2)11(OC2H4)6OCH2COOH) to improve colloidal stability in solution. Specifically, 100–200 μL, 7.93 mM thiol solution was added to 2 mL silver plate solution collected from the growth cycle. This 7.93 mM thiol solution was prepared by dissolving 5 μL thiol as received from Prochimia in 1 mL water. The reaction solution was then allowed to sit overnight without stirring to complete the ligand exchange. After that, the plates with exchanged ligands sediment to a thin layer at the bottom of the vial. The supernatant was carefully removed, and 2 mL water was added to the sediment. After 5 h of additional sedimentation, the supernatant was removed and the final sediment product was stored in water in an 8 mL glass vial.The silver plates obtained above were coated with citrate ligands. These ligands were exchanged with carboxylate‒thiols (HS(CHThe shape and size distribution of silver plates and the assembled 3D lattice structures were characterized using SEM (Hitachi S4800). The plate thickness was measured using either tapping-mode AFM (Asylum Research Cypher) or SEM when the plates were vertical to the viewing angle. The silicon wafer used in the AFM measurement was first sonicated with acetone and isopropanol each for 15 min, and then treated with oxygen plasma (Harrick Plasma Cleaner PDC-23G) at medium power for 1 min to render the wafer surface hydrophilic. Then one drop of silver plate solution (~5 μL) was pipetted on the wafer and allowed to dry under ambient conditions before the AFM measurements. The 3D lattice structures formed in solution in the optical microscopy chamber were controllably dried in the chamber under ambient conditions, to maximally maintain the structure for SEM imaging.Approximately 150 μL silver plate suspension after thiol modification was added to an eight-well chamber . Bright-field optical microscopy imaging was performed using a Zeiss inverted microscope (Axiovert 200) with a ×100 oil objective (NA = 1.45) and ×1 or ×1.6 post magnification. Optical microscopy movies were recorded using a complementary metal-oxide-semiconductor camera (Edmund Optics 5012 M GigE) at a rate of 17.2 or 21.8 frames per second (fps).http://www.esrf.eu/computing/scientific/FIT2D) was used to analyze the two-dimensional (2D) scattering plots and obtain the intensity (I) and scattering vector (q) profile  were overwhelmed by the profile of the unscattered beam. Consequently, the q values of the first two peaks were derived from higher-order peaks  as SAXS measurements were conducted on the truncated plate sample (ource 1.5 Å/8 keV Etot(d) was evaluated as a sum of van der Waals attraction35 between silver in water and electrostatic repulsion between charged ligands34, Etot(d) = EvdW(d) + Eel(d), whered is the face-to-face spacing between two neighboring plates, tplate is the plate thickness, and S is the plate basal plane area. In the electrostatic potential, ε is the relative permittivity of water, ε0 is the vacuum permittivity, ζ is the zeta potential of the thiol-coated silver plates in water, and b is the Debye length, given (in nanometers) for an aqueous solution of sodium chloride salt as I (in moles per liter). In addition, tligand is the thickness of the carboxylate–thiol monolayer on the plate surface34. Here we assumed that the two plates are in a perfectly aligned, face-to-face configuration.In Fig. Because the plates in this system achieve large interparticle separations, the Hamaker constant was treated as an “effective” Hamaker constant in such a way that the retardation of the van der Waals attraction at large separations is accounted for. In other words,a is constant describing the geometry of the interaction  and λ is a parameter describing the onset length scale for retardation. H0 is the Hamaker constant for silver and silver interacting across water at zero separation (4.0 × 10–19 J)60. This model was previously shown to be a simple but robust approximation for more complicated retardation theories61. The value of λ can be approximated byh is Planck’s constant, c is the speed of light, and ΔE is a “characteristic energy” in the electronic spectrum of the material, commonly taken to be the first ionization energy. For silver, λ is therefore about 164 nm.Here Net interaction potentials and the locations of their secondary minima at different ionic strength conditions are indicated in Supplementary Fig. N = 21,392 to 26,744 beads per plate (depending on the extent of truncation) with beads 17.5 nm in diameter , pairwise interaction model to evaluate the effect of relative plate rotation on the interaction potential Figs.  and 4. Ai in one plate and beads j in the other. Similarly, the plate–plate electrostatic interaction was evaluated asThe van der Waals interaction between anisometric plates was taken as the sum of all bead–bead van der Waals interactions, i.e.,A1, A2, B1, and B2. The sum of these two curves, Etot, was used to determine the position of the secondary energy minima, using Mathematica. We also evaluated the bead size effect by calculating the van der Waals interaction using a smaller bead size (8.75 nm in diameter) and the results are consistent with different bead sizes (Supplementary Fig. Note that for the electrostatic interactions, the ligand length was taken into account in determining the interbead distance, depending on whether the beads were on the near, opposite, or same faces of the plates. Net van der Waals and electrostatic interactions were determined in this manner for a series of interparticle distances. The two potentials were then fit in Origin to curves of standard form for interparticle interactions:To assess the validity of the CG approximation, we first compared the analytical and CG models assuming no rotation between the plate pair. A comparison of interaction curves based on the two calculations at various ionic strengths is given in Supplementary Fig. Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6


The diffusion length (Ld) of the precursors have been measured with unique nucleation control methods. It was revealed that the Ld reaches up to ~750 μm. This ultra-long diffusion can be attributed to precursor droplets observed during in-situ monitoring of WS2 growth. The integrated synthesis of >35,000 single crystals and monolayer WS2 was achieved at the wafer scale based on this model. Our findings are highly significant for both the fundamental study of droplet-mediated crystal growth and the industrial application of integrated single-crystal TMDs.Transition metal dichalcogenides (TMDs) attract intence attention due to its unique optoelectrical features. Recent progress in production stage of TMD enables us to synthesis uniform and large area TMD with mono layer thickness. Elucidation of growth mechanism is a challenge to improve the crystallinity of TMD, which is regargeded as a next crutial subject in the production stage. Here we report novel diffusion and nucleation dynamics during tungsten disulphide (WS Recent progress with molten salt-assisted growth5 in the production stage enabled us to synthesise uniform polycrystalline films on a large scale. Attaining better crystallinity in single crystal films with large domain sizes, uniform edge structures, and fewer vacancies is our next challenge. For this purpose, a deep understanding of the TMD crystal growth mechanism is crucial. The effects of the sulfur/molybdenum (S/Mo) ratio6 in MoS2, the step edge of the substrate7, the carrier gas8 and nucleation promoters9 have been investigated. A theoretical model has also been established using a thermodynamic approach11. However, little is known about TMD growth dynamics, their precursors and nucleation in particular, which are important for the synthesis of high-quality single crystals. Herein we report novel diffusion and nucleation dynamics during tungsten disulfide (WS2) growth. Accurate nucleation-controlled growth enabled us to determine the diffusion length (Ld) of the precursors. Ld reached ~750 μm, nearly two orders of magnitude longer than that of conventional semiconductors such as Si12, GaAs13 and SiC14. This ultra-long diffusion was attributed to precursor droplets observed during in-situ monitoring of WS2 growth. The integrated synthesis of >35,000 single crystals and monolayer WS2 was achieved at the wafer scale based on this model. Our findings are highly significant for both the fundamental study of droplet-mediated crystal growth and the industrial application of integrated single-crystal TMDs.Transition metal dichalcogenides (TMDs) are among the most well-known layered materials. They have various features that are desirable in semiconductors, including stable neutral and charged excitons, valley polarisation capability, and superconductivity16 is useful for studying the growth dynamics of 2D crystals. In this study, we used Au dots to precisely control WS2 nucleation and site density  was 20 μm. Triangular WS2 crystals were grown at the Au sites, thereby demonstrating that precise control of the nucleation sites and their density was possible.Control of nucleation sitesLd), an important kinetic parameter of crystal growth, was determined experimentally  increased with growth time. LWS2 was related to (Γefft)0.5 in our model, where Γeff and t were the effective precursor flux to the growth edge of WS2 and growth time, respectively. Γeff was proportional to πΓ0Ld2, where Γ0 was the influx of vapor-phase precursors to the substrate per unit area , so LWS2 would change as a function of LBW  within the region where the temperature decreased from ~721 °C to ~654 °C. The maximum Ld of ~750 μm was observed at 721 °C (P1) and 698 °C (P2)  Fig. . The obsLAu Fig. . This wa14. When we tried to explain the ultra-long diffusion of WS2 precursors with this model, the distance covered in a single jump was over 100 times larger than the distance between NN sites. Although ‘long jumping’ during the diffusion of W on a substrate surface has been reported, the longest jumps were only a few times longer than the distance between NN sites18. Ultra-long jumps (100 × NN length) have not been reported for any materials, suggesting that WS2 precursors diffuse by a different mechanism. A possible mechanism for ultra-long precursor diffusion will be discussed later.Hopping transport is the predominating model for conventional semiconductors, in which precursors jump between nearest-neighbour (NN) stable sites. Diffusion occurs over a few micrometres within the limited traveling time2 growth dynamics, understanding the nucleation phase is important. We varied Au dot shape and diameter (DAu) in a combinatorial experiment (see Methods) to gain insight into the nucleation dynamics of WS2  than it was when DAu ≈ 4 μm  > 6  equation21:P is the difference between the droplet pressure (Pin) and vapor pressure (Pout) at the interface; γ is the surface tension of the droplet; and r is the droplet radius. For the droplet to be stable, ΔP should be as small as possible. This means that materials with low γ are more stable, especially at the nanoscale (r < 500 nm). The γ of molten metal droplets can be reduced by increasing the temperature22, reducing droplet size23, and mixing with oxygen24. It can be surmised that nanoscale droplets containing WxSyOz at high temperatures would exhibit very low surface tension, making them candidate precursor droplets. Since the physical properties of nanoscale droplets themselves are not yet fully understood25, the identification of precursor droplet components is an important step for achieving a detailed understanding of WS2 growth dynamics.Based on these results, we concluded (i) precursor diffusion length was ultra-long (~750 μm); (ii) single crystals could grow on Au dot structures with various shapes; and (iii) there was a clear correlation between single crystal concentration and Lors Fig. . Since ture Fig. , we surmin-situ monitoring  growth model was reported for the catalyst-guided growth of MoS2 nanoribbons26. In the growth of 2D WS2 demonstrated in our study, it was not a catalyst but the precursor itself in liquid form that diffused over long distances. This differed significantly from the mechanism of nanoribbon growth.To confirm the unique growth dynamics of droplet-induced nucleation, we attempted to observe the nucleation phase directly via ved Fig. . At the ved Fig. . These cved Fig. . This wates Fig. . This estes Fig.  and 4g–iDAu, LAu and growth temperature were made in a combinatorial experiment . Measurement by atomic force microscopy (AFM) revealed the thickness of WS2 was approximately 0.8 nm  intensity map in Fig. 2 contained two peaks separated by 61 cm−1  on an ELS-7500 EBL system  and thermal evaporation of Au (see Supplementary Information for more details). The thickness of each Au dot was fixed at 50 nm.The Au dot arrays were fabricated on a SiO2 was synthesised by thermal CVD using WO3 as a tungsten source. Ar was used as the carrier gas at a flow rate of 150–450 sccm. Sulphur (0.5 g) was placed in the CVD oven  on a quarts boat was set 15 cm downstream in the centre of the CVD furnace. For in-situ monitoring, NaCl (6 mg) was mixed into the WO3 to enhance evaporation.WS2/Si substrate  and Ar (488 nm) lasers were used for excitation. Mapping was performed in steps of 200–500 nm.The optical microscope was set above the quartz tube between the main electrical furnace and additional heater (AH). To maintain the elevated temperature of the substrate, an AH was placed outside the main electrical furnace. The substrate was set between the main furnace and the heater, enabling optical observation of the substrate surface during CVD growth (Supplementary Fig. Supplementary DatasetSupplementary Information


This study determined whether there is a statistically significant difference between efficient and inefficient Organization for Economic Co-operation and Development (OECD) countries in terms of health indicators using fuzzy data envelopment analysis (FDEA).In the study, FDEA was performed with three input variables directly affecting health, four environmental factors considered to indirectly affect health, and two output variables. Literature research was used to determine appropriate variables. In FDEA, three different α-cut levels were used. The hypotheses regarding whether there was a statistically significant difference between efficient and inefficient countries in input and output variables were tested for all α-cut levels of upper bound efficiency values.In terms of health indicators, 17 countries were efficient at α-cut 0 and 0.5. At α-cut 1, 18 countries were efficient. There was only a statistically significant difference between the efficient and inefficient countries in “the number of physicians.”This study shows the number of physicians was the most important determinant affecting the efficiency of a country’s healthcare system. Inefficient countries had a greater mean for number of physicians. Thus, inefficient countries consume more resources than efficient ones. Countries are under obligation to use healthcare resources effectively without compromising on quality.4There are many factors, health indicators, and indicator definitions developed by national and international organizations, reference groups, and academicians that affect the efficiency of health systems.7Events or situations that occur in real life might be indefinite in various aspects. Imprecise data can also be encountered due to uncertainty.11This study determined whether there is a statistically significant difference between efficient and inefficient OECD countries in terms of health indicators using FDEA.2 emissions (H6),”“number of physicians (H1),” “number of total hospital beds (H2),” “current expenditure on health (H3),” tobacco consumption % of population aged 15+ who are daily smokers (H4),” “measles immunization % of children immunized (H5),” “CO and “school enrollment, secondary (% gross) (H7).” The two chosen outputs were “life expectancy total population at birth (H8)” and “infant mortality, no minimum threshold of gestation period or birthweight (H9).”In this study, each of the 36 OECD countries was referred to as a decision making unit (DMU) and included in the analysis. Data were obtained from the OECD databaseData from 2015 were used where data availability was highest. If 2015 data was unavailable, the data for the nearest year was used. According to Retzlaff-Roberts, Chang, & Rubin,The implementation part of the study consisted of two steps. In the first step of the analysis, the health efficiencies of OECD countries were determined using FDEA. For efficiency measurement with FDEA, Wang, Greatbanks, and Yang’s modelThis model was preferred because it is widely used in the literature. Solutions have been made by creating interval data in accordance with the α-cut level approach of Zimmermann.After efficiency values of countries at different α-cut levels were calculated, the second step determined whether there was a statistically significant difference between efficient and inefficient countries in the selected input and output variables. Therefore, the normality of data was examined. Comparisons between groups were done using independent samples t-test for variables with normal distribution and the Mann-Whitney U test for variables with non-normal distribution. The null hypothesis was rejected at the 5% level.Each variable was assessed for a statistically significant difference between efficient and inefficient countries. The data acquired for α = 0, α = 0.5, and α = 1 cut levels of upper bound efficiency values were used. The upper limit was used because the lower limit and upper limit values are the same at the α = 1 level. In addition, there was no efficient country at the lower limits of α=0 and α=0.5. This makes it impossible to compare efficient and inefficient countries with lower boundaries. Statistical data generated by the hypothesis tests are presented in “the number of physicians” (H1). The mean value of inefficient countries was higher than efficient countries. The proper distribution of healthcare resources is very important. Thus, inefficient countries consumed more resources than efficient ones. No other hypotheses were accepted at any alpha level showing that “total number of hospital beds,” “current expenditures on health,” tobacco consumption % of population aged 15+ who are daily smokers,” “measles immunization % of children immunized,” or “CO2 emissions” (Hypothesis 1 was accepted for each of the three alpha cut levels. In other words, there was a statistically significant difference between efficient and inefficient countries in issions” .Similarly, the hypotheses with non-normal data were not accepted at any alpha level. Therefore, there was not a statistically significant difference between efficient and inefficient countries in “school enrollment,” “life expectancy,” or “infant mortality” .The healthcare system has a major impact on society, just as society impacts healthcare.Especially in recent years, international comparisons of health system efficiencies have attracted the attention of health policy makers.DEA, which is a relative performance measurement tool,The hypotheses about whether there is a statistically significant difference between the efficient and inefficient status of countries in input and output variables, which constitute the main purpose of the study, were tested by statistical methods. According to the results, a significant difference was found between efficient and inefficient countries only in “the number of physicians” at all three α-cut levels. Similarly, in the study of Bal & Bilge,This study not only contributes to the literature but also provides guidance to health managers, planners, policy makers, decision makers, and academicians who are interested in the measurement of health efficiencies and making cross-country comparisons in healthcare. It has provided evidence of significant differences in countries efficiencies and the important variable for further focus. Future studies should analyze different environmental variables to determine countries’ health efficiencies.


Here we report that they independently adjust photosynthesis on the basis of analyses using single and higher order loss-of-function mutants. In short experiments of photosynthetic response on transition from dark to low light, we reveal a sequential functioning of VCCN1 and CLCe in the activation of photoprotection and of KEA3 in its downregulation to a low steady state while adjusting the electron transport. On transition from low to high light, VCCN1 accelerates the activation of photoprotection, whereas KEA3 slows it down on transition from high to low light. Based on parallel electrochromic band shift measurements, the mechanism behind is that VCCN1 builds up a pH gradient across the thylakoid membrane, whereas KEA3 dissipates this gradient, which affects photoprotection. CLCe regulates photosynthesis by a pH-independent mechanism likely involving Cl− homeostasis. Nevertheless, all genotypes grow well in alternating high and low light. Taken together, the three studied ion channels/transporters function independently in adjusting photosynthesis to the light environment.In variable light environments, plants adjust light use in photosynthetic electron transport and photoprotective dissipation in the thylakoid membrane. In this respect, roles of the K Rapid and abrupt changes in light conditions affect electron transport through photosystem II (PSII), cytochrome b6f and photosystem I (PSI) complexes in the thylakoid membrane2. To overcome the constraints imposed by sunlight, energy capture and dissipation of excess light energy are tightly regulated during photosynthesis. In order to avoid damage to photosystems, excess energy is quenched by dissipation as heat in a mostly pH-dependent process known as qE-type of non-photochemical quenching (NPQ)3. NPQ undergoes activation and downregulation in response to changes in light intensity, and is controlled by the pH in the thylakoid lumen, the pH sensor protein PsbS and the carotenoid zeaxanthin4. The rates of NPQ activation and in particular of its downregulation are rather slow (order of minutes) as compared to natural light fluctuations (order of seconds), and are likely limited by the slow kinetics of pH changes in the thylakoid lumen5. Several Arabidopsis thaliana npq mutants have been discovered, but they grew similarly to wild-type plants under constant laboratory light conditions7. Nevertheless, experiments in field conditions with the same mutants indicated that NPQ confers increased plant tolerance to fluctuating light1. Moreover, tobacco mutants engineered to accelerate NPQ response to fluctuating light showed improved photosynthesis and biomass production8.Plants harvest sunlight and convert the captured energy into biochemical products. For the energy conversion to take place, several processes occur in a timely manner in what is generically called photosynthetic light-dependent reactions. Sunlight is not only the energy source for photosynthesis but also an important limiting factor due to its fluctuations in intensity10. In the thylakoid membrane of Arabidopsis, the two-pore K+ channel TPK3 is essential for activation of NPQ11, whereas the K+/H+ antiporter KEA3 accelerates its downregulation and stimulates the electron transport12. The mechanism behind the action of both proteins is thought to be an altered K+:H+ stoichiometry in the lumen, which in turn changes the lumenal pH. Armbruster and colleagues13 provided experimental evidence for the activation of KEA3 by its C-terminus upon sudden shifts to low light intensities. The Cl− channel/transporter CLCe regulates the electron transport15, whereas the voltage-dependent Cl− channel VCCN1 accelerates the activation of NPQ in conditions of increasing light intensities17. To explain these observations, Cl− influx is thought to electrically balance the H+ uptake into the thylakoid lumen, which in turn favors additional H+ uptake and hence lumen acidification. In addition, ion channels/transporters are important for thylakoid ultrastructure18, that may also influence photosynthetic energy conversion19.There is increasing evidence that NPQ is also regulated by ion channels/transporters able to modulate the kinetics of pH changes in the lumen+ uptake into the lumen, generating an electrochemical gradient  across the thylakoid membrane. The PMF is composed of an electric field (∆Ψ) and a pH gradient (∆pH), both driving the synthesis of ATP20. They have also distinct roles since a high ∆pH activates NPQ and inhibits electron transport4, whereas a high ∆Ψ enhances the rate of charge recombination and PSII photodamage21. Loss-of-function of each of the identified thylakoid-located channels/transporters altered the PMF partitioning into ∆pH and ∆Ψ which influenced the balance between light use in electron transport and photoprotective heat dissipation18. Moreover, since the KEA3 loss-of-function maintains high NPQ only in low light conditions12, whereas the lack of VCCN1 and CLCe diminishes NPQ and lowers electron transport, respectively, at all light intensities17, distinct mechanisms in the action of the three proteins can be hypothesized. In this study, we characterized single, double and triple loss-of-function mutants in Arabidopsis and show that KEA3, CLCe and VCCN1 function independently to fine-tune photosynthesis in response to changes in the light environment.Photosynthetic electron transport is coupled with Hclce-2, kea3-1 and vccn1-1 single loss-of-function mutant lines18, hereafter named clce, kea3 and vccn1, with those of newly generated clcekea3, clcevccn1, kea3vccn1 double and clcekea3vccn1 triple mutant lines.To better understand the role of CLCe, KEA3 and VCCN1 and their possible interdependency in the regulation of PMF and photosynthesis, we compared the phenotypes of −2 s−1) and displayed similar shoot fresh weight at 5 weeks  plants under standard light conditions  kinetics on intact leaves of dark-adapted plants. At 5 weeks, the curve in wt had a polyphasic shape, while lower fluorescence levels at the J and I steps, previously reported in clce15, were also reproduced in this study and retained in clce double and triple mutant lines , associated with electron transport at PSI22. Double normalization to O and J reflecting PSII antenna size and activity resulted in much smaller differences between wt and the clce lines as compared to those in the I peak , the photosynthetic performance index , and the time to reach maximal fluorescence intensity (tFm) were similar to wt in all mutants with the exception of clce lines  and found a more pronounced reduction of P700+ in dark-incubated clce single and clcekea3vccn1 triple mutant lines than in wt  light were indistinguishable from wt in P700+ reduction kinetics ) as measure of the effective electron transport of PSII was slightly but significantly reduced under these conditions  as based on the non-significant differences from the wt for the Chl fluorescence parameters (Supplementary Table 10. We further found that when tap water was used (1–5 mM Cl−), 6-week-old clce lines showed Chl fluorescence and tFm values indistinguishable from wt  displayed normal Chl fluorescence and tFm similar to wt  throughout the illumination and a lower Y(II) than wt when reaching the steady state of NPQ. The corresponding parameters in vccn1 were similar to wt, whereas the double and triple mutant lines displayed intermediate patterns. Based on the determined Y(II) and Y(I) differences , KEA3 and CLCe stimulated electron transport adjusting it to the NPQ on transition from dark to low light.Photosynthetic electron transport measured as PSII and PSI quantum yield (Y(II) and Y(I), respectively) in the same experiment, was reduced in vccn1 lines, whereas Y(II) and Y(I) were not largely affected .To further investigate the role of CLCe, KEA3 and VCCN1 in photosynthetic regulation, we undertook experiments under fluctuating light (transitions from low intensity to high intensity and back to low intensity). On transition from low to high light, NPQ was induced slower in ate Fig. . NPQ in wer Fig. . Y(I) anwer Fig. insets.Fvccn1 lines and similar to wt in the other lines . These data are consistent with a major role of VCCN1 in NPQ dynamics on transition to high light.The calculated halftime of NPQ induction was 1.5-2-fold higher in left panel). The ∆pH significantly decreased in vccn1 lines, whereas in the other genotypes ∆pH was similar to wt . PMF size was elevated only in vccn1 lines . Taken together, these data indicate that on transition from low to high light, VCCN1 functions in building up a significant pH gradient to rapidly activate NPQ without largely affecting the electron transport through photosystems. CLCe and KEA3 do not play a major role in photosynthetic regulation under these conditions and only fine-tune the NPQ produced by VCCN1.In parallel experiments we determined PMF size and partitioning from electrochromic shift (ECS) recordings at the end of the transition from low to high light .Y(II) and Y(I) under the same conditions were lower in nes Fig. . Based ones Fig. insets.kea3 lines was 3-fold higher than in all other genotypes including wt , consistent with a role of KEA3 on transition from high to low light. In parallel experiments with ECS recordings at the end of the transition , only the kea3 lines displayed a significantly higher ∆pH  while preserving the wt PMF size . Taken together, these data indicate that during the high to low light transition, KEA3 functions in the dissipation of pH gradient leading to NPQ relaxation, while enhancing PSII and PSI efficiency. CLCe also affects PSI and PSII electron transport, and this happens likely by a ∆pH-independent mechanism.The halftime of NPQ relaxation in −2 s−1 followed by 3 min at 70 µmol photons m−2 s−1). The plants of all genotypes had similar rosette size at 4 and 6 weeks . No major changes in the overall structure relative to wt in either dark- or light-adapted plants were observed at either scale of 1 μm or 200 nm  and different protocols for fixation and embedding of the samples between this and previous reports may impact the intra-thylakoidal ionic strength, which was demonstrated to result in structural rearrangements in vccn117. Taken together, the TEM data indicate wt-like thylakoid ultrastructure in all mutant lines, that could not explain the short-term alterations in NPQ and photosynthesis observed in this study.The thylakoid membrane undergoes dynamic changes in its overall shape depending on the light environment, and this may control important processes in photosynthesis+ and Cl− fluxes across the thylakoid membrane have been predicted to function in membrane depolarization and regulation of photosynthesis26. In this study, we bring evidence that three molecular players in these fluxes, namely CLCe, KEA3 and VCCN1, act independently and by distinct mechanisms in the adjustment of photosynthesis to the light environment.K− channel in electrophysiological experiments using planar lipid bilayers17, and was therefore proposed to mediate Cl− import into the thylakoid lumen. CLCe is believed to transport Cl− as well14, but to date, no electrophysiological evidence has been put forward for its substrate or mode of regulation. The mechanism of Cl− transport may be different since CLCe modulates photoprotection and electron transport in different manners and time scales from VCCN1 in the studied conditions. In dark- and growth light-adapted plants, CLCe is likely required for an optimal electron transport activity . Our results support the view of different modes of action for CLCe and VCCN1 in regulation of photosynthesis.VCCN1 and CLCe play different roles also in fluctuating light conditions, since VCCN1 acts in the activation of NPQ on transition to high light, whereas CLCe stimulates the electron transport on transition to low light Fig. . In suppon Table –5. The mon Table , support13 where KEA3 is inactive in high light and is rapidly activated upon decreasing light intensity to export H+ out of the lumen thus dissipating the pH gradient. KEA3 also favors the relaxation of the NPQ formed on transition from dark to low light, with a maximum at 1 min of illumination , clce-2 (SALK_010237) and vccn1-1 (SALK_103612) T-DNA insertion mutant lines were obtained from the Arabidopsis Biological Research Center (https://arabidopsis.org/abrc/) and were previously characterized18. Single mutant lines were crossed to generate double and triple mutant lines. For genotyping of the mutants, genomic DNA was isolated from 4-week-old plants and used for PCR. Gene-specific primers are listed in Supplementary Table The Arabidopsis thaliana plants of accession Columbia-0 (wt) and mutant lines were grown in a growth chamber  for 5–8 weeks in soil using a daily cycle of 8 h of light (120 µmol photons m−2 s−1) at 21 °C and 16 h of dark at 19 °C (unless otherwise stated). The plants were watered once every week with deionized water (resistivity approx. 15 MΩ cm−1) for all experiments if not otherwise indicated. In some experiments, the plants were watered once every week with tap water (1–5 mM NaCl) or with deionized water supplemented with 0.5 mM or 5 mM NaCl. In fluctuating light experiments, plants were grown for 6 weeks using alternating high light and low light (3 min at 660 µmol photons m−2 s−1 followed by 3 min at 70 µmol photons m−2 s−1) and watered twice a week with deionized water.v/v) ethanol at 65 °C for 10 min and its content was determined by spectrophotometry28.Chl was extracted from leaf discs in 96% (OJIP) kinetics were recorded on 30 min dark-adapted plants by applying a saturating actinic red pulse of 3,600 µmol photons m−2 s−1 for 1 s using a Handy-PEA  fluorometer. Where indicated, detached leaves were first incubated for 30 min in deionized water or in deionized water supplemented with 150 mM KCl under growth light conditions, then removed from the solution and dark adapted for 15 min before the OJIP kinetics were recorded14. The maximal quantum yield of PSII photochemistry (Fv/Fm), the photosynthetic performance index based on equal absorption (PIABS), and the time to reach Fm (tFm) were calculated using Hansatech PEA Plus v1.10 software according to Strasser and colleagues29. PIABS consists of 3 partial performances, namely those related with the concentration of reaction centers, the maximal energy flux reaching PSII centers, and the electron transport at the onset of illumination30. Chl fluorescence was also measured on plants during steady-state illumination (4 h in growth light) using a MultispeQ instrument, and the PSII quantum yield (Y(II) was calculated according to Kuhlgert and colleagues31, (http://photosynq.org).Fast Chl fluorescence (−2 s−1 (low light) or 660 μmol photons m−2 s−1 (high light) for 10 min. For experiments in fluctuating light, attached leaves of 30-min dark-adapted plants were first illuminated for 10 min with low light, then with 3 min of high light and then again 3 min of low light. NPQ and Y(II) were calculated based on changes in Chl fluorescence according to Genty and colleagues32, whereas Y(I) was calculated from absorbance changes at 830 nm (reflecting the redox state of P700) according to Klughammer and Schreiber33. The intensity of the saturating actinic red pulse applied to determine NPQ, Y(II) and Y(I) was 5,000 µmol photons m−2 s−1 and of 800 ms duration. NPQ, Y(II) and Y(I) differences were calculated as the difference of average value in wt minus average value in each mutant. Calculation of NPQ halftime (t1/2) was performed in GraphPad. For this purpose, NPQ induction or relaxation data points were fitted with a single exponential decay and the decay constant (t1) was used to calculate the t1/2 as follows: t1/2 = t1 * ln(2), where ln(2) is natural logarithm of the number 2.Slow kinetics of Chl fluorescence induction and P700 oxidation-reduction were simultaneously recorded with a pulse-amplitude modulated fluorometer DUAL-PAM 100 equipped with DUAL-DB and DUAL-E emitter-detector module . The kinetics were first recorded on attached leaves of 30 min dark-adapted plants using actinic red light of 70 μmol photons m3. Before the measurements, the plants were adapted to growth light for at least 1 h. The P700 signal was recorded during exposure of attached leaves to actinic red light of 120 µmol photons m−2  and during exposure to far-red of 128 µmol photons m−2  after 4 min in darkness. To monitor the P700+ reduction kinetics, one saturating actinic red pulse  was applied before AL was switched off and another one at the end of darkness and FR illumination. Where indicated, plants adapted to growth light were also used to record light response curves of Y(I) with 1 min exposure at increasing light intensity via P700+-absorbance changes at 830 nm33.P700 oxidation-reduction kinetics were recorded based on absorbance changes at 830 nm using DUAL-PAM 100. The P700 signal calculated as the difference in transmittance at 875 nm and 830 nm is displayed as P700 ΔI/I * 105 at the end of the transition at the given light intensity. PMF size was calculated as the difference between the ECS signal in light and the minimum value of the ECS signal after the light was turned off  that were 16-h dark adapted or 3 h after the onset of illumination were cut into small squares (2 × 2 mm) under dim green light and fixed in Karnovsky’s solution  in vacuum for 4 h in darkness. The fixed leaves were washed three times with 0.1 M sodium cacodylate, pH 7.3. The leaves were post-fixed in 1% osmium tetroxide for 2 h. The samples were washed twice with 0.1 M sodium cacodylate buffer followed by water and thereafter dehydrated with a series of graded acetone. The leaves were infiltrated and embedded in Spurr’s low viscosity resin. Leaf pieces of at least two different plants per treatment were analyzed independently. Ultrathin sections of 80 nm were made by a diamond knife using Leica (EM-UC7) or Reichert-Jung (Ultracut E) ultramicrotomes and mounted on formvar/carbon coated 200 mesh nickel grids. All the samples were contrasted by negative staining with readymade solutions of uranyless and lead citrate  or by lead citrate. Electron micrographs were taken by Philips CM100 or JEOL JEM 1011 microscopes equipped with Morada high resolution digital camera. ImageJ software was used to measure granum diameter on the micrographs. Calculations were done on 250–300 randomly chosen grana originating from 20–25 chloroplasts taken randomly from 10–12 different mesophyll cells per treatment and genotype. The overall structure was investigated for >50 chloroplasts taken randomly from 15–20 mesophyll cells per treatment and genotype.Leaves harvested from 7-week-old plants (age when the Statistics were performed in GraphPad software using the Tukey one-way ANOVA test. For all data, P < 0.05 was considered significant.Supplementary information