We found that the same functional group does not result in the same performance trends for different nanostructured carbons. Drastically different behavior was observed for the amino-group functionalization, where a significant improvement is observed for MWCNT, while worse performance compared to non-functionalized material is obtained for RGO. On the other hand, hydroxyl and carboxyl group functionalization improves the lead adsorption regardless of the form of carbon. The best performing RGO sample, namely carboxyl group functionalized one, exhibited maximum lead adsorption capacity of 298.49 mg g Functionalization with same functional group results in varied lead adsorption performance for different nanostructured carbon materials. Consequently, they have been extensively studied, alone or in various composites.1,44 since it improves the hydrophilicity of the nanocarbon, and it can also enable increased adsorption of the target contaminant via electrostatic interactions.1 In the case of Pb2+, it was reported that the adsorption mechanisms include interaction with oxygen containing and/or negatively charged functional groups, cation–π interaction, and adsorption on defect sites.1 In general, physisorption,  and chemisorption (surface complexation) processes coexist.16 In some cases, specific mechanisms have been found to be dominant. For example, surface complexation rather than ion exchange was identified as main adsorption mechanism for lead for oxidized MWCNTs,26 while another study identified that the dominant mechanism of lead adsorption was chemisorption involving phenolic groups.25 In the case of graphene oxide (GO), it was proposed that hydroxyl and carboxyl groups located at the edges of GO sheets were main participants in lead ion complexation.38,41 It is not clear, however, whether those findings are generally applicable due to large variation in reported experimental conditions for lead removal.It is well known that surface functionalization plays an important role in heavy metal removal,1 In addition, for some materials such as graphene oxide (GO), very wide range was reported for Pb2+ adsorption capacity (36–789.9 mg g−1).1,41 Part of the reason for that could be differences in the synthesis method of GO, which is known to affect the adsorption capacity.8 Other reasons may include differences in pH, ionic strength, and experimental conditions in general .1 Thus, direct comparisons of different materials from the published data would be difficult. Nevertheless, it is very clear that surface functionalization and/or preparing nanostructured carbon-based composites can result in efficient heavy metal removal from water. A wide range of surface functionalizations, morphologies, and different composites has been reported,5–29 including those based on carbon nanotubes (CNTs),5–7,10–13,15–17,23–26,37,40 graphene oxide (GO),8,9,14,18–22,27,30,31,36,38–41 and reduced graphene oxide (RGO).34Furthermore, while the carbon nanomaterials typically display higher adsorption capacity compared to conventional adsorbents, the reported results on the adsorption affinity of divalent heavy metals can be contradictory.1,10,38,41 Carboxylic group functionalization in particular was found to result in high efficiency of Pb removal by multiwall carbon nanotubes (MWCNTs) and graphene oxide.13,20,38,41 Similar finding, i.e. significant enhancement of lead adsorption, has also been reported for amino group, and the contribution of functional groups to the total lead adsorption was estimated to exceed 80%.16 However, molecular dynamics simulations of single wall CNTs functionalized with carboxyl, hydroxyl, and amide (–CONH2) functional groups predicted that an improvement in lead adsorption with carboxyl functionalization is significantly higher compared to hydroxyl or amide groups.17 Thus, there is an obvious interest in conclusively establishing what type of functional groups and what type of nanostructured carbon will yield superior lead adsorption performance by experimental investigation, before proceeding with more complex functionalization and/or composite syntheses.It is well recognized that –COOH and –OH functional groups result in negative surface charge in aqueous solution, which enhances the adsorption of positively charged metal ions.16 the comparisons of different functional groups on different forms of nanostructured carbon have been lacking. Here we compare the lead adsorption on MWCNTs and reduced graphene oxide (RGO) for samples without functionalization, as well as samples functionalized with hydroxyl, carboxyl, and amino group. The samples have been commercially obtained, since commercial samples are relevant for practical use. In addition, we have prepared graphene oxide (GO) samples by modified Hummers' method, and compared the performance to different commercial RGO samples. All the samples have been comprehensively studied, and their performance in lead adsorption has been compared not only in terms of adsorption kinetics and isotherms but also in terms of lead removal by filtration. Differences in the performance for amino-group functionalization for MWCNT and RGO are discussed, since amino-group functionalization improved the lead adsorption capacity of MWCNTs, but reduced the lead adsorption of RGO. Unlike amino-group, hydroxyl- and carboxyl-groups result in performance improvements regardless of the type of nanostructured carbon. Thus, these functional groups would be most relevant for the preparation of composite materials for practical applications.While there have been comparisons of lead adsorption performance as-prepared, oxidized and amine-functionalized MWCNTs,4 AR), ferric chloride (FeCl3 99%), and sodium hydroxide (NaOH AR) was purchased from Dieckmann. Lead(ii) nitrate (Pb(NO3)2 99%) was purchased from Alfa Aesar. Sulphuric acid (H2SO4 AR) was purchased from RCI Labscan. Hydrogen peroxide (H2O2 30%), hydrochloric acid (HCl 37%), and nitric acid (HNO3 68%) were purchased from VWR Chemical. Phosphoric acid (H3PO4 85%) was purchased from Lancaster Synthesis. RGO and carbon nanotubes with different functionalizations were purchased from Times Nano. The properties of different nanostructured carbon samples are summarized in ESI, Tables S1 and S2.†Graphite (325 mesh) was purchased from Aladdin. Potassium permanganate . The solution was then filtered by 0.22 μm syringe filter, and the filtrate was collected for ICP-OES analysis.10 mg of sorbent was placed in a 20 mL glass vial, and then 10 mL of 50 ppm Pb2+ solution was added to the vial and stirred with a magnetic stir bar for 90 minutes. The solution was then filtered by 0.22 μm syringe filter, and the filtrate was collected for ICP-OES analysis.10 mg of sorbent was placed in a 20 mL glass vial. For each vial, 10 mL, 25 ppm, 50 ppm, 100 ppm, 250 ppm, and 500 ppm Pb2+ solution was then allowed to flow through the filter under vacuum. After that, one extra mL of the feeding solution was passed through the filter and was collected for ICP-OES analysis.Laboratory wipes was first cut into 5.5 cm × 5.5 cm pieces. One piece was pressed into a cone with about 5 mm height and pushed into a 1 mL pipette tip. The sorbent was then loaded into the pipette tip. Finally, another piece was pressed into a ball and pushed into the pipette tip covering the sorbent. 50 mL of 10 ppm PbLead ion content in water was determined by Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) measurements using PE Optima 8300 ICP-OES. Scanning electron microscopy (SEM) images were taken by Hitachi S4800. Fourier transform infrared spectroscopy (FTIR) measurements were performed using a PerkinElmer Spectrum Two IR Spectrometer. XPS was performed using an EscaLAB 205 xi XPS system equipped with a Al Kα X-ray source. Brunauer–Emmett–Teller (BET) surface areas of the samples were characterized by a Quantachrome QUADRASORB EVO analyzer at −195.8 °C.3,7,13 The Langmuir model assumes that the adsorption is localized in a monolayer and that there is no interaction among adsorbed species, and describes the adsorbed quantity per quantity of sorbent material qe as:3,13qm is the maximum adsorption capacity, Ce is the equilibrium concentration, and KL is the enthalpy of adsorption.3 This equation can be approximated as:13The experimental data of lead adsorption on different samples are modeled with Freundlich and Langmuir isotherm models.3,7,13KF is a Freundlich constant,3,7 and n is a constant related to isotherm nonlinearity.7Freundlich model is an empirical model which assumes the involvement of different sites with several adsorption energies:r2 values ranging from 0.972 to 0.996. This implies that in these samples, inherent assumptions of the model are fulfilled, i.e. that the monolayer coverage is achieved and that there is no interaction between the adsorbates.13 Obtained qm values indicate that GO-HM, OH-RGO, and especially COOH-RGO samples are very promising for lead adsorption, with obtained qm values of 118.42 mg g−1, 123.76 mg g−1 and 298.49 mg g−1, respectively. Surprisingly, NH-RGO sample shows even worse performance compared to RGO samples, indicating that amino-group functionalization does not enhance lead adsorption on RGO. The obtained values of maximum adsorption capacity fall into the previously reported range for GO between 36 mg g−1 to 659 mg g−1.1 Different from high lead adsorption capacity previously reported for amino-functionalized graphene of 461 mg g−1,4 the lowest maximum adsorption capacity in our work is obtained for NH-RGO samples. However, obtained result is in agreement with a report that GO-NH2 had lower lead adsorption capacity compared to GO.3,30 Furthermore, good fit to Freundlich model is obtained for RGO and GO-HM samples, while the obtained r2 values are lower for RGO samples with different functional groups . Better fit to a Langmuir isotherm  compared to Freundlich isotherm is in agreement with a previous report on the adsorption of metals on GO nanosheets.3,30,38 It can indicate that the adsorption surfaces of OH-RGO, COOH-RGO, and NH-RGO are likely homogeneous, and it also indicates a possible chemical adsorption process,30,38 which would be expected for the interaction between the functional groups and lead ions.Linear fitting to 16 and 29.9 mg g−1), while the obtained value for amino-functionalized MWCNT samples is in good agreement with a previous report for diethylenetriamine-functionalized MWCNTs (58.26 mg g−1).16 It was also previously reported that the maximum adsorption capacity of oxidized MWCNT was 17.5 mg g−1, which was slightly higher than that of MWCNT 16.9 mg g−1.16 Generally, lead adsorption in MWCNTs increases with increased oxygen content.25 This is in agreement with the obtained increase in the maximum adsorption capacity for higher OH-group content of MWCNT samples, so that the performance of 5.5% OHCNT is comparable to that of NHCNT samples.In the case of MWCNT, we can observe that good fit to both adsorption isotherm models is obtained for all the samples except MWCNT and COOH-CNT. The CNT-based samples in general exhibit rather poor dispersion in water, which is the likely reason for inferior fit of the data despite stirring of the solution. RGO-based and GO-HM samples exhibit significantly better dispersion in water, as shown in Fig. S4 and S5.3,7,13,16,21,25,38k2 is the rate constant of the adsorption, and qe is the equilibrium adsorption capacity, qt is adsorption capacity at a time, and t is time. For all the samples, excellent fits to the pseudo-second order kinetic model are obtained, as shown in Adsorption kinetics, which provides information on the adsorbate uptake rate, was also studied. Pseudo-second order kinetic model is commonly used to describe kinetic data for lead adsorption on nanostructured carbon samples, and it is given by a following equation:−1 due to OH groups,13,23,29,38 peaks attributed to –CH2 and CH3 vibrations at 2948 cm−1 and 2848 cm−1,7,23,29 and 1716–1726 cm−1 C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O vibration in COO–.8,20,29,38 Other commonly observed peaks in nanostructured carbon (CNT or GO) include vibrations attributed to CC and CO carbonyl groups 1620–1634 cm−1,8,20,29,38 C–O–C and C–O groups at 1217–1220 cm−1 and 1047–1050 cm−1,8,20,38 C–O stretching 1097 cm−1,29 1373 cm−1 C–O carboxyl vibration,8 deformation of C–H bond at 1450 cm−1,7 and tertiary C–OH groups deformation at ∼1400 cm−1.38 Samples containing amine groups may also exhibit amide carbonyl stretching at 1650 cm−1,29 1580 cm−1 and 1180 cm−1 N–H in plane and C–N bond stretching,29 1117 and 1052 cm−1 C–N bond stretching,7 and 1630 and 1587 cm−1 bending vibrations of N–H.7Thus, we can consistently observe worsening of the lead adsorption for RGO samples with amino-functionalization, while the lead adsorption is significantly improved in amino-functionalized MWCNT. To investigate the adsorption mechanisms in more detail, FTIR measurements have been performed, and the obtained results are shown in 16 For example, the interactions with carboxyl group can lead to the changes in intensity of the peaks at 1726 cm−1 and 1260 cm−1,16 as well as peaks at 1635 cm−1 and 1384 cm−1 which correspond to asymmetric and symmetric COO– vibrations.16 Changes in the peak at 1097 cm−1 corresponding to phenol group are also sometimes observed.16 In addition to the interaction with carboxyl group, lead adsorption can result in the shifts of stretching vibrations of amino- and hydroxyl groups.12 In amino-group functionalized samples, shifts of the peaks at 1650 cm−1 and 1580 cm−1 corresponding to amide I and N–H in plane stretching have been reported, as well as changes in the intensity of bands corresponding to C–N stretching and out-of-plane NH2 bending mode at 1180 cm−1 and 800 cm−1.16 It was previously reported that the lead adsorption resulted in the reduction if C–O stretching vibration at 1280 cm−1, as well as 1647 cm−1 peak in 1640–1750 cm−1 band corresponding to CO vibrations, while hydroxyl peak did not exhibit significant changes, indicating stronger interaction between lead and carboxylic and/or carbonyl groups compared to hydroxyl groups.9In general, the comparisons of FTIR spectra of samples before and after lead adsorption can result in observations of changes in peak intensity, peak shifts and the appearance or disappearance of peaks.−1, and such an increase is considerably more pronounced in OH-CNT 5.5% sample, indicating that in these samples there is an interaction between lead ions and hydroxyl groups, resulting in an increase of vibration attributed to C–OH groups deformation at ∼1400 cm−1.38 This is consistent with physisorption16 being dominant mechanism on CNT-based samples, with the exception of those containing –OH groups where chemisorption related to hydroxyl groups can be observed. In NH-CNT samples, no significant changes are observed, other than small increase in the peak close to ∼1400 cm−1.The obtained FTIR spectra are shown in −1 and ∼1200 cm−1, which can be attributed to carbonyl and C–O vibrations.8,20,29,38 In OH-RGO, NH-RGO, and GO-HM samples, we observe an increase corresponding to C–OH groups deformation at ∼1400 cm−1,38 while NH-RGO also exhibits an increase in the feature at ∼1160 cm−1 which can be attributed to C–N stretching.16 A prominent increase in the wavenumber range of 1050–1100 cm−1 is observed for GO-HM samples, which is probably assigned to C–O groups. Thus, we can observe that amine functionalization in RGO samples results in an obvious interaction between lead ions and the amino groups, while no such feature is observed in NH-CNT samples. Thus, the adsorption mechanism of lead ions on functionalized carbon nanostructures is dependent not only on the functional group but also on the morphology of the nanocarbon, which may affect the accessibility of the functional groups (for example carboxyl and hydroxyl groups at the edges of GO sheets were found to interact strongly with lead ions38). Similar trends in terms of the effect of nanocarbon morphology, i.e. improved adsorption performance of samples containing GO compared to those with CNTs, were previously observed in Cr(vi) removal, although the mechanism has not been clarified.50On the other hand, in GO-based samples exhibiting high performance, such as COOH-GO we can observe a more significant change in the FTIR spectra, with prominent increase in features at ∼1600 cmC bonding, 285.1–285.4 eV to C–C bonding, 285.8–286.4 eV to C–O bonding, 286.8–287.6 eV to CO bonding, and 288.8–289.5 eV to O–CO bonding.8,14,20,27,30,34,39,55–60 For O 1s peak, peak at 533.1–533.6 eV corresponds to C–OH bonding, while peak at 530.6–532.4 eV corresponds to carboxyl (–COO) and carbonyl (CO) bondings.27,30,37,56–58 In addition, Pb signal was detected in all the samples after the lead adsorption . Despite the fact that COOH-RGO exhibited the highest lead adsorption capacity based on Langmuir model isotherm fitting, no distinct mechanisms compared to other COOH- and OH-containing samples can be observed. Thus, we can conclude that the presence of oxygen containing functional groups enhances the lead adsorption performance of all nanostructured carbon samples, with the degree of enhancement dependent on the oxygen content and sample geometry, as well as how well the samples can be dispersed in water. It should also be noted that while in CNT-based samples some correlation can be observed between BET surface area and lead adsorption, in RGO/GO-based samples there is a complete lack of correlation between the BET surface area and lead adsorption performance. Thus, we can conclude that BET surface area measurement performed based on nitrogen gas adsorption is not necessarily a good predictor of the metal ion adsorption performance in solution. It should also be noted that XPS revealed differences in behavior of NHCNT and NH-RGO samples, in agreement with FTIR results. In NHCNT samples, the N 1s peak at 399.6 eV corresponding to amino group30 disappears after lead adsorption, while the peak at 285.3–285.4 eV corresponding to C–N bonding30,59 can still be observed. In NH-RGO samples, N 1s signal can be observed both before and after lead adsorption, with small changes in peak components corresponding to imino- and amino-groups at ∼398.7 eV and ∼399.7 eV.30,33To obtain further insights into the lead adsorption process, XPS measurements have been conducted. The obtained results are summarized in −1). In the case of RGO, the best performance was obtained by COOH-RGO with maximum lead adsorption capacity of 298.49 mg g−1, while OH-RGO and GO samples synthesized by modified Hummers' method exhibited similar performance with maximum lead adsorption capacities of 123.76 mg g−1 and 118.42 mg g−1, respectively.We investigated the lead adsorption for different types of nanostructured carbon  and different functional groups . We found that while amino-group functionalization significantly enhanced lead adsorption in MWCNT samples, it resulted in deterioration of the performance of RGO. On the other hand, hydroxyl and carboxyl group functionalization resulted in improved performance regardless of the starting form of carbon. The lead adsorption performance of MWCNT improved with increased number of hydroxyl functional groups, and for higher –OH content approached that of the best performing amino-functionalized samples (58.547 mg gThere are no conflicts to declare.RA-008-C8RA02264J-s001


Plant height is a key factor in the determination of rice yield since excessive height can easily cause lodging and reduce yield. Therefore, the identification and analysis of plant height-related genes to elucidate their physiological, biochemical, and molecular mechanisms have significant implications for rice breeding and production.qPH9), which was mapped to a 2.02-Mb region of Chromosome 9. Local QTL mapping, which was conducted using 13 single nucleotide polymorphism (SNP)-based Kompetitive allele-specific PCR (KASP) markers for the qPH9 region, and traditional linkage analysis, facilitated the localization of qPH9 to a 126-kb region that contained 15 genes. Subsequent haplotype and sequence analyses indicated that OsPH9 was the most probable candidate gene for plant height at this locus, and functional analysis of osph9 CRISPR/Cas9-generated OsPH9 knockout mutants supported this conclusion.High-throughput quantitative trait locus (QTL) sequencing analysis of a 638-individual F2:3 mapping population resulted in the identification of a novel height-related QTL (OsPH9 was identified as a novel regulatory gene associated with plant height in rice, along with a height-reducing allele in ‘Dongfu-114’ rice, thereby representing an important molecular target for rice improvement. The findings of the present study are expected to spur the investigation of genetic mechanisms underlying rice plant height and further the improvement of rice plant height through marker-assisted selection.The online version contains supplementary material available at 10.1186/s12284-022-00576-2. Plant height is a key factor in determining rice yield since excessive height can easily cause lodging and reduce yield. As such, the breeding and large-scale promotion of semi-dwarf rice varieties, which has been conducted since the 1950s, has increased rice yield by 20–30% and has, accordingly, been hailed as the "green revolution" in rice production. The first major breakthrough was attributed to the successful application of a semi-dwarf gene , abscisic acid (ABA), and various brassinosteroids  was subjected to BSA-seq analysis and fine-mapping strategy, along with haplotype and sequence analysis, to identify potential candidate genes. Further, CRISPR/Cas9 gene editing was used to develop a knockout mutant to assess the function of the putative height-related genes.2:3 population of 638 individuals. In the spring of 2019, ‘DF114’ (n = 48), ‘LY11’ (n = 48), and F2:3 (n = 638) individuals were planted in four rows under natural conditions in paddy fields at Acheng Experimental Station , and 5 plants from the center of each plot were selected for plant height evaluation.The japonica varieties ‘Dongfu 114’ (‘DF114’) and ‘Longyang 11’ (‘LY11’) were obtained from Northeast Agriculture University  and used as female and male parents, respectively, to generate an F2:3 individuals were collected separately for total genomic DNA extraction, which was performed using the Cetyltrimethylammonium bromide (CTAB) method , with minor modifications, and the isolated DNA was quantified using a Nanodrop 2000 spectrophotometer . After quantification, a Qubit 2.0 fluorimeter  was used to pool equimolar amounts of genomic DNA from the 30 shortest and 30 tallest individuals .All flag leaves of the 638 F‘mem -M’ setting, where -M is an option used to mark shorter split-alignment hits as secondary alignments. Variant calling was performed using the GATK Unified Genotyper , and both SNPs and indels were filtered using the GATK Variant Filtration function with proper standards . All mutations for genes, functions, and genomic regions were annotated using ANNOVAR . The resulting libraries were sequenced by Genedenovo  using the HiSeq X10 NGS platform (Illumina). To achieve quality trimming, thereby ensuring high-confidence variant calling, raw reads were filtered by removing reads with ≥ 10% unidentified nucleotides, with > 50% bases with shared quality scores of ≤ 20, or aligned to the barcode adapter. To identify SNPs and indels, filtered reads were aligned to the ‘Nipponbare’ reference genome sequence . The Logarithm of the odds (LOD) score threshold for confirming a significant QTL was established using a permutation test with 1000 replicates and a significant level of P < 0.01.KASP markers were designed using Premier 5.0 CRISPR/Cas9 gene-editing vector construction was conducted as described by Li et al. . Two tarhttp://www.r-project.org/) and Origin 2018 .Differences between parent and progeny plant height were detected using SPSS18.0 . Data represent means ± standard deviations. Graphs were drawn using edgeR  peaks in the ∆(SNP-Index) distribution spanned 12.14-Mb (4.78–16.92 Mb) and 5.98-Mb (11.82–17.80 Mb) intervals (qPH7 and qPH9) on chromosomes 7 and 9, respectively, and significant peaks in G-value distribution completely covered the results of ∆(SNP-Index) analysis, whereas those in the ED and Fisher’s exact test P-value distributions completely covered the interval on Chromosome 9 alone. Furthermore, only the region identified in the Fisher’s exact test P-value distribution (2.02 Mb of qPH9) was included in the intersection of genome regions identified by all four methods. It is also worth noting that, in the Δ(SNP-Index) and ED value distributions, the peak values observed for qPH9 were higher than those observed for qPH7, resulted in the identification of 15 candidate genes , 15,915,683 (A > G/R), 15,915,701 (T > G/K), 15,915,717C (C > T) and 15,915,782 (T > C) in the promoter region, and 15,916,129 (C > T), 15,916,186 (C > A/M) and 15,916,244 (T > C/-) in the CDs region, respectively. A total of 11 haplotypes were obtained based on the different combinations of 8 SNPs. As shown in Fig. OsPH9 is closely related to the change in plant height. Among them, 15,916,244 (T > C) is consistent with resequencing results , was conducted using the CRISPR/Cas9-generated osph9-mutant lines (A467-3 and A467-12) in which the motifs GGG and GC were deleted at the 26 and 247 bp of the CDs region, respectively  leading to varying degrees of dwarfing  in a large rice population with an effect equivalent to that of SD1. In the present study, BSA-seq was used to identify a major QTL (qPH9), and regional linkage mapping analysis was used to rapidly reduce the implicated interval, which was responsible for 20.50% of plant height variation, from 2.02 Mb to 126 kb. Interestingly, previous studies have identified plant height loci that are either near or inclusive of qPH9, and qPH9 also overlaps with qSL28-9 and qPH9-1 (RZ422-RZ12), which are 3.49 and 7.62 Mb intervals linked to plant height  and 295 japonica cultivars from northeast China to analyze the allelic variation in SD1, which further enriched the allele types related to SD1 plant height. This study found that there are multiple haplotypes in OsPH9 in the 3 k germplasm resources and are closely related to rice plant height. It can be inferred that the mutation of OsPH9 in CDs region 15,916,244 (T > C) is the main cause of decrease in ‘DF114’ plant height.During the past decade, genotype datasets for many rice accessions have been released and used to identify several loci associated with important agronomic traits  are very similar, especially H3 and H4 histones  related with the height of a mature rice plant. Experimental verification by haplotype and sequence analysis confirmed OsPH9 as a candidate gene of the qPH9 region, and the function of OsPH9 was further verified using gene-editing technology. Although further work is needed to elucidate the mechanism underlying the role of OsPH9 in plant height, the present study provides resources for breeding programs aimed at improving rice plant height.Investigating the genetic basis of plant height is important for breeding purposes. In the present study, BSA-seq and linkage-map analysis were used to identify a QTL  and Indels detected in the samples.  Table S3: Statistics of variation in the 2.02 Mb interval of qPH9 based on resequencing data.  Table S4: Primers used for the 13 KASP markers.  Table S5: Further mapping information for qPH9 using the 13 KSAP markers.  Table S6: Annotated genes within the 126-Kb interval.  Table S7: Haplotype and plant height difference of Os09g0433600.  Table S8: Haplotype and plant height difference of chr9:15916244 T.  Table S9: Information on 26 germplasm resources of Hap6 haplotypes ofOs09g0433600.Additional file 2. Figure S1: The sequence information of DEP1 in ‘Dongfu 114’ and ‘Longyang 11’.Additional file 3. Figure S2: The sequence information of Os09g0433600 in ‘Dongfu 114’ and ‘Longyang 11’.


The scarcity of livers for transplantation is rising, and new strategies to extend the donor pool are being explored. One solution is to use marginal grafts from extended criteria donors, presenting, for example, liver steatosis. As current preservation solutions  were mainly designed for static cold storage (SCS) only, IGL-2, a modified version of IGL-1, was developed to be suitable for SCS and dynamic preservation, such as hypothermic oxygenated perfusion (HOPE). In this study, we investigated the combined effect of IGL-2, SCS, and HOPE and compared it to the most used preservation solution (UW and Belzer MPS). Four experimental groups with six rats each were designed using Zucker rats. All groups underwent 24 h of SCS (in IGL-2 or UW) + 2 h of normothermic machine perfusion (NMP) at 37 °C to mimic transplantation. HOPE (IGL-2 or Belzer MPS) was performed before NMP on half of the rats. The IGL-2 group demonstrated lower transaminases and a significantly low level of glycocalyx proteins, CASP3, and HMGB1 in the perfusates. These data suggest the protective role of IGL-2 for fatty livers in preserving the endothelial glycocalyx, apoptosis, and inflammation. Surgeons and researchers have explored new strategies to expand the pool of donors and shorten the patient waitlist for liver transplantation . One of + and low Na+) and the presence of hydroxyethyl starch (HES), an oncotic agent. Even though the efficacy of HES in the reduction of edema has been widely demonstrated in multiple organs  rats, aged 9–10 weeks, were purchased . The rats were housed in separate cages with a closed ventilation system, two rats per cage, in a room with controlled humidity and temperature and a 12 h light cycle. Water and food were provided ad libitum. The rats presented a rate of steatosis between 5% and 50%. Rats were chosen randomly.®, Fresenius, France). Before procurement, the liver was flushed with 20 mL of PS through the portal vein at 5 °C ± 3 °C.Liver procurement was performed in a sterile room, under general anesthesia, through a silicon mask at 2 L/min airflow and isoflurane at 4% for induction and then at 2% for maintenance. The gastrointestinal tract was covered with wet gauze through a midline incision and placed to the left. The hepatic ligaments were cut on the left section of the diaphragm, and then the right suprarenal vein was closed with 8–0 non-absorbable polypropylene sutures. Heparin (0.3 UI/gr) was injected intravenously. The liver was rinsed through the abdominal aorta with 50 mL of preservation solution (PS) using a syringe pump , and the gold standard of dynamic preservation, PERF-GEN® . BEL-GEN and PER-GEN have the same composition as the original ones developed at the University of Wisconsin by F.O. Belzer and J. Southard, respectively, named UW and UW-MPS.Four experimental groups (n = 6/group) were designed  to compaFor static preservations groups, livers were preserved for 24 h in static cold storage (SCS) using IGL-2 (IGL2.SCS) or BEL-GEN (BELGEN.SCS) at 5 °C ± 3 °C. For dynamic preservation groups, livers underwent 2 h of hypothermic oxygenated perfusion (HOPE) at 5 °C ± 3 °C using IGL-2 (IGL2.HOPE) or PERFGEN (PERFGEN.HOPE) after 24 h of SCS at 5 °C ± 3 °C.All livers were rinsed with 20 mL of Ringer lactate through the portal vein cannula to meet the clinical practice. To mimic liver transplantation, all livers underwent 2 h of normothermic oxygenated perfusion (NMP) using a perfusate composition previously described in Berendsen et al. . The perHypothermic machine perfusion was performed according to previous studies using an in-house machine perfusion system ,59. We u® Chemistry Systems AST and ALT reagents.Analysis of perfusate alanine aminotransferase (ALT)-aspartate aminotransferase (AST) was performed at the clinical laboratory at our institution using Siemens Healthcare Diagnostics-ADVIAQuantification of syndecan-1 , heparan sulphate proteoglycan , high mobility group protein B1 , and active caspase 3  in perfusates were obtained according to the manufacturer’s instructions. Optical density at 450 nm was quantified using a Modulus II Microplate Reader .Liver tissues were fixed in 10% paraformaldehyde and 3 μm sections were stained with haematoxylin-eosin saffron (HES) according to standard procedures. The damage grade score was based on necrosis and coagulation, acute necrosis, disorganization, granular precipitate, sinusoidal dilatation, eosinophilic deposits, number of cells remaining in vessels, and inflammation. An expert pathologist carefully measured each component and each was given a mark between 0–3 . Histologic slides were scanned using Lamina  and analyzed with CaseViewer 1.4 software .p < 0.05 was considered as significant. .Data from the damage grade scores were compared statistically using a nonparametric one-way ANOVA test  with Dunn’s post hoc analysis. All data are expressed as means (± SD or ± SEM). Data were compared statistically using multiple pairwise t-tests .


Elaeis guineensis Jacq.) production. As oil palm breeding takes a long time, molecular markers of genes related to drought tolerance characteristics were developed for effective selection. Two methods of gene identification associated with drought, differential display reverse transcription polymerase chain reaction (DDRT-PCR) and pyrosequencing platform, were conducted before developing the EST-SSR marker. By DDRT-PCR, fourteen out of twenty-four primer combinations yielded the polymorphism in leaf as 77.66% and root as 96.09%, respectively. BLASTN and BLASTX revealed nucleotides from 8 out of 236 different banding similarities to genes associated with drought stress. Five out of eight genes gave a similarity with our pyrosequencing sequencing database. Furthermore, pyrosequencing analysis of two oil palm libraries, drought-tolerant, and drought sensitive, found 117 proteins associated with drought tolerance. Thirteen out of sixty EST-SSR primers could be distinguished in 119 oil palm parents in our breeding program. All of our found genes revealed an ability to develop as a molecular marker for drought tolerance. However, the function of the validated genes on drought response in oil palm must be evaluated.Drought is a major constraint in oil palm ( Elaeis guineensis) is a perennial monocotyledonous crop plant producing the most oil in the world; its production is equal to 10 t ha-1 or 13 t ha-1 year-1 palm oil under favorable conditions [Oil palm  and single nucleotide polymorphisms (SNPs) . PyroseqMany efforts have elucidated the mechanisms of drought tolerance in plants through genomics approaches, and several genes associated with drought stress have been reported ,18,19,20Phoenix dactylifera, Elaeis guineensis, and others at 64%, 16%, and 20%, respectively. Eight out of twenty-five sequences had similarities to genes associated with stress conditions  and 18.82%  could be translated to protein, respectively. The mapped proteins were both associated and non-associated with the dehydrated condition. After filtrate, 117 proteins were associated with drought tolerance, which could be separated into 11 biochemical groups as follows: (1) abscisic acid, (2) antioxidant, (3) compatible compound, (4) ethylene, (5) heat shock protein, (6) helicase, (7) mannitol, (8) proline, (9) senescence/ripening, (10) transcription factor, and (11) trehalose .−5 to 1 × 10−50 (37%), followed by 1 × 10−51 to 1 × 10−100 (35%), and 1 × 10−151 to 1 × 10−180 (11%) (Elaeis guineensis), followed by date palm (Phoenix dactylifera) at 4%, and the other at 4%, respectively  a. The nuectively b. Gene Oectively . http://pantherdb.org/, accessed on 28 May 2020) also found the largest functional group of thioredoxin (PTHR10438), similar to the analysis with InterPro protein signature databases.Further, functional analysis of proteins obtained from the InterPro Scan technique and InterPro protein signature databases found 142 genes out of 192 protein functions. The twenty top-hit were the proteins in domain function. Thioredoxin-like fold (domain) (IPR012336) was the highest gene (five genes), followed by thioredoxin domain (domain) (IPR013766) (three genes), AP2/ERF domain (domain) (IPR016177) (three genes), and DNA-binding domain (domain) (IPR015943) (three genes), respectively . The anaMoreover, KEGG analysis involved 11 major metabolic pathways, 31 secondary pathways, and 15 enzymes. Lipid metabolism was the highest number of enzymes and the highest number of genes , followed by amino acid metabolism  and carbohydrate metabolism  .All eight genes were mapped with both MPOB and NCBI databases. However, five of eight genes from DDRT-PCR were mapped with a transcriptome sequencing database. They contained histone H2A (99% similarity), cysteine proteinase (Cys) (100% similarity), pentatricopeptide repeat-containing protein (32% similarity), trehalose-6-phosphate synthase (87.5% similarity), and serine/threonine-protein phosphatase PP1 (92% similarity) . NeverthOne hundred and forty-two genes were used for sequence distribution analysis for molecular maker development via the EST-SSR technique. The highest and the lowest repetitions were 3-base and 5-base, respectively . Then, 1Moreover, 23 primers were tested with 119 oil palm parents in our breeding program. All primers were able to distinguish between drought-tolerant and drought-sensitive oil palms. The genetic correlation of oil palm parents had a coefficient of 0.57, divided oil palm samples into five groups with three independent samples  A. The oiArabidopsis, and it was found that the overexpression of the H2A variant gene TaH2A.7 from wheat significantly enhanced drought tolerance [Ruta graveolens [The identification of the drought-tolerant gene in oil palm via DDRT-PCR and pyrosequencing technology was conducted in this study. DDRT-PCR revealed polymorphisms at cDNA levels in assuming drought at different time periods when compared between the leaf and root of four oil palm varieties with twenty-four primer combinations. Meanwhile, the different periods of drought stress generated genetic changes among the tissue and oil palm varieties. However, pyrosequencing technology provided more details of the genes involved in the drought trial between the four oil palm varieties, tolerance and sensitivity, and the analysis of the different gene expressions between both trials, resulting in interesting genes that could be used for molecular marker development. By using these two techniques, 8 genes from DDRT-PCR and 117 proteins from pyrosequencing were associated with drought tolerance. By these, five of eight genes from DDRT-PCR were mapped with the gene in the pyrosequencing database . These eolerance . The supolerance . The expolerance ,25,26, aolerance . The greolerance ,29,30. Solerance . The basolerance . The forolerance . ATP-depolerance ,35,36, eolerance . There iolerance ,39. Fromolerance , chickpeolerance , corn [4olerance , rice [4olerance , and Rutaveolens . FurtherCapsicum annuum L.). Still, the total sequence data were longer than pepper [E. guineensis WRYK transcription factors, including EgWRKY07, 26, 40, 52, 59, 73, and 81, which displayed multiple roles in the drought response of the oil palm seedling leaf [E. guineensis, pisifera), EgWRKY07 and EgWRKY52, with high similarity to AtWRKY30 in Arabidopsis, it was reported that they are strongly upregulated to salinity stress [For 454 pyrosequencing, two sstDNA libraries were shorter than the results obtained from pepper . Interestingly, this gene had no previous report on plant species’ drought tolerance or abiotic response. A further study must be conducted on this gene’s abiotic response. Additionally, 13 primers tested with 10-year-old oil palm parents in our breeding program revealed a close genetic relation to Lamé. Thirty percent of oil palm samples in this group demonstrated bunch production collaboration with drought tolerance markers. Moreover, drought stress is controlled by genetics, the environment, and interaction between the two with plants of different ages. Thus, some markers obtained from our study could not accurately determine drought stress for qualitative characteristics. Further, our new finding genes and more molecular markers must be determined.For EST-SSR, 142 genes were screened. Because 1 gene consisted of 2 to 3 isotigs, 125 positions obtained from 142 genes were used for primer design. Among the 60 primers designed, most tri-nucleotides revealed the most frequent type of SSR motif. This finding aligns with the results reported in cereals , A. mongngolicus , and C. . annuum . Five ouFour oil palm commercial varieties at 12 months old, Lamé, Surathani 2 (Deli × Lamé), Ghana, and Surathani 1 , were grown in composite soil  in 200L fiberglass tank in the greenhouse at 28 ± 2 °C with the relative humidity nearly 80% at Walailak University, Nakhon Si Thammarat, Thailand. The oil palm variety, Lamé, is known for its drought tolerance. In contrast, the Ghana variety is known for its drought sensitivity, both obtained from CPT-Agrotech Co., LTD, Chumporn province, Thailand. The oil palm variety Surathani 1 is expected to be well-watering. At the same time, the variety Surathani 2 is known for its drought tolerance. Both were obtained from Surathani Oil Palm Research Center, Surathani province, Thailand. Well-watering oil palm plants conducted water stress until the tensiometer reached 0 centibars, then irrigation stopped until 90 days. This experiment used five oil palm plants for irrigation and no irrigation.On the contrary, for control, well-watering was performed to keep the tensiometer at 45 centibars for 90 days. For DDRT-PCR, leaf and root tissue were harvested at 15-day intervals until 90 days. Leaf samples were collected from the second leaf stalk. Conversely, root samples were collected from the adventitious root at 15–20 cm depth from the soil. For the pyrosequencing study, the second leaf of Lamé and Surathani 1 at 45 days after dehydration was collected for RNA extraction. All samples were immediately placed in liquid nitrogen and kept at −80 °C until usage.The total RNA at different dehydration times was extracted according to the instructions of the Total RNA Mini Kit Plant . The number of replications was three. The successful RNA extraction was confirmed with Eppendorf BioPhotometer Plus Model #6132  and 1% agarose gel electrophoresis. Then, RNA was transcribed to cDNA using a Transcriptor High Fidelity cDNA Synthesis Kit , followed by the instructions. A total of 20 μL cDNA synthetic reaction consisted of 2 μL total RNA (2 μg), 1.0 μL oligo (dT) primer (2.5 μmolar), 4.0 μL reaction buffer, 0.5 μL protector RNase inhibitor (40 unit/μL), 2.0 μL deoxynucleotide mix (dNTP), 1.0 μL reverse transcriptase, and 8.5 μL sterile dH2O. The reaction was performed in Eppendorf  by incubating at 65 °C for 10 min, followed by quick chilling on ice until it cools off, then continuous incubating at 45 °C for 30 min, 85 °C for 5 min, and terminated by heating at 95 °C for 5 min. All cDNA samples were stored at −80 °C until further use. For qualitative examination, the obtained cDNA was amplified with 18 S rRNA under the following conditions: 95 °C/4 min, 95 °C/30 s, 55 °C/30 s, 75 °C/1 min for 35 cycles, 75 °C/7 min, and 4 °C before running on 1.5% agarose gel.http://www.ncbi.nlm.nih.gov/BLAST/, accessed on 16 June 2019). Sequence alignments were performed with CLUSTAL_X.Differential Display RT-PCR (DDRT) was performed as described by  with ming, 4 °C for 45 min; the supernatant was put into an ultracentrifuge tube, 5.7 M cesium chloride was added and centrifuged in an ultracentrifuge  at 25,000 rpm, 20 °C for 20 hr. After discarding the supernatant, the pellet was washed with 70% ethanol, dried at room temperature for 1 h, dissolved with 200 ul RNase-free water, put on ice for 1 h, and homogenized regularly. The total RNA was transferred into a new 1.5 mL tube and kept at −80 °C before usage. The mRNA was removed from the extracted RNA via PolyATtract® mRNA Isolation System I and II  following the manual’s instructions.The total RNA was extracted from the leaf tissue of each sample at 45 days to monitor the molecular response of water deficiency. A 3–5 g leaf was ground in liquid nitrogen before putting the fine powder into a 50 mL tube, 25 mL extraction buffer  was added and heavily shaken before being kept in ice for 10 min. The mixture was centrifuged at 4000× http://www.cmbi.ru.nl/cdd/biovenn/, accessed on 6 June 2020) and area-proportional Venn diagrams [The GS- FLX Titanium, the 454 Life Sciences sequencing platform , discovered the nucleotide sequence of an interested mRNA. RNA-sequence data from each library were assembled with 454/Roche’s newbler v2.6. The water stress variety (Lamé) assembly library was compared with the water stress sensitive (Surathani 1) library. Then, the combined group of both samples was annotated using Blast2GO against the plant non-redundant database. Gene Ontology distribution was assigned using the Blast2GO function  and redudiagrams  were usediagrams .http://bioinfo.ut.ee/primer30.4.0/, accessed on 8 November 2020) and Oligo-Nucleotide Properties Calculator . The primer was designed as 18–23 bp with the PCR product size of 200–300 bp, 40–60% CG content, and 50–70 °C salt adjusted with both forward (F) and reverse (R). The amplified reaction of 10 μL contained 1.0 μL cDNA (diluted 10 times), 5 μL Toptaq buffer, 0.25 μL oligo (dT) primer (1.0 μmolar), 0.25 μL arbitrary primer (1.0 μmolar), and 3.5 μL sterile dH2O. Gene expression was performed via Real-Time PCR  by using EvaGreeen® Super Mix  as a supermix. 18 S rRNA was used as a reference gene. The RNA from the leaf and root at 0, 15, 30, 45, 60, 75, and 90 days after dehydration was used. The PCR condition was as follows; 1 cycle at 95 °C/3 min, 95°C/30 s, 60 °C/30 s for 35 cycles, 72 °C/7 min, and keep at 4 °C. The triplet replication was used with 18 s rRNA as a reference with the standard DNA as 10−1, 10−2, 10−3, and 10−4 times. A melting curve was used to confirm the PCR product. Expression data were analyzed as cited [The nucleotide sequence from BLASTx of DDRT was aligned with the isotigs from the pyrosequencing and searched against NCBI and MPOB databases before being selected for the RT-PCR study. Primers of those selected genes were designed using Primer3 Input . The repeated sequences were compared for primer design, and genes were selected only in drought-tolerant libraries. Sixty primer pairs were designed from 40 sequences of replicated genes using Primer3 Plus and the Oligonucleotide Properties Calculator program. After scanning PCR conditions with Surathani 2, the primers were amplified with 119 oil palm parents under a CPI Agrotech Co., Ltd., Thailand breeding project. The volume of all primer reactions was set as 10 μL; 2 μL DNA, 5 μL Toptaq buffer, 0.25 μL primer forward, 0.25 μL primer reverse, and 2.5 μL sterile dH2O. Gradient temperatures annealing were performed for suitable PCR condition: 94 °C/3 s, 94 °C/30 s, 40 cycles at 40–60 °C/30 s, 72 °C/60 s, and 72 °C/7 min before being kept at 4 °C. The PCR products were run in 1% agarose gel for 30 min and dyed with ethidium bromide before being counted and calculated to distinguish the genetic diversity of all oil palm samples.A repeating base sequence was searched from the pyrosequencing database for the SSR marker. The expressed sequence tag (EST) under dehydrated conditions of the drought-tolerant and drought-sensitive library was set for repetition base as two bases (10 replications), three bases (6 replications), four bases (5 repetitions), five bases (4 repetitions), and six bases (3 repetitions) via WebSat (E. guineensis), similar to primers isotig03937, are engaging for further study in response to drought and abiotic stress in oil palm. Moreover, 5 of 60 EST-SSR primers were identified as drought-tolerant and drought-sensitive varieties. Although 23 primers tested with 119 oil palm parents could separate the oil palm sample into 5 groups with Group 2 and Subgroup 2, genetically closely related to the drought tolerance sample, only 30% of brunch production in these groups responded to our marker. In parallel, identifying target gene expression using DDRT-PCR and 454 sequencing technology is considered a potential procedure for gene selection in improving the tolerance of oil palm trees. Our result might be the basis for an in-depth genomics study of oil palm for selecting genes for drought tolerance.Drought is one of the natural phenomena affecting yield in oil palm, considered a quantitative trait involving the participation of a complex set of genes. In this study, we produced some new transcriptomic information for oil palm drought stress via DDRT-PCR and 454 sequencing technology for further marker development in our oil palm breeding program. DDRT-PCR exposed eight genes associated with drought stress in plants. In contrast, the pyrosequencing of two libraries, drought-tolerant and drought-sensitive varieties, revealed 117 proteins related to drought tolerance. The drought tolerance library had a repeated sequence of 142 genes for annotation with GO ontology, InterPro Scan technique, InterPro protein signature database analysis, and KEGG analysis. Five of eight genes, namely histone H2A, cysteine proteinase, pentatricopeptide repeat-containing protein, trehalose-6-phosphate synthase, and serine/threonine-protein phosphatase PP1, from DDRT-PCR were mapped with transcriptome sequencing database. In contrast, bHLH106, ATP-dependent DNA helicase PIF1-like, and probable LRR receptor-like serine/threonine-protein kinase At3g47570 were found in DDRT-PCR only. Notably, the WRKY transcription factor in our pyrosequencing data differed from that reported in oil palm seedlings under drought stress. These genes, together with spliceosome-associated protein 130 A (


DCT]) rises, reduces the ionized calcium concentration in that segment ([Ca++]DCT), and thereby induces increased secretion of parathyroid hormone (PTH) to maintain normal calcium reabsorption. In patients with CKD, we previously documented correlations between [PTH] and phosphate excreted per volume of filtrate (EP/Ccr), a surrogate for [P]DCT. In the present investigation, we estimated [P]DCT from physiologic considerations and measurements of phosphaturia, and sought evidence for a specific chemical phenomenon by which increased [P]DCT could lower [Ca++]DCT and raise [PTH].Secondary hyperparathyroidism (SHPT) complicates advanced chronic kidney disease (CKD) and causes skeletal and other morbidity. In animal models of CKD, SHPT was prevented and reversed by reduction of dietary phosphate in proportion to GFR, but the phenomena underlying these observations are not understood. The tradeoff-in-the-nephron hypothesis states that as GFR falls, the phosphate concentration in the distal convoluted tubule ([P]2 (mean 29.9 ± 9.5) and 27 controls (“CTRL”) with eGFR > 60 mL/min/1.73m2 (mean 86.2 ± 10.2). In each subject, total [Ca]DCT and [P]DCT were deduced from relevant laboratory data. The Joint Expert Speciation System (JESS) was used to calculate [Ca++]DCT and concentrations of related chemical species under the assumption that a solid phase of amorphous calcium phosphate (Ca3(PO4)2 ) could precipitate. Regressions of [PTH] on eGFR, [P]DCT, and [Ca++]DCT were then examined. At filtrate pH of 6.8 and 7.0, [P]DCT was found to be the sole determinant of [Ca++]DCT, and precipitation of Ca3(PO4)2  appeared to mediate this result. At pH 6.6, total [Ca]DCT was the principal determinant of [Ca++]DCT, [P]DCT was a minor determinant, and precipitation of Ca3(PO4)2  was predicted in no CKD and five CTRL. In CKD, at all three pH values, [PTH] varied directly with [P]DCT and inversely with [Ca++]DCT, and a reduced [Ca++]DCT was identified at which [PTH] rose unequivocally. Relationships of [PTH] to [Ca++]DCT and to eGFR resembled each other closely.We studied 28 patients (“CKD”) with eGFR of 14–49 mL/min/1.73mDCT increases, chemical speciation calculations predict reduction of [Ca++]DCT through precipitation of Ca3(PO4)2 . [PTH] appears to rise unequivocally if [Ca++]DCT falls sufficiently. These results support the tradeoff-in-the-nephron hypothesis, and they explain why proportional phosphate restriction prevented and reversed SHPT in experimental CKD. Whether equally stringent treatment can be as efficacious in humans warrants investigation.As [P] The parathyroid hormone concentration ([PTH]) rises as the glomerular filtration rate (GFR) falls in patients with chronic kidney disease (CKD) . This phDCT), where PTH regulates reabsorption of ionized Ca (Ca++) [DCT reduces [Ca++]DCT, and [PTH] rises to maintain Ca++ reabsorption at a rate compatible with normocalcemia DCT if fractional delivery of filtrate to the DCT is assigned a constant value  and EP/Ccr were demonstrated under multiple conditions, but a chemical mechanism to explain these correlations was not investigated DCT under various modeling scenarios, of which the most germane proved to be ones that excluded or included possible precipitation of amorphous calcium phosphate (Ca3(PO4)2 ). Regressions of [PTH] on [Ca++]DCT were then performed at pH 6.6, 6.8, and 7.0, which are representative values over the documented pH range in the DCT DCT reduced [Ca++]DCT in advanced CKD to values associated with increased [PTH]. Relationships of [PTH] to [Ca++]DCT and to eGFR were virtually identical.The Joint Expert Speciation System (JESS) employs a compilation of thousands of equilibria to predict concentrations of ions and other chemical entities under defined conditions ,27. In t the DCT . If prec2 and 30 patients with eGFR 14–49 mL/min/1.73m2. GFR was estimated with the 4-variable MDRD formula. All participants were normocalcemic . Two patients with CKD were excluded from the present study, one because the serum ultrafilterable calcium concentration ([Cauf]s) was reported to be lower than the ionized calcium concentration ([Ca++]s)–a physiologic impossibility–and one because the association of [PTH] of 169 pg/mL with [Ca++]s of 1.35 mM implied autonomy of PTH secretion. A control subject who failed to collect a 24-hour urine specimen was also excluded. The study sample in the present report therefore includes 28 patients with CKD (denoted as “CKD”) and 27 control subjects (denoted as “CTRL”). CKD and CTRL were not matched for age, race, or gender.Between 2010 and 2013, data were collected from 28 control subjects with estimated GFR (eGFR) > 60 mL/min/1.73m++]s, [Cauf]s, and plasma [PTH]1–84 ([PTH]) were measured as previously described DCT and [Ca]DCT using thermodynamic relationships and equilibrium constants described in the chemical literature. However, it is necessary in such calculations to stipulate a priori which if any precipitates may be formed. Such stipulations are based on certain empirical rules of thumb, particularly Ostwald’s Rule of Stages, as described below.Phosphate and calcium exist in filtrate of the DCT as either free ions  [uf]s [f) to the DCT is 0.2 in controls and 0.35 in subjects with CKD [DCT was computed as 0.1(eGFR)[Cauf]s/(0.35)eGFR in CKD and as 0.1[(eGFR)[Cauf]s/(0.2)eGFR in CTRL. Similarly, total [P]DCT was calculated as (24h EP)/0.35(eGFR) in CKD and as (24h EP)/0.2(eGFR) in CTRL. Equations for total [Ca]DCT and [P]DCT thus simplified to 0.1[Cauf]s/FDf and (24h EP)/{FDf(eGFR)}, respectively.Total [P]rus (EP) ; the ratP) [uf]s ; and frawith CKD –33. Accof at normal and reduced GFR DCT, [CaHPO40]DCT, [Cacit–]DCT, [Caox0]DCT, [CaHCO3+]DCT, and [CaSO40]DCT were calculated as concentrations of the most likely pertinent species of dissolved calcium.In the present work, possible solid phases in the DCT included brushite 2 , including eGFR, [PTH], 24h EP, 24h ECa, [P]s, [Ca++]s, [Cauf]s, [P]DCT, and [Ca]DCT. For each of these parameters, normality of distribution was examined with plots and with the Shapiro-Wilk test. When distributions were judged to be normal, differences between means were assessed with t-tests for unpaired values . When distributions were skewed, differences between medians were assessed with the Mann Whitney U test. Results were considered to be statistically significant at p < 0.05.In each subset of subjects, mean values were determined for parameters not affected by pH or precipitation of Ca++]DCT induced by [P]DCT determines [PTH] in stages G3 and G4 CKD. For comparative purposes, we examined least-squares regressions of total [P]DCT and total [Ca]DCT on eGFR, and regressions of [PTH] on total [P]DCT, total [Ca]DCT, and eGFR (3(PO4)2 .The hypothesis investigated in the present study was that DCT (“determinants”) were sought with least-squares regressions of [Ca++]DCT on total [P]DCT and total [Ca]DCT. The tradeoff-in-the-nephron hypothesis was tested with regressions of [PTH] on [Ca++]DCT at pH 6.6, 6.8, and 7.0, assuming precipitation of Ca3(PO4)2  if logSI was ≥ 0 2  if logSI was ≥ 0 (To investigate the possibility that CaHPO and 7.0 . To dete was ≥ 0 .Ca) was 0.1 in both CKD and CTRL, we also considered the possibility that in CKD, a higher FDCa might increase the effect of total [Ca]DCT on [Ca++]DCT. To investigate that possibility, we performed regressions of [Ca++]DCT on total [Ca]DCT, [Ca++]DCT on [P]DCT, and [PTH] on [Ca++]DCT at FDCa 0.15 and 0.2 and pH 6.6, 6.8, and 7.0  .The research project that provided the data reported herein ,13 was a3(PO4)2  in the DCT.We present our results in accordance with the order of questions posed in 3(PO4)2 .DCT varied inversely with eGFR in CKD but not CTRL, and total [Ca]DCT was unrelated to eGFR in each group. [PTH] rose with [P]DCT in CKD but not CTRL  against eGFR (x) in the combined groups appeared to depict a hyperbola described in part by the formula xy = k (y) on 100/eGFR (100/x) displayed a linear relationship (y) as a power function of eGFR (x) in the form y = kx–1+ c. The hyperbola described by the function is included in a xy = k . Accorditionship . The assth-75th percentile range, and upper and lower limits of logSICa3(PO4)2  in CKD and CTRL under four combinations of conditions. LogSICa3(PO4)2  was 0 at saturation and > 0 at supersaturation. In 3(PO4)2 .3(PO4)2  was predicted in no subjects with CKD and in five CTRL (3(PO4)2  were predicted in the top two quartiles of CKD and in all but four CTRL 2  occurred at pH 6.73 2  did not occur  against [Ca++]DCT (x) appeared to depict a hyperbola described in part by the formula xy = k (y) on 10/[Ca++]DCT (10/x) was accordingly documented (y) as a power function of [Ca++]DCT (x) in the form y = kx–1+ c. The hyperbola described by the function is included in At pH 6.8, [PTH] varied inversely with DCT was again entirely dependent on [P]DCT in both groups, but relationships were curvilinear rather than linear  on 10/[Ca++]DCT (x) was accordingly demonstrated (y) as a power function of [Ca++]DCT (x) in the form y = kx–1+ c. The hyperbola described by the function is included in At pH 7.0, results resembled and accentuated those obtained at pH 6.8. [Can linear . [Ca++]D [Ca]DCT . [PTH] rL x 10−4 . Again, n xy = k . A signinstrated , and the++]DCT were visually similar  is not amenable to log transformation, the general formula y = kxn–a simpler power function of x–transforms to the linear equation logy = logk + n(logx).Because relationships of [PTH] to eGFR and to  and x = eGFR or [Ca++]DCT at pH 6.8 or 7.0.++]DCT x 104). These results indicate that in addition to the hyperbolic formulas in Figs y = kxn related [PTH] (y) to both eGFR and [Ca++]DCT (x) in combined CKD and CTRL. In ++]DCT x 104) was assigned a z-score. After this standardization procedure, slopes of lines relating log[PTH] to either log(eGFR) or log([Ca++]DCT x 104) were virtually identical, as is indicated by the extreme overlap in confidence intervals.3(PO4)2  was assumed in most scenarios, we also considered the possibility that formation of CaHPO40 might cause a reduction of [Ca++]DCT and an elevation of [PTH] as [P]DCT rose 2 , we examined regressions of [Ca++]DCT on concentrations of Cacit−and other Ca complexes to ascertain whether these compounds could reduce [Ca++]DCT and raise [PTH] secondarily. [Ca++]DCT varied inversely with [CaHPO40]DCT, presumably because of the relationship of [CaHPO4]DCT to [P]DCT  could have been higher in CKD than the assigned value, 0.1, with the result that total [Ca]DCT exerted an effect on [Ca++]DCT that was not evident at FDCa 0.1. We therefore examined regressions of [Ca++]DCT on total [Ca]DCT, [Ca++]DCT on total [P]DCT, and [PTH] on [Ca++]DCT at FDCa 0.15 or 0.2. At pH 6.6, 6.8, and 7.0, logSICa3(PO4)2  was essentially zero in all subjects at both FDCa values (data not shown), and therefore indicated universal precipitation of Ca3(PO4)2  at the increased values of FDCa. At pH 6.6, a previously significant relationship of [Ca++]DCT to total [Ca]DCT disappeared  and plasma membrane calcium ATPase 1b (PMCA1b) DCT also falls in response to increased total [P]DCT; if [P]DCT reduces [Ca++]DCT sufficiently, [PTH] rises to preserve Ca reabsorption and maintain normocalcemia DCT [++]DCT is related to total [P]DCT in CKD and CTRL 2 is posited in states of supersaturation, the results imply that in CKD, [PTH] increases as [P]DCT rises and [Ca++]DCT falls. At FDCa 0.1 and pH 6.8 or 7.0, the upward trajectory of [PTH] is accentuated at a sufficiently reduced [Ca++]DCT eGFR[Cauf]s/{FDf(eGFR)}, where FDf = fractional delivery of filtrate to that segment. This formula simplified to total [Ca]DCT = (0.1)[Cauf]p/FDf. We assumed FDf of 0.2 in CTRL and 0.35 in CKD [uf]s was not different in CKD and CTRL, FDf was solely responsible for differences in total [Ca]DCT in the two groups. In CKD, higher FDf lowered total [Ca]DCT; this consequence could possibly have obscured an effect of higher FDCa on [Ca++]DCT, but when FDCa was increased to 0.15 or 0.2, [Ca++]DCT remained independent of total [Ca]DCT, and total [P]DCT continued to be the sole determinant of [Ca++]DCT 2  occurred in the DCT. When no precipitation, FDCa 0.1, and pH 6.8 were assumed 2  was substantially negative, the DCT was not saturated with this solid (with five exceptions in CTRL), and relationships of [Ca++]DCT to [Ca]DCT and [P]DCT resembled those predicted in the absence of precipitation at pH 6.8 2  was more prevalent, and [P]DCT emerged as the sole determinant of [Ca++]DCT 2  was universal at pH 6.6, 6.8, and 7.0, and [Ca++]DCT was determined exclusively by [P]DCT (DCT on [Ca++]DCT is dominant when precipitation of Ca3(PO4)2  occurs and negligible when precipitation does not occur. By inference, tradeoff-in-the-nephron is not applicable when FDCa is ≤ 0.1 and pH is ≤ 6.6 simultaneously, but it is applicable under the more likely conditions that either pH is ≥ 6.7 (Ca is ≥ 0.1 (3(PO4)2  may dissolve downstream in a more acidic milieu DCT became the only plausible phosphate-containing determinant of [Ca++]DCT, but JESS calculations rendered this scenario unlikely; [Ca++]DCT was consistently predicted to be an order of magnitude higher than [CaHPO40]DCT, and [Ca++]DCT varied substantially with pH at a given [CaHPO40]DCT 2 determined [Ca++]DCT, and [Ca++]DCT determined complex concentrations other than [CaHPO40]DCT. Low relative concentrations of these complexes preclude the possibility that [Ca++]DCT was suppressed by substances such as citrate or oxalate.We also investigated the effect of anions other than phosphate on DCT was the primary determinant of [Ca++]DCT, and substantial elevations of [PTH] were predicted over a narrow range of [Ca++]DCT (3(PO4)2  occurred at pH 6.8 or 7.0, [P]DCT was the sole determinant of [Ca++]DCT; [PTH] increased to abnormal levels over a wider and more plausible range of [Ca++]DCT, and a continuous hyperbolic relationship between [PTH] and [Ca++]DCT emerged if CKD and CTRL were considered together 2 . If [Ca++]DCT is sufficiently reduced, [PTH] rises to maintain normal Ca++ reabsorption. The similarity of relationships of [PTH] to [Ca++]DCT and [PTH] to eGFR suggests that the first relationship causes the second 2  at pH ≥ 6.73 (DCT determined [Ca++]DCT by inducing precipitation of Ca3(PO4)2 . [PTH] rose in curvilinear fashion as eGFR and [Ca++]DCT fell 2 . Since supersaturation was not universal in CKD at FDCa of 0.1 2 in the DCT  DCT am., s.) .Ca was reduced in proportion to eGFR in CKD 2 . Although this phenomenon occurs in both CKD and CTRL, [Ca++]DCT appears to fall sufficiently to raise [PTH] in CKD only. Whether they are defined by the equation y = kx–1+ c or the simpler power function y = kxn, relationships of [PTH] to [Ca++]DCT and [PTH] to eGFR are so similar that the former seems likely to cause the latter. Our observations strongly suggest that the tradeoff-in-the-nephron hypothesis explains SHPT in stages G3 and G4 CKD. They also suggest that humans with SHPT may be treated successfully by reducing phosphate influx in proportion to the reduction in GFR.We describe an examination of the tradeoff-in-the-nephron hypothesis with the Joint Expert Speciation System. At pH values typical of the DCT, we present evidence that [P]S1 Fig(TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S3 Fig(TIFF)Click here for additional data file.S4 Fig(TIFF)Click here for additional data file.S5 Fig(TIFF)Click here for additional data file.S6 Fig(TIFF)Click here for additional data file.S7 Fig(TIFF)Click here for additional data file.S1 Table(TIFF)Click here for additional data file.S2 Table(TIFF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(PDF)Click here for additional data file.S5 File(PDF)Click here for additional data file.S6 File(PDF)Click here for additional data file.S7 File(PDF)Click here for additional data file.S8 File(PDF)Click here for additional data file.S9 File(PDF)Click here for additional data file.S10 File(PDF)Click here for additional data file.


Vitamin D and osteoporosis in Graves’ disease (GD) have been examined in cross-sectional studies with divergent results. Here, we prospectively studied vitamin D metabolism and bone health in patients with newly diagnosed GD.Thirty consecutive patients with de novo overt thyrotoxicosis diagnosed with GD were included. At diagnosis, none of the patients were treated with vitamin D or anti-osteoporotic drugs. All patients were initially treated with antithyroid drugs. Blood samplings were taken at baseline and at 6 weeks, 3, 6, 12 and 24 months after treatment start. Serum levels of 25OHD3, 1,25OH2D3, calcium, parathyroid hormone (PTH), and C-terminal telopeptides of Type I collagen (CTX-I) were analysed. Bone mineral density (BMD) was measured at baseline, and 1 and 2 years after treatment initiation.p = 0.002, < 0.001 and 0.005 in the spine, left total hip and left femoral neck, respectively.At diagnosis, patients with GD did not have vitamin D deficiency. There were no significant correlations between levels of 25OHD3 and thyrotoxicosis. Upon treatment of the thyrotoxicosis, serum calcium fell transiently, and PTH and 1,25OH2D3 increased. 25OHD3 fell within the normal range and stabilised at 6 months. CTX-I fell over 12 months, BMD increased significantly up to 2 years, The present data underline that thyrotoxicosis has a negative impact on bone health and demonstrate fine-tuned dynamics in bone and vitamin D metabolism. Upon treatment, bone health improved over a follow-up period of 24 months despite rising PTH. Increased conversion of 25OHD3 to 1,25OH2D3 occurs during treatment of GD. Graves’ disease (GD) is an autoimmune form of hyperthyroidism with an annual incidence of 21 in 100, 000 individuals . Hyperth. have also reported vitamin D deficiency in 40% of women and in 18% of men having GD from Japan [. showed that 35% of patients with thyrotoxicosis had vitamin D deficiency [Epidemiological studies have reported associations between vitamin D 25OHD3) deficiency and the risk of developing a variety of diseases, such as cardiovascular diseases, cancer, diabetes and infectious diseases –19. In aOHD3 defificiency . Other sficiency –29. Howeficiency . All stuIn the present study, we have prospectively monitored vitamin D metabolism and calcium homeostasis in patients with newly diagnosed GD, taking into consideration the severity of thyrotoxicosis and changes in bone mineral density.: pregnant or planning to be pregnant during the study, secondary osteoporosis, corticosteroids treatment in the last 2 years as well as vitamin D and/or calcium supplementation. TRAb was below the reference range  in one patient. This patient otherwise had laboratory findings and symptoms typical of GD, with a high and homogeneous uptake at scintigraphy and was included in the study. Two patients started with vitamin D supplementation without prescription after the baseline visit. One patient had osteoporosis at time of diagnosis and started treatment with bisphosphonate and calcium with vitamin D. These three patients were excluded from the follow-up analyses.Patients with de novo GD, diagnosed by decreased levels of thyroid stimulating hormone (TSH) and positive autoimmune antibodies against TSH-receptors (TRAb), were recruited at the Uppsala University Hospital from February through November 2017. Inclusion criteria: males and females, aged 30–80 years, with newly diagnosed GD. Exclusion criteriaThe study comprised six visits. During the first visit (baseline), all patients underwent an examination, including recording of demographic characteristics (sex and age), medical history, family history of fracture, concomitant medication, weight and height, and a physical examination. Blood samplings were taken to measure TSH, freeT4, freeT3, TRAb, CTX-I, 25OHD3, calcium, phosphate and PTH at baseline and at 6 weeks, 3, 6, 12 and 24 months after diagnosis and treatment start. We also measured 1.25OH2D3 at the first four visits. Vitamin D-binding protein (DBP) was measured at visits 1, 5 and 6, and sexual hormone binding globulin (SHBG) was measured at visits 1 and 6. During visit 1, the patients completed a questionnaire regarding risk factors for osteoporosis. Dual-energy X-ray absorptiometry (DXA) was performed on the hip and lumbar spine within 2 weeks after visit 1 and then 1 and 2 years after study inclusion. All 30 patients included in the study received conventional block and replace treatment. Anti-thyroid drugs, methimazole or propylthiouracil, were initiated in conjunction with visit 1, started with 10–20 mg methimazole doses or 150–300 mg propylthiouracil daily. Thyroxine 50–100 μg was added daily when thyroid hormone levels fell into the normal range, and TSH levels were kept in the low normal range. During follow-up, four patients received radioiodine (RAI) because of persistently elevated TRAb after 10–15 month treatment. One patient received RAI after 5 months at the request of the patient. Three patients underwent total thyroidectomy, two patients due to neutropenia, which developed directly after the start of methimazole in one case and after 10 months on methimazole in the other. One female patient underwent surgery after 5 months because of desire to become pregnant. Out of the remaining 22 subjects, 21 patients received ATD for 18–24 months until negative TRAb. One patient had a spontaneous recovery before treatment initiation.Graves` orbitopathy (GO) was defined as signs and eye symptoms related to GD and classified as ‘mild’ GO with symptoms, such as gritty sensation and tearing due to dry eyes, caruncle swelling and/or redness, upper eyelid retraction, or as ‘moderate to severe’ in the instance of redness and/or swelling of the eyelids, pressure or pain in the eyes, exophthalmos, chemosis, diplopia or signs of optic nerve compression. The eye signs and symptoms were detailed in the medical records by the attending endocrinologist as well as by the nurse at all visits.® (CTX-I) was analyzed using Cobas® pro instruments and reagents . An ELISA was used for the quantification of C-terminal telopeptides degradation products of Type I collagen , according to manufacturer (BioVendor). Plasma calcium (reference 2.15–2.5 mmol/L); serum PTH ; serum 25OHD3 (deficiency < 25 nmol/L and insufficiency < 50 nmol/L); serum 1,25OH2D3 ; plasma phosphate ; SHBG levels  were all measured per routine methods used at the Department of Clinical Chemistry at the Uppsala University Hospital. Vitamin D binding protein was measured by colorimetric sandwich-ELISA .Plasma TSH , free T4 , free T3 , TRAb (reference < 1.75 IE/L), and CrossLapst tests compared with visit 1 using Bonferroni-adjusted alpha levels. For each analysis, normal distribution was assessed by Shapiro–Wilkes test at each timepoint. If the data were not normally distributed, they were log transformed (base 10), after which the assessment was reiterated. No extreme outliers were found after visual inspection of the data and the residuals. Due to violation of normality even after log transformation, changes in free T3 and DBP were analysed using Friedman’s test with post-hoc Bonferroni-adjusted Wilcoxon signed rank tests. For the same reason, changes in SHBG levels were analysed using Wilcoxon signed rank test. P values < the Bonferroni-adjusted alpha level  were considered significant. SPSS version 27  was used for the analyses.Data are presented as median (range) unless otherwise indicated. Changes in plasma levels of bone turnover markers and bone mineral density (BMD) were analysed using repeated-measures ANOVA with Greenhouse–Geisser correction. We performed post-hoc paired Thirty subjects with overt hyperthyroidism without vitamin D supplementation or treatment against osteoporosis at diagnosis of GD were included in the study. Median age was 55 years (range 35–72 years), all but one was of Scandinavian origin, two were smokers, 29 were women. The baseline data present all 30 included patients Table . Twenty-p = 0.042). Otherwise, there were no correlations between 1,25OH2D3 and free T3, TSH, TRAb or free T4 (data not shown).At baseline, levels of 25OHD3 were 66.8 37.3–130) nmol/L (Table 30 nmol/Lp = 0.77), whereas SHBG declined from 172 (65–634) at baseline to 69.5 (14–299) at visit 6 (p < 0.001) (data not shown).During treatment, the levels of DBP did not change  = 0.28. Although a positive trend but the relationship between the variables is not significant.When calculating calcium values regarding fT4 at baseline using Spearman’s Rho the value of 2 in the spine, 0.95 (± 0.14) g/cm2 in the left total hip and 0.91 (± 0.13) g/cm2 in the left femoral neck  g/cm.91 ± 0.1 g/cm2 inp = 0.002), in the left total hip 7.5% (± 7.9) (p < 0.001) and in the left femoral neck 4.4% (± 7.0) (p = 0.005)  (p = 0.5). Nor was there any difference of 1,25OH2D3, 59 (17–155) and 102 (22–136), respectively (p = 0.17).During the study 17 patients developed GO. Three patients with moderate to severe orbitopathy received steroids, two were treated with oral steroids and one with iv steroids. At baseline, there was no significant difference regarding the levels of 25OHD3 during the trial and risk of GO, 66.1 (37.3–99.2) and 67.1 (41.3–130) for GO and non-GO group, respectively  at baseline and after 12 months with ATD. The bone mineral density increased significantly in the lumbar spine and femoral neck . Two morIn this study, we observe an increase in BMD concomitant with declining levels of 25OHD3. There are, however, results from randomised clinical studies, finding that calcium and vitamin D can be used in the treatment of osteoporosis . FurtherRegarding the levels of vitamin D, the inclusion of patients was from February through November and blood test collected six times during the follow-up period for 2 years. The vitamin D levels in this study were similar to those measured in a cohort of 870 healthy Uppsala residents   and highThe limitation of this study is the relatively small sample size. The strength, however, is the prospective design and the fact that not only 25OHD3 was analysed but also other parts of the calcium homeostasis. This includes the active form of vitamin D, 1,25OH2D3, which hitherto is less investigated in GD. Considering this, and that the reported findings reach statistical significance, we believe that the study offers sufficient strength for the conclusions presented.In summary, no vitamin D deficiency was observed at time of diagnosis of Graves’ disease, and levels of 25OHD3 were significantly decreased at follow-up. Lower levels of 25OHD3 are likely to occur secondary to a higher conversion to 1,25(OH)D3 due to increased PTH levels after treatment of thyrotoxicosis.GD has a significant impact on the skeleton, and treatment of thyrotoxicosis leads to improved BMD, which continues to rise for 2 years after treatment start.


Nicotiana tabacum L.) production and quality worldwide. However, the role of magnesium (Mg) nutrition in plants has been frequently overlooked, especially under cold stress, and Mg deficiency adversely affects plant growth and development. Here, we evaluated the influence of Mg under cold stress on tobacco morphology, nutrient uptake, photosynthetic and quality attributes. The tobacco plants were grown under different levels of cold stress, i.e., 8°C, 12°C, 16°C, including with a controlled temperature of 25°C, and evaluated their effects with Mg (+Mg) and without Mg (–Mg) application. Cold stress resulted in reduced plant growth. However, the +Mg alleviated the cold stress and significantly increased the plant biomass on an average of 17.8% for shoot fresh weight, 20.9% for root fresh weight, 15.7% for shoot dry weight, and 15.5% for root dry weight. Similarly, the nutrients uptake also increased on average for shoot-N (28.7%), root-N (22.4%), shoot-P (46.9%), root-P (7.2%), shoot-K (5.4%), root-K (28.9%), shoot-Mg (191.4%), root-Mg (187.2%) under cold stress with +Mg compared to –Mg. Mg application significantly boosted the photosynthetic activity (Pn 24.6%) and increased the chlorophyll contents (Chl-a (18.8%), Chl-b (25%), carotenoids (22.2%)) in the leaves under cold stress in comparison with –Mg treatment. Meanwhile, Mg application also improved the quality of tobacco, including starch and sucrose contents, on an average of 18.3% and 20.8%, respectively, compared to –Mg. The principal component analysis revealed that tobacco performance was optimum under +Mg treatment at 16°C. This study confirms that Mg application alleviates cold stress and substantially improves tobacco morphological indices, nutrient absorption, photosynthetic traits, and quality attributes. In short, the current findings suggest that Mg application may alleviate cold stress and improve tobacco growth and quality.Cold stress is one of the major constraints limiting the productivity of many important crops, including tobacco ( Nicotiana tabacum L.), belonging to the family Solanaceae, originated from tropical and subtropical America but is now widely cultivated in China (Tobacco (in China . In soutin China . TobaccoNicotiana tabacum L.) is one of the most important economic crops in south China and often poses cold stress and hinders early sowing (Glycine max) decreases the number of pods and seeds per plant and results in low yield and poor quality (Gossypium hirsutum L.), the cold stress for short time during later growth stage encounters the leaf spot lesions (Zea mays) encounters a cold injury during emergence to maturity, it results in poor germination rate and growth of leaves, inhibits the photosynthetic activity, slow filling rate and accumulation of dry matter, and finally low yield and poor quality  increases the enzyme activity and nutrient content of cucumber seedlings and enhances the tolerance of cucumber seedlings to cold stress . GlycineMagnesium (Mg) is an essential micronutrient for plant growth and development, as it is involved in various physiological and biochemical processes such as photosynthesis and enzymatic activities . Mg is cNicotiana tabacum L. cv. CB-1) were surface sterilized with 75% ethanol and 30% hydrogen peroxide (1:1) for 5 min. The seeds were then rinsed several times with sterilized water before being vernalized at 4°C for 12h in the dark 2·4H2O, 397μM MgSO4·7H2O and 282μM KH2PO4. Micronutrients: 22.5μM Fe(II)-EDTA, 46.2μM H3BO4, 14.3μM MnCl2·4H2O, 0.76μM ZnCl2, 0.32μM CuCl2·2H2O, 0.13Mμ Na2Mo4·2H2O). While, hydroponic pots of –Mg were supplemented with modified Hoagland’s nutrient solution without magnesium 2·4H2O, 147μM Na2SO4, and 282μM KH2PO4; micronutrients: 22.5μM Fe(II)-EDTA, 46.2μM H3BO4, 14.3μM MnCl2·4H2O, 0.76μM ZnCl2, 0.32μM CuCl2·2H2O, 0.13μM Na2Mo4·2H2O). The growth chambers were set to photosynthetically active radiation of 700 μmol·photon m-2·s-1, the relative humidity is 80%, 12 hours: 12 hours for light: dark cycle. The nutrient solution was replaced weekly. The experiment was harvested after eight weeks of temperature and Mg treatment.The hydroponic pots of each growth chamber were divided into two sets (6 hydroponic pots for each). One set was treated with magnesium addition (+Mg) and four different temperatures, i.e., 8°C, 12°C, 16°C and 25°C, and named as +Mg8, +Mg12, +Mg16 and +Mg25, respectively. The other set without magnesium (–Mg) and the abovementioned temperatures are –Mg8, –Mg12, –Mg16 and –Mg25, respectively. It is important to investigate the effect of Mg because the main tobacco-producing areas of Fujian Province have an uneven distribution of exchangeable Mg in the soil, and some areas are severely deficient in Mg (The tobacco plants were harvested and separated into shoots and roots. Shoot and root were weighed to obtain the fresh biomass of shoot fresh weight (SFW) and root fresh weight (RFW). To determine the shoot dry weight (SDW) and root dry weight (RDW), the plant material was oven-dried for 48h at 70°C .2SO4) and 4.0 mL of hydrogen peroxide (H2O2) on digestion tubes. The digestion tubes were kept in the digestion block for approximately 2h, at a temperature of 200 ± 20°C until about 2ml of clear digestive fluid without granules was obtained. Then the deionized water was added to the digestive fluid and filtered using a 0.45-micron filter, and the extracts were stored at 4°C until nutrient determination (3): perchloric acid (HClO4) in a ratio 5v:1v on the hot plate at 180°C until clear aliquot was obtained. The deionized water was added to the digestive fluid and filtered using a 0.45-micron filter, and the extracts were stored at 4°C for further analysis , phosphorous (P), and potassium (K) minerals analysis, 0.05 g of tobacco plant material of each root and shoot was digested in 5 mL of concentrated sulfuric acid . The samples were put into 25 mL 80% acetone solution. They were then kept in darkness at room temperature overnight to allow for the extraction of the leaf pigments. The mixture of chlorophyll and 80% acetone solution was examined at 663 nm, 645 nm, and 470 nm to determine chlorophyll-a, chlorophyll-b, and carotenoids, respectively. The calculation method was as follows:2 concentration (Ci), were measured for each plant with a CIRAS-3 portable photosynthesis system . Measurements were made on an upper, third fully expanded leaf of an individual plant representing each monoculture tank, typically between 09:00 and 11:00 local time, and were measured once Pn was stable after 2 to 4 min and under the following conditions: 400 μmol·mol-1·CO2, 1000 μmol·m-2·s-1 photosynthetically active radiation, 25°C leaf temperature , stomatal conductance (Gs), and intercellular COperature .m (photosystemII- PSII) . The following equations were used to calculate the Fv/FI- PSII) ;0= initial fluorescence, Fv = variable fluorescence, Fm = maximum fluorescence, and Fv/Fm denotes the maximal quantum yield of PSII.Where FP<0.05. The principal component analysis (PCA) was performed to evaluate the differences in biomass, minerals, nutrient content, photosynthetic traits, and quality parameters between the +Mg and –Mg under varying levels of low temperature . The results were visualized with Origin Pro 2021 .All the statistical analyses were performed using IBM-SPSS version 21.0 . Two-way analysis of variance (ANOVA) was performed to analyze the effect of Mg, temperature, and their interaction effect of tobacco morphological traits, minerals nutrients concentration in roots and shoots, photosynthetic traits, and quality parameters. All the measurements are presented as means with their standard errors (Mean ± SE). The statistically significant mean differences among various treatments were identified by using Duncan’s test at a significance level of Application of Mg significantly improved the tobacco morphological traits, including shoot fresh weight (SFW), root fresh weight (RFW), Shoot dry weight (SDW), and root dry weight (RDW) as compared to –Mg under cold stress Figure 2-1) and +Mg25 (2.07 g·plant-1)  Table 1.Application of Mg under varying levels of temperature altered the nutrients uptake Figure 3Similarly, for nitrogen (N), a significant difference was observed for aboveground plant parts , and shoot N contents under +Mg treatment were significantly higher than –Mg at all temperatures and showed maximum N contents at 25°C Table 2.The potassium (K) contents of the shoot were not significantly affected among +Mg and –Mg treatments at 8°C, 12°C, 16°C, and 25°C temperature, while temperature had a significant effect. However, it significantly increased with increasing temperature. Maximum shoot K content (683.2 mg) was found at +Mg25 Table 2.The +Mg significantly increased the shoot phosphorous (P) contents at all four temperature levels compared with –Mg. Shoot P contents significantly increased with increasing temperature, and maximum shoot contents (117.5 mg) were observed at +Mg25 Table 2.-1 and 26.68 mg·plant-1, respectively. The increase rates were 91.9% and 88.5%, respectively, compared with –Mg8. Thus, shoot and root Mg contents were significantly affected by Mg, temperature, and their interaction effect , intercellular carbon dioxide concentration (Ci), transpiration rate (Tr), and photosynthetic rate (Pn), were significantly affected by the Mg application under cold stress. The Gs for both +Mg and –Mg decreased with the decrease in temperature level. The Gs under +Mg tobacco plants increased 36.9%, 62.0%, 9.0% and 29.2% compared to –Mg treatment at 8°C, 12°C, 16°C and 25°C, respectively. The highest value of Gs was recorded at 25°C, i.e., 420.67 mmol·m at 25°C Table 3.Chl-a) content was significantly affected by Mg at 12°C, 16°C and 25°C, and maximum Chl-a content was recorded at +Mg25 (Chl-b) (Chl-a+b) , gas exchange parameters , carbohydrate  and photosynthetic rate (Pn), correlation analysis was conducted between chlorophyll , gas exchange parameters  carbohydrate  and photosynthetic rate (Pn). As shown in Chl-a, Chl-b, Chl-a+b, sucrose, starch and reducing, respectively.The above results indicated that the photosynthetic system of tobacco plant had changed to some extent. With the increase of temperature, the chlorophyll of tobacco plants increased and the carbohydrate produced by the photosynthetic system also increased. In order to further clarify the relationship between chlorophyll  per active reaction center (RC) and the heat dissipation (DIo/RC) were increased, suggesting that after partial RC deactivation caused by cold stress, the energy dissipation efficiency of the remaining active RC improved, which increased the burden of active RC, to resist cold environmental stress. However, +Mg treatment significantly alleviates this procedure. Additionally, cold stress decreased the excitation energy used to reduce QA (TRo/RC) and the electron transfer capacity (ETo/RC) of a single RC in tobacco, while +Mg treatment significantly alleviated the decline of ETo/RC, causing more energy to enter the electron transfer, which was conducive to maintaining the normal operation of the PSΠ linear electron transfer and increasing the tobacco’s resistance to adversity, especially cold stress.The phenomenological energy fluxes per the exciting cross sections (CS) of the grapevine leaves during the growing season are presented in Chl-a, Chl-b, Chl-a+b, Cart, ABS/RC, DIO/RC, TRO/RC, ETO/RC, ABS/CSm, DIO/CSm, TRO/CSm, ETO/CSm, PIabs, DFabs), and quality attributes (sucrose and starch), and overall these showed the best performance in under +Mg at 25°C.The principal component analysis (PCA) clustered the input variables of temperature and Mg into different groups  because Mg application generally increases CO2 fixation, transportation, and distribution of photoassimilates that contribute to improved plant growth (Changes in photosynthetic characteristics in response to stress have received much attention in recent years. Hence, in this study, we also characterize the photosynthetic traits. We found higher chlorophyll contents, including Chl-s and Mg . We founs and Mg . The inct growth . This ist growth Figure 5t growth , indicatt growth . It has t growth . These fSucrose and starch are known as important tobacco quality indicators. It has been reported that temperature also efficiently induces sucrose and starch biosynthesis . Plant MThe comprehensive analysis of PCA also showed the variation in morphological, the concentration of mineral nutrients, photosynthetic traits, and quality parameters for +Mg and –Mg treatments under cold stress Figure 9Mg is an essential nutrient for plant growth and development, as it is involved in various physiological and biochemical processes such as photosynthesis and enzymatic activities. However, the role of Mg in improving plant growth and development under cold stress has not been fully elucidated. This study confirmed that the addition of Mg improved the photosynthetic system and nutrient uptake of tobacco plants under cold stress. The addition of Mg increased the chlorophyll content and photosynthetic rate, and increased the carbohydrate to improve the tolerance of tobacco plants. These results confirmed that magnesium application had significant protective effect on cold stress of tobacco. Further investigation is needed in tobacco fields under various cold stress scenarios by considering the Mg application to improve the tobacco yield and quality.The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding authors.JL: Experimental set-up and parameters determination, data analysis, writing-original draft, software, visualization. MM: writing and reviewing. AS, QG and YW: Parameters determination, data curation and software. HZ: Writing-reviewing. WL: Investigation, conceptualization, writing-reviewing. CZ: Conceptualization, data curation, writing-reviewing and editing. All the authors contributed to the article and approved the submitted version.