Artemisia ordosica.Non-structural carbohydrates (NSC) play a significant role in plant growth and defense and are an important component of carbon cycling in desert ecosystems. However, regarding global change scenarios, it remains unclear how NSCs in desert plants respond to changing precipitation patterns. [Methods] Three precipitation levels  and two precipitation intervals levels (5 and 15 d) were simulated to study NSC (soluble sugar and starch) responses in the dominant shrub A. ordosica. The effect of precipitation on NSC content and its components depended on extended precipitation interval. With lower precipitation and extended interval, soluble sugar content in roots increased and starch content decreased, indicating that A. ordosica adapts to external environmental changes by hydrolyzing root starch into soluble sugars. At 5 d interval, lower precipitation increased the NSC content of stems and especially roots.Precipitation level and interval interact to affect the NSC (both soluble sugar and starch components) content of A. ordosica follows the “preferential allocation principle” to preferentially transport NSC to growing organs, which is an adaptive strategy to maintain a healthy physiological metabolism under drought conditions. The findings help understand the adaptation and survival mechanisms of desert vegetation under the changing precipitation patterns and are important in exploring the impact of carbon cycling in desert systems under global environmental change.The online version contains supplementary material available at 10.1186/s12870-023-04512-4. Water is an important environmental limiting factor for plant growth that significantly impacts plant survival, growth, and distribution, particularly in nutrient- and water-limited desert ecosystems , 2. In tThe regulation of non-structural carbohydrate (NSC) allocation is a central plant response strategy to precipitation changes. NSCs are important for plant metabolism and the main storage form of energy , 7. SoluNumerous studies have examined how NSC content responds to precipitation changes. Some studies have suggested that precipitation reduction can increase plant NSC content , 21, whiArtemisia ordosica is a dominant and constructive species that covers 31% of the Mu Us Sandy Land of Northwestern China and plays an important role in the stability of the ecosystem in the region. Therefore, in this study, taking A. ordosica as the research object, we conducted a field control experiment simulating three levels of precipitation  and two levels of precipitation intervals  in a desert shrubland in the Mu Us Desert of northern China and examined the effects of changes in precipitation patterns on NSC storage at the whole-plant and organ levels. The study addresses the following three question: (1) Will the alteration in precipitation patterns have an effect on the NSC, starch and soluble sugar content of A. ordosica? (2) Do soluble sugars and starch transform each other to adapt to precipitation change? (3) Does the distribution ratio of plant NSC among different organs change due to precipitation change?A. ordosica, Hedysarum mongolicum, Salix psammophila, Leymus secalinus, Heteropappus altaicus, Pennisetum centrasiaticum, Corispermum puberulum, Setaria viridis, and Cynanchum thesioides. We chose A. ordosica as our study species. It is a deciduous dwarf shrub, the average height of mature plants is between 50 and 100 cm, and the branches are mainly yellowish brown or blackish grey. Normally we divide the branches into vegetative twigs and reproductive twigs by the maturity of their growth. In semi-fixed and fixed sands, the root system of A. ordosica can usually penetrate the soil layer mainly 20–45 cm deep, and in mobile sands, the root system of A. ordosica penetrates 100–200 cm, and can keep the normal growth of the plant by absorbing the deep soil groundwater. A. ordosica is an excellent sand-fixing half-shrub plant endemic to arid and semi-arid desert areas in China, widely distributed in fixed and semi-fixed sandy areas in Inner Mongolia, Ningxia, Shaanxi, Gansu, and other provinces, exhibiting characteristics such as resistance to wind erosion and barrenness, and playing an important role in ecological construction of sandy area vegetation.The experimental area was at the Yanchi Research Station in Ningxia City on the southwest edge of Mu Us Sandy Land, China . The area has a typical temperate continental monsoon climate with average annual precipitation and evaporation of 280 and 2024 mm, respectively. Precipitation is mainly concentrated in summer and autumn, with 83.3% of annual precipitation occurring from May to September. Annual average temperature is 8.1 ℃, annual sunshine duration is approximately 2867.3 h, and the frost-free period is approximately 165 d. The prevailing wind is northwesterly, with average annual wind speed of 2.8 m/s, and mainly concentrated from November to April of the following year. The experimental site has a loose soil structure and is dominated by aeolian sandy soils. The main plant species in this region are A. ordosica seeds were sown in each PVC bucket. After the plants grew, any excess A. ordosica were removed at seedling stage, leaving only one per bucket. The height of the PVC buckets was sufficient for A. ordosica roots to grow, as the roots of A. ordosica are mainly distributed between 0 and 50 cm depth. After two months of slow growth, precipitation treatment started in early June.The experiment was conducted from March to October in 2020 and 2021 at the Yanchi Research Station in Ningxia City. In late March 2021, the sandy soil in the study area was screened as cultivation soil, loaded into PVC buckets , and buried in the soil to prevent the influence of soil moisture on the experimental treatment. In addition, 10 The experiment used a two-factor  level combination, and the precipitation amount treatment set the precipitation gradient according to the average monthly precipitation amount during the regional growing season (May–October). Regional precipitation statistics show that average annual precipitation amount from 1990 to 2017 was 311 mm, varying by approximately 30% around this annual average . The experiment therefore tested three corresponding precipitation levels: annual mean natural precipitation amount (W), W decreased by 30% (W-), and W increased by 30% (W+). The annual precipitation data (1990–2017) show that precipitation frequency is generally stable within the study area, with the largest proportion of precipitation events (62.09%) occurring within a time interval of 0–5 d. Precipitation intervals in the arid and semi-arid regions of Northwest China are expected to increase in the future. Therefore, in this experiment, a precipitation interval of 5 d was selected to simulate the natural precipitation frequency (T). To account for the increased precipitation levels associated with prolonged rainfall events, this interval was extended to 15 d to simulate periods of heavy rainfall (T++). The experiment involved six treatment levels: W-T, WT, W + T, W-T++, WT++, and W + T++. The WT level represents the treatment with natural precipitation amount and precipitation frequency and served as the control group in this experimental series. Our experiment used the randomized complete block design with 3 replicates per treatment. The gradients of total monthly precipitation amount and precipitation interval were controlled by artificial irrigation following the experiment design.A. ordosica plants were removed from the buckets to ensure the plants are intact. Each plant was then divided into three parts: leaves, stems, and roots, placed in an oven at 120 °C for 30 min, and then dried at 70 °C to a constant weight. After crushing, the material was passed through a 0.4 mm sieve and stored prior to determining the starch and soluble sugar contents. Soluble sugar and starch contents were determined from the absorbance at 625 nm of the same anthrone reagent in a spectrophotometer. The soluble sugar content was based on the regression equation of a standard solution of glucose, while the glucose content multiplied by a conversion factor of 0.9 was calculated as the starch content. Soluble sugar and starch contents were shown as a percentage of dry matter [A. ordosica plants were calculated following Eq.  of NSCs and their carbon components (soluble sugar and starch) in the whole plant and different organs of A. ordosica, respectively. BOrgan represents the biomass (g) of each organ of A. ordosica.where A. ordosica plants by precipitation amount and precipitation intervals. One-way ANOVA was used to test for differences in the contents of NSCs and their components among different precipitation amounts, precipitation intervals, and plant organs. Duncan’s test was used to make multiple comparisons. Origin 2022b data analysis and graphing software  was used to provide graphical representations of the data.Microsoft Excel 2019 was used to input and collate the experimental data, and SPSS software  was used for statistical analysis. Two-way ANOVA was used to test the differences in the contents of NSCs and their components in whole A. ordosica in stems and roots followed the sequence: W- > W > W+. The content of NSCs in stems was as follows: W- > W > W+, whereas the contents of soluble sugar and NSCs in leaves and the content of NSCs in roots did not show significant differences among different precipitation amount treatments. At the level of T++, the contents of soluble sugar and NSCs in each organ showed non-difference under the different precipitation amount treatments, and the content of starch in stems was as follows: W- < W < W+. In other words, at natural precipitation frequency, the decrease in precipitation amount increased soluble sugar content of stems and total NSCs. However, at longer precipitation intervals, the decrease in precipitation amount increased the starch content of stems.As shown in Fig. A. ordosica was not affected by the precipitation interval, and the contents of starch and NSCs in stems were in the order of T > T++; the starch and NSC contents of the leaves and roots were not affected by the precipitation interval. At the W level, the starch and NSC contents in each organ showed non-difference under different precipitation intervals, and the soluble sugar content in the roots of A. ordosica was lower at T than at T++. At the W + level, the contents of soluble sugar and NSCs in each organ of A. ordosica were not affected by the precipitation interval, and the starch content in roots was higher at T than at T++. In conclusion, under the background of natural precipitation amount, extended precipitation intervals increased the soluble sugar content of the roots of A. ordosica. With 30% reduction in the precipitation amount, longer precipitation interval was associated with lower starch content in stems of A. ordosica. However, when the precipitation amount increased by 30%, longer precipitation interval decreased the starch content in the root system.At the W- level, the soluble sugar content of each organ of A. ordosica stems and roots (P < 0.05), whereas precipitation amount had no significant effect on the content of NSCs or their components in each organ. The interaction between precipitation amount and precipitation interval had significant effects on the total content of soluble sugar, starch, and NSCs in the stems of A. ordosica (P < 0.05), and had a significant effect on the soluble sugar content of roots (P < 0.05).As shown in Table A. ordosica plants followed the sequence: W- > W > W+, that is, the soluble sugar, starch, and NSC contents of the whole A. ordosica plant increase when precipitation decreases. At the level of T++, the starch content of the whole A. ordosica plants followed the order of W + > W > W-. The soluble sugar and NSC contents of A. ordosica plants were non-difference under different precipitation treatments. These results suggest that the longer the precipitation interval and the lower the amount of precipitation, the lower the starch content of the whole A. ordosica plant.It can be seen from Fig. A. ordosica plants showed significant differences according to precipitation interval, being lower at T than at T++. At W- and W + levels, no significant difference was observed in the effect of precipitation intervals on soluble sugar content in the whole plant. At the W- level, the contents of starch and NSCs in A. ordosica plants were higher at T than those at T++, that is, the reduced whole-plant starch and NSC content was associated with longer precipitation interval. However, at the W and W + levels, longer precipitation interval did not result in significant changes in whole-plant starch content.At natural precipitation levels (W), the soluble sugar content of A. ordosica plants (Table A. ordosica plants (P < 0.05).Precipitation amount had significant effects on the contents of soluble sugar and NSCs in whole ts Table , whereasA. ordosica significantly increased in stems and roots; the most significant increases were detected for soluble sugar and NSC contents of the roots, suggesting that A. ordosica plants allocated more carbon to roots in order to absorb more soil water in response to low precipitation amount. W + T treatment significantly reduced the soluble sugar content and increased the starch content in all organs, reduced the total NSC content in stems, and increased the NSC content in other organs, indicating that significant reduction in NSC content under drought stress occurred only in some organs. W-T++, WT++, and W + T + + treatments significantly reduced the root starch content but significantly increased the root soluble sugar and total NSC contents , whereas precipitation amount and its interaction with precipitation intervals showed no significant effect on the soluble sugar–starch ratio (P > 0.05)  Table .A. ordosica did not show significant differences under the different precipitation amount treatments. In other words, under the background of natural precipitation frequency, the decrease in precipitation amount increased the soluble sugar–starch ratio in roots and whole plants, whereas with longer precipitation interval, the decrease in precipitation had no effect on the soluble sugar–starch ratio in organs and whole plants.At the T level, under different precipitation levels at T frequency, the soluble sugar–starch ratio in the leaves and stems of A. ordosica did not show significant differences and those in the roots and whole plants were significantly different in the order W- > W > W+ Fig. c, d. At A. ordosica showed non-difference at different precipitation intervals. At the W level, the soluble sugar–starch ratio in the whole A. ordosica plants was significantly different under different precipitation intervals, being higher at T + + than at T; however, at the W + level, the soluble sugar–starch ratio in each organ of A. ordosica only differed significantly in the root system, being higher at T + + than at T. In the context of natural precipitation, the longer the precipitation interval, the higher the soluble sugar–starch ratio of the whole A. ordosica plant. In the context of a 30% increase in precipitation amount, the longer the precipitation interval, the greater was the soluble sugar–starch ratio in the root system of A. ordosica. With prolonged precipitation interval, the soluble sugar–starch ratio in the roots of A. ordosica was greater than one, and the amount of soluble sugar in the roots of the plant was always greater than that of starch. However, the soluble sugar–starch ratio in whole A. ordosica plants was less than one . Compared with the control (WT), extended precipitation interval  resulted in an increase in the soluble sugar content of the roots of A. ordosica together with a decrease in starch content, and the soluble sugar–starch ratio was greater than 1 (Figs. A. ordosica for adaptation to the changing external environment initiated by water stress due to extended precipitation interval. By hydrolyzing root starch, A. ordosica supplements the plant’s demand for soluble sugars, increases the concentration of osmotic regulators, maintains the water balance of cells, and ensures the root system’s ability to absorb water and nutrients [A. ordosica to save energy and maintain normal physiological metabolism. Meanwhile, the root system was found to be the major part of the regulation mechanism of carbohydrate distribution in A. ordosica.Water resources affect the carbon assimilation yield of plants and thereby change the NSC content in plants . Climate of NSCs , 22, 23.utrients , 18, whiA. ordosica increased dramatically, especially soluble sugar in roots. This suggests that under drought stress A. ordosica allocates more carbon to underground parts to maintain the survival of the root, which was consistent with the hypothesis of optimal allocation [A. ordosica to cope with changes in the drought environment.At the same time, the allocation of NSCs among different plant organs can buffer the different synchronisms of carbon supply and demand among different organs under stress and maintain plant osmoregulation, growth, and development . The alllocation , 46. Prelocation , 48. Simlocation , 49. Aftlocation , 51, whiA. ordosica plants. The effect of precipitation amount on the content of NSCs and their components in A. ordosica depends on the variability in precipitation interval. At natural precipitation frequency, with a decrease in precipitation amount, the contents of soluble sugar in roots and starch in stems of A. ordosica gradually increased. In contrast, under prolonged precipitation interval, the starch content of the whole plant decreased as precipitation decreased.The interaction between precipitation amount and interval had significant effects on the content of NSCs and their components in A. ordosica to cope with changes in precipitation. Lower precipitation amount and longer precipitation interval led to an increase in the soluble sugar and starch fractions of whole A. ordosica plant, and NSC prefer to be allocated more in the roots systems. These results suggest that root system was the main regulatory site of carbohydrate distribution in A. ordosica.The interconversion of NSC components and coordinated allocation between organs are adaptive strategies for This study provided new ideas for the adaptation mechanisms of plants in deserts to global environmental changes, and offered a new insight into the vegetation structure in desert regions under global change scenarios.Below is the link to the electronic supplementary material.Supplementary Material 1


Reserve starch from seeds and tubers is a crucial plant product for human survival. Much research has been devoted to quantitative and qualitative aspects of starch synthesis and its relation to abiotic factors of importance in agriculture. Certain aspects of genetic factors and enzymes influencing carbon assimilation into starch granules remain elusive after many decades of research. Starch phosphorylase (Pho) can operate, depending on metabolic conditions, in a synthetic and degradative pathway. The plastidial form of the enzyme is one of the most highly expressed genes in potato tubers, and the encoded product is imported into starch-synthesizing amyloplasts. We identified that the genomic locus of a Pho1a-type starch phosphorylase is duplicated in potato. Our study further shows that the enzyme is of importance for a normal starch granule phenotype in tubers. Null mutants created by genome editing display rounded starch granules in an increased number that contained a reduced ratio of apparent amylose in the starch. The relArabidopsis, PHS1, a homolog of Pho1 in Arabidopsis, is suggested to be part of the core set of evolutionary conserved genes, involved in starch granule initiation  by maintenance of plastidial maltodextrin pools . In Arabitiation . Pho1 isitiation . Other citiation . The enzitiation . The direr disks . Reserveer disks . The higer disks .Tst1) has been reported in the fifth intron of Pho1a  in leaves, but only as homodimer in tubers . The selected target, 5′-GCTGTTGCAAAGAATGCCTT-3′, was located on the negative strand on exon 2, adjacent the PAM site 5′-AGG-3′   for 5 months and were regularly fertilized with SW Bouyant RikaS 7-1-5 + mikro .A primary analysis for screening of induced indels was made using high-resolution fragment analysis (HRFA hereafter) of PCR amplicons spanning the target site as described previously  using prego.com) Figure 22.2in silico characterization of the genomic Pho1a loci, a reference mRNA sequence of Pho1a (GenBank accession no. X52385.1) was blasted against high confidence gene models of long-read chromosome-scale genome assembly of doubled monoploid potato S. tuberosum Group Phureja DM 1-3 516 R44 Genome Assembly v6.1 (DM v6.1 hereafter) using blastn with default parameters (http://spuddb.uga.edu/blast.shtml) (Pho1a loci in DM v6.1 assembly.For the t.shtml) . Top hitPho1a genomic sequences were aligned with previously reported Pho1a transcripts based on PGSC v4.03 assembly  to refine exon/intron structure and deduce full length genomic sequence of Pho1a loci. Additionally, genomic sequences of homologous Pho1a loci from 24 wild potato species, representative of tuber-bearing clade of Petota section, two non-tuber-bearing wild potato species from the neighboring section Etuberosum, and two landraces of S. tuberosum Group Phureja (http://solomics.agis.org.cn/potato/tool/blast) to detect the presence of duplication and insertion of Tst1 in respective species. Specific genomic regions were amplified by PCR using primers listed in Pho1a loci on chromosome 3 of S. tuberosum L. cv Desirée.These putative assembly , Pho1a g library , and copry (Tst1  using Clol/blast) and S. ld.shtml;  were retPho1a in cv. Desirée based on HRFA and ICE analysis was examined by copy number estimation by Real Time quantitative PCR (RT-qPCR) on QuantStudio3 thermocycler  using Maxima SYBR Green/ROX qPCR Master Mix (2X) . StGBSS1 (GenBank accession no. A23741.1), a known single-copy gene, was used as reference gene  using VeriFlex , and the initial copy number in 10 ng gDNA sample was calculated using following formula:The duplication of nce gene . The prih and Tm Table 1 h and Tm . The optnX is PCR product after cycle n, 0X is initial copy number, E is amplification efficiency, and n is cycle number.where 2.32O for 20–30 min), stained with Lugol’s solution (2% KI (w/v) and 1% I2 (w/v) for 3 min), and visualized under a light microscope  equipped with an Infinity X-32 digital camera  as described previously  to compare growth rate characteristics and individual leaf samples from greenhouse cultivated events were collected (middle and end of light phase) to evaluate transitory starch characteristics. Briefly, the leaf samples were fixed by immersion into fixative solution  for 24 h at 37°C, dehydrated and decolored (50% (v/v) ethanol for 24 h, and 96% (v/v) ethanol for 2 × 24 h; both steps at 37°C) and stored at 4°C. Afterwards, the leaf samples were rehydrated (50% (v/v) ethanol for 30–60 min; dHeviously .2O, and photographed (Cannon 450D DSLR) on a light table to record distribution of starch granules across tuber axis(s) in triplicates. These tuber samples were further sliced into 0.5-mm-thin sections using a mandolin and visualized under a light microscope after staining with Lugol’s solution as previously described . Novex gel was run for 120 min at 200 V at 4°C. The glycogen containing gel was run for 120 min at 250 V at 4°C. Subsequently, both gels were washed in an incubation buffer  for 10 min before being moved to a substrate buffer  and incubated at room temperature (approximately 22°C) for 3 h. After incubation, the gel was briefly rinsed in dH2O before staining for 5–10 min in 0.1× Lugol’s solution and imaged using a photocopier .Tuber extracts from a subset of mutational events (based on above phenotypic characterization) and Desirée (control) were assessed for starch phosphorylase activity using a modified version of . In tota2.5Freeze-dried samples from five full knockout events were homogenized in a Retsch Mixer Mill MM400 , at 30 Hz, for 30 s, and 50 mg of homogenized material was utilized to determine total starch content using Total Starch Assay Kit  following the manufacturer’s instructions. Briefly, total starch in the samples was converted into maltodextrins by thermostable α-amylase , which were subsequently quantitatively hydrolyzed into D-glucose by amyloglucosidase . The resulting D-glucose was measured in a colorimetric reaction employing glucose oxidase/peroxidase (GOPOD) reagent, and absorbance was measured at 510 nm . All analysis were performed in triplicates; total starch concentration was calculated using Mega-Calc™  and reported as mean values of percentage on dry weight (DW) basis.2.6g, 20 min). The supernatant was discarded, and the pellet was dried overnight. For each sample, 8 mg of purified starch was used for analysis. Samples were suspended in water, solubilized with 5M NaOH, and incubated at room temperature with intense agitation for 1.5 h. After solubilization, samples were neutralized with 3M HCl and then buffered by addition of 50mM sodium phosphate buffer. Samples were stained using iodine dissolved in 85% Dimethyl sulfoxide (DMSO), incubated for 10 min at room temperature, and the absorbance was measured at 620 and 550 nm . Amylose content was calculated as percent of total starch content using the following formula.Amylose content was measured in starch from tubers of nine mutational events and Desirée (as control) by a colorimetric method, as described previously . In shorStGBSS1 was estimated in tuber samples of selected events by RT-qPCR on a QuantStudio3 thermocycler  using Maxima SYBR Green/ROX qPCR Master Mix (2X) , using primers listed in StTUBB1 (NM_001288449.1) was used as reference gene as described above in Section 2.2. The samples were run in triplicates, and the fold change was calculated by 2–δδCt as described previously  following the manufacturer’s instructions. A total of 50 mg of homogenized material from flash-frozen tubers samples was utilized to determine the initial free sugar content in tuber samples at the time of harvest. The free sugar analysis was repeated on 50 mg of homogenized tuber samples, from tubers stored at 4°C for 3 months. In brief, the assay involved pH-dependent conversion of D-glucose and D-fructose into glucose-6-phosphate (G-6-P) intermediates using a hexokinase/phosphoglucose isomerase/glucose-6-phosphate dehydrogenase-based reaction and subsequent stoichiometric quantification of Nicotinamide adenine dinucleotide phosphate (NADPH) by absorbance at 340 nm . The sucrose level was calculated from the difference in D-glucose concentration before and after the hydrolysis by β-fructosidase. D-Glucose and sucrose standards were used to ensure accuracy of spectrophotometer measurements and effectiveness of the β-fructosidase hydrolysis reaction by comparing D-glucose to D-fructose ratio, respectively. All analyses were performed in triplicates; free sugar levels were calculated using Mega-Calc™  and reported as mean values of percentage on fresh weight (FW) basis.33.1Pho1a gene in the auto-tetraploid potato cv. Desirée. In total, 140 regenerated events were selected for HRFA screening for indels, and 129 out of the 140 events had induced mutations in at least one allele (92%) (data not shown). A total of 14 events, 5 events from Group 1 , 5 events from Group 2 , and 4 events from Group 3 , were selected for greenhouse cultivation and further characterization  against DM v6.1 genome assembly resulted in five hits on chromosome 3 with high sequence identity, namely, Soltu.DM.03G007710 to Soltu.DM.03G007760   was found to align with the genomic sequence between Soltu.DM.03G007710 and Soltu.DM.03G007720, and Soltu.DM.03G007750 and Soltu.DM.03G007760 with high similarity  and Pho1.2a  loci. Furthermore, the genomic sequence up to 7.7 kbp upstream of Pho1.1a and Pho1.2a coding sequence was nearly identical  covering Pho1.1a and approximately 23 kbp region (Chr03:16827879.16851055) encompassing Pho1.2a, were delimited by approximately 300-bp-long conserved sequence, which may have served as recombination hotspots  against the above-mentioned genome assemblies resulted in the identification of putative 1 or 2 Pho1a loci in respective assemblies  single Pho1a locus with insertion of Tst1, 3) duplicated Pho1a loci without insertion of Tst1, and 4) duplicated Pho1a loci with insertion of Tst1  during cultivation in greenhouse, except for SPD-9, which had an aberrant phenotype  Figure 83.7The total starch content in four of the five FKO events was found slightly lower as compared to Desirée (control)   where no significant change was detected in sucrose content between fresh and cold stored tubers (The higher average sucrose level of cold-stored FKO group tubers compared to Desirée (control), concomitant with a lower glucose level of cold-stored FKO group tubers compared to Desirée (control), could indicate that Pho1a has a role in cold sweetening of potato tubers.4Pho1a.The role of plastid starch phosphorylase is still under investigation in plants. Even though it is one of the most highly expressed genes in potato tubers and one of the most prominent enzymatic activities in amyloplasts, its role, if any, for reserve starch synthesis is still unclear. In this manuscript, we identified that the major form, Pho1a, is encoded by a duplicated gene locus, and we further characterized its role for reserve starch composition and phenotype by observing CRISPR/Cas9 mutants of Pho1a locus was found by the detection of up to seven differently mutated alleles in a single regenerated potato event. A plausible cause for this could be chimeric regeneration consisting of differently mutated somatic cells. However, applying RNP CRISPR/Cas9 tools to potato protoplasts generally avoids this problem from the transient nature of the application. Stable transformation for CRISPR/Cas9 mutations carries a higher risk of creating chimeric plants as mutations can occur at any time during the regenerative and vegetative phase. Copy number estimation of the Pho1a locus by RT-qPCR confirmed that more than one copy exists in the parental variety Desirée. A duplication of the Pho1a locus was further verified, analyzing the current available potato genome assembly DM v6.1. In addition, duplication of the Pho1a locus was also evident in multiple assembled wild potato genomes. The Pho1a copies on chromosome 3 were found to have highly conserved sequences in the DM v6.1 assembly. A ~7.5-kbp upstream region of both copies was also highly conserved. With the exception of a ~1.3-kbp long upstream genomic segment, ~25 kbp long genomic segment harboring Pho1.1a and ~23 kbp long genomic segment encompassing Pho1.2a were nearly identical. Both segments were delimited by ~300-bp-long conserved sequences, which were only detected in Pho1a loci from cultivated tetraploid potato cultivars. The presence of these conserved sequences may act as recombination hotspots during meiosis.The first indication of a duplicated Pho1a (X52385.1) spanned across 15 exons that were highly conserved in wild and cultivated potato assemblies, indicating an evolutionary constraint. However, the intronic regions were less conserved and contained species specific insertions. As a result, the length of a putative Pho1a locus ranged from 8,440 to 16,228 bp, depending on insertion and length of Tst1 in intron 5 and species-specific insertions in intron 2, 9, 11, and 13.A reference mRNA sequence of Pho1a locus is miss-annotated in the DM v6.1 assembly where Pho1.1a locus is annotated as Soltu.DM.03G007710 and Soltu.DM.03G007720. Both genes share a high sequence identity with exons 1–5 and exons 6–15 of reference mRNA sequence of Pho1a. Similarly, Pho1.2a locus is annotated as Soltu.DM.03G007750 and Soltu.DM.03G007760, which share high sequence identity to exons 1 and 2, 3–5 and exons 6–15 of reference mRNA sequence of Pho1a, respectively. A comparison of putative Pho1a loci among DM v6.1, DM v8.1, DM1S1, and S. tuberosum Group Phureja assemblies of E86-69 and E4-63 indicated probable sequencing errors leading to putative stop codons and thus resulting in mis-prediction of coding sequences of above genes. Furthermore, these sequencing errors are resolved in DM1S1, and duplicated Pho1a loci are annotated as Soltu.DM1S1.03G006810, i.e., Pho1.1a and Soltu.DM1S1.03G006830, i.e., Pho1.2a.The duplicated Pho1a has previously been reported to contain the transposon sequence Tst1 in the intron between exons 5 and 6  is the enzyme responsible for amylose synthesis, and the expression of the corresponding gene was assessed. The relative expression of Pho1.1a and Pho1.2a regarding contribution to Pho1a activity. Consistent with the literature  in potato leaves (Pho1a did not alter the number or size of starch granules in leaves of mutational events.Pho1 has been suggested to play a role in the transitory starch degradation due to high [Pi] concentration . Howeverbidopsis  and Pho1o leaves  did not o leaves . In agrePho1a has been suggested to be involved in the regulation of potato response to changes in temperature; however, effects of cold stress and cold storage of tubers have opposite effects . While p5Pho1a was shown to be tandemly duplicated in potato and thus named Pho1.1a and Pho1.2a. The gene duplication does not exist in tomato but can be found in species more related to Solanum tuberosum. Both genes encoding Pho1a were mutated using CRISPR/Cas9, and the impact on tuber development and starch accumulation was investigated in full knockouts. The results showed that lack of Pho1.1a and Pho1.2a have a large impact on tuber shape, size, and number per plant. Furthermore, an increase in small starch granules and more round granule shape was found, while the overall apparent amylose content was decreased. A differential, although low effect from absence of Pho1a, was observed upon cold storage of tubers that may indicate a role for Pho1a in starch degradation and cold sweetening. This study firmly establishes Pho1.1a and Pho1.2a as an important enzymatic factor in forming the starch granule phenotype and structure in reserve starch accumulation of potato tubers.The original contributions presented in the study are included in the article/PH conceptualized the study. PH, MA, and SS planned the study. SS, MF, MA, PV, HT, and NO performed experiments. All authors analyzed results. SS and PH wrote the manuscript. SS, PH, MA, and MF edited the manuscript. All authors approved the manuscript in its final form.


In this paper, a miniaturized weighing cell that is based on a micro-electro-mechanical-system (MEMS) is discussed. The MEMS-based weighing cell is inspired by macroscopic electromagnetic force compensation (EMFC) weighing cells and one of the crucial system parameters, the stiffness, is analyzed. The system stiffness in the direction of motion is first analytically evaluated using a rigid body approach and then also numerically modeled using the finite element method for comparison purposes. First prototypes of MEMS-based weighing cells were successfully microfabricated and the occurring fabrication-based system characteristics were considered in the overall system evaluation. The stiffness of the MEMS-based weighing cells was experimentally determined by using a static approach based on force-displacement measurements. Considering the geometry parameters of the microfabricated weighing cells, the measured stiffness values fit to the calculated stiffness values with a deviation from −6.7 to 3.8% depending on the microsystem under test. Based on our results, we demonstrate that MEMS-based weighing cells can be successfully fabricated with the proposed process and in principle be used for high-precision force measurements in the future. Nevertheless, improved system designs and read-out strategies are still required. The measurement of small forces is indispensable for various applications in different fields that range from nanoscale metrology to microbiology and quantum physics ,2,3. TheHowever, there are some aspects of a microcantilever that must be considered critically. The knowledge of the cantilever stiffness is essential for different reasons. Firstly, the stiffness has a linear influence on the measurement accuracy, which is also true for absolute force measurements, and therefore needs to be determined precisely. Furthermore, the stiffness must fit the input forces to be measured so that the beam deflects adequately. If the deflections are too large, resulting from insufficient stiffness, the measurement accuracy can be affected by occurring nonlinearities or even result in cantilever breakage. Therefore, knowledge of the cantilever stiffness is essential. Nevertheless, the stiffness variation of microcantilevers due to the manufacturing process is typically in the range of only 30%.To precisely measure macroscopic forces in a wide range, the principle of an electromagnetic force compensation (EMFC) balance is widely used ,7,8. AddInspired by the macroscopic force-compensation weighing cells, a microscopic force compensation mechanism is presented and its stiffness is characterized in correlation with microfabrication. The focus is not only on the system stiffness in the direction of motion but also in out-of-plane direction, which has not been investigated in other microscopic force compensation mechanisms so far. Furthermore, various aspects such as the analytical and numerical stiffness calculation, the microfabrication process, the influence of real system geometries on the stiffness and first stiffness measurements based on a force-displacement measurement are discussed. The comparison of the measured stiffness values to the calculated values shows an agreement with a deviation to each other from −6.8 to 3.8% depending on the investigated system and adapted geometry parameters.s, which is limited to 5 µm by inherent mechanical stops. The guiding mechanism resembles a parallelogram linkage with an upper (1) and a lower lever (2) and four flexure hinges (A–D). The deflection s is transferred to a transmission lever with a coupling element (4) and two flexure hinges , and amplified to an output deflection The basic mechanism studied in this work is inspired by macroscopic EMFC balances, as described in detail in . The impn in in-plane direction, which can generate comparatively high forces. The force of the actuator To detect the deflected shuttle position, a differential electrostatic comb sensor is used because of its very high resolution, which transduces the position into an electrical voltage signal. The controller is used to determine the required microactuator voltage, to move the shuttle back into its zero position. The utilized differential arrangement enables the operation of the sensor independently of environmental influences like temperature or air humidity. The microactuator is an asymmetric electrostatic plate actuator with several fingers A the overlapping area of the actuator plates, Here c, the resolution of the position sensor The resolution of the weighing cell, as per Equation (2), depends on the stiffness of the system The stiffness of the MEMS-based weighing cell is an important parameter for the achievable resolution of the system as shown in Equation (2). Therefore, a stiffness evaluation of the mechanism is required. It is first determined analytically using a rigid body approach and then also modeled numerically using the finite element method. The comparison of both approaches and the results are presented in the result The calculation of the stiffness of the single flexure hinges of the MEMS-based weighing cell, see in For the analytical stiffness calculation of the entire mechanism, a rigid body model is used to describe the MEMS-based weighing cell like Reference . The thiThe basis for the analytical description is the Lagrange equation, which is the equation of motion of a mechanical system (Equation (2)):y-direction, the degree of freedom of the system is φ2 of the transmission lever  from which the elastic potential energy of the hinges in the entire mechanism is obtained according to Equation (7):The force equation can be calculated by the first partial derivative of the elastic potential energy of the system according to the deflection angle. With the second partial derivative, the spring stiffness of all torsion springs in the entire mechanism can be calculated by:To derive the overall system stiffness, a conversion according to:Finite element method (FEM) simulations are state of the art for the numerical calculation of complex geometries and were therefore used for comparison of the analytical calculation and further investigations. For the numerical stiffness calculation, the mechanical Ansys Parametric Design Language solver of the software ANSYS was used for a static structural analysis.3 and in the rigid parts around 3400 µm3. Parameter studies showed that these meshing combinations yield a convincing compromise between computational time and model accuracy. As model material, anisotropic silicon  was used directly from the ANSYS material library. The parts connected to the frame are secured with fixed supports and the force is introduced on the upper part of the shuttle, the probing area as indicated in s is calculated at the point of the force application. The system stiffness was derived assuming a force of For the simulation, the flexure hinges were modeled as separate solid bodies in the computer-aided design program (Autodesk Inventor) and then connected with surface-surface-contacts in ANSYS a. The elThe microfabrication process of the previously described MEMS-based weighing cell is based on well-established silicon-technology and thin film processing. The workflow and the material choices for the fabrication are shown in the schematic overview in For the realization of the contact pads on the device layer, an aluminum layer of 800 nm is used  process (Oxford ICP PlasmaPro100 Estrelas) and an approximately two micrometer thick resist mask   acts on the loading button (3), the aluminum-based balance (2) deflects from its zero position. This deflection is detected by means of a slit aperture (1) and a differential interferometer (9) (from SIOS Messtechnik). The deflection signal in the form of an analog voltage or a digital quadrature signal is transmitted to the controller that compensates the deviation from the set position. This is done by transferring an according compensation voltage to a voltage-current converter, which is connected to the coil (7) in a constant magnetic field (6). Due to the occurrence of Lorentz forces, the balance returns to its zero setting. The compensation current is read-out with a multimeter and finally used to determine the stiffness. The main advantage of this measuring device is the linearity of forces. The elastic force of the MEMS-based weighing cell is calculated with the following Equation:x, y, z  to record the displacement of the weighing balance signal. The positioning of the MEMS-based weighing cell relative to the loading button in the directions To evaluate the stiffness as a function of the microsystem geometry, systems as shown in The stiffness of the single hinges obtained with the detasFlex software, which were used for the analytical calculation are shown in The analytical and numerical results differ with a maximum of 3.8%. The stiffness ratio is a means of describing the relation of the stiffness in out-of-plane motion to the stiffness in the direction of motion. As the stiffness of the individual hinges increases in the direction of motion, the stiffness in the out-of-plane direction also increases, but not to the same degree. Depending on the hinge parameters the stiffness ratio varies from 1.8 for the stiffest system to 10.8 for the system with the lowest stiffness in direction of motion.As discussed before, geometry deviations have a significant influence on the stiffness of the weighing cell. Therefore, the lateral and vertical geometry deviations of the fabrication process are analyzed in detail in the following section and an overview of the measured geometries are shown in The lateral geometry is around 0.5 to 1 µm smaller than specified in the design a,d,e. A zR = 0.14 µm) of the roughness of the lower 50 µm etch depth (zR = 0.57 µm). Since the etch rate decreases in depth with longer etching and ends at 200 cycles for the 100 µm thick component layer, the etch riffles got smaller the deeper the structures are etched.Vertical geometry deviations resulting from the DRIE process are exemplarily shown in Due to the manufacturing process of deep reactive ion etching (DRIE), see To evaluate the influence of a sidewall angle, as marked in Comparing the numerical stiffness in direction of motion of a single flexure hinge with a sidewall angle of 90 ° determined by means of FEM simulation with the calculation results based on the detasFLEX software, deviations of −2.3 to 1.1% are obtained. The studies have also shown that the stiffness decreases with decreasing sidewall angle as shown in Furthermore, it was observed that the smaller the height of the flexure hinge, the larger are the deviations resulting from different sidewall angles in a range of just one degree. For instance, for a hinge height of 15 µm the influence of a deviation of one degree in sidewall angle results in a deviation of 30% for the stiffness. For a hinge height of 5 µm, the deviations are already around 65%. Regarding the out-of-plane motion direction, the same behavior can be observed for the correlation between stiffness and etching angle b. AdditiThe stiffness of the MEMS-based weighing cell is measured using the previously described force-displacement measuring device a. The weThe movement of the shuttle of the MEMS-based weighing cell and the compensating current of one of the cycles during the long-term measurement of system W 3-1 C1 are shown in The difference between the loading and unloading force is measured to be approximately 25 µN per cycle c. A sligx-direction could not be done with the same precision based on the setup design, which was originally built for measurements in one direction only. The results of the eccentric test of system W 3-1 is exemplarily shown in The guiding system of the MEMS-based weighing cell mechanism has been designed in such a way that the force measurement is invariant to the force application point on the probing area. Accordingly, the stiffness measurement of the MEMS-based weighing cell should be independent of the force application point as well. To verify this assumption a so-called eccentric test was conducted. For this, four additional force application points are used for measuring the stiffness, as shown in Based on the previous measurements done in the The measurements showed that the fabricated flexure hinges have slightly lower hinge heights than specified in the original design, cf. The deviations between the calculated stiffnesses of the MEMS-based weighing cells and the experimentally determined stiffnesses range from 6.8 to 70%, .During the stiffness measurements, it is not possible to observe the movement of the shuttle of the MEMS-based weighing cell. Accordingly, any system deterioration, such as breakage of a hinge or even of the entire system, cannot be monitored and thus not prevented. Subsequent inspections of the systems have shown a damaged flexure hinge A W3-1 and W2-1 and a complete breakage of the systems W 3-1 C2 and W 3-2. The later occurred during the out-of-plane stiffness measurement. Therefore, the following discussion is partly based on theoretical assumptions.h = 15 µm, sufficient agreement between the calculated and measured stiffness was demonstrated with a deviation of only 0.1%. System W 3-1 C2 and W 3-1 C1 are expected to have the same stiffness due to their identical geometry. However, this could not be shown in the measurements , and dueThe design, fabrication and mechanical analysis of functional MEMS-based weighing cells was demonstrated. For microfabrication, silicon-on-insulator substrates and deep reactive ion etching (DRIE) with optimized process parameters were used to fabricate, for example, the required comb capacitors, shuttle and bending hinges. The system was particularly studied in terms of in-plane and out-of-plane stiffness, which is a crucial parameter. A rigid body model for the stiffness calculation was presented and discussed to efficiently determine the stiffness of the entire microsystem and for a reduced MEMS-based weighing cell mechanism. Numerical simulations based on the finite element method were performed for comparison purposes. The system stiffness analysis was performed taking into account the manufacturing deviations. The analytical results were in convincing agreement with the simulations, with deviations ranging from 0.04 to 3.8%. The microfabricated MEMS-based weighing cells were furthermore also experimentally characterized with respect to in-plane and out-of-plane stiffness using a force-displacement measurement approach. The measured stiffness in the direction of motion showed with deviations in the range of 0.1 to 6.7% sufficient agreement with the analytical results.However, an improved experimental characterization approach is needed to avoid breakage of MEMS components and to improve the overall robustness of the measurement. Nevertheless, we demonstrated that MEMS-based weighing cells can be successfully realized in analogy to their macroscopic counterparts, which provides a solid basis for further research in this field.


Oryza sativa L.) quality has been paid more and more attention recently. Cooking and eating quality (CEQ) is an important indicator of rice quality. Since CEQs are quantitative traits and challenging for measurement, efforts have mainly focused on two major genes, Wx and SSIIa. Chalkiness and floury endosperm significantly affect the eating quality of rice, leading to noticeable changes in CEQ. Due to the easily observable phenotype of floury endosperm, cloning single gene mutations that cause floury endosperm and evaluating changes in CEQs indirectly facilitate the exploration of the minor genes controlling CEQ. In this study, eight mutants with different degrees of floury endosperm, generated through ethylmethane sulfonate (EMS) mutagenesis, were analyzed. These mutants exhibited wide variation in starch morphology and CEQs. Particularly, the z2 mutant showed spherical starch granules significantly increased rapid visco analyzer (RVA) indexes and urea swelling, while the z4 mutant displayed extremely sharp starch granules and significantly decreased RVA indexes and urea swelling compared to the wild type. Additionally, these mutants still maintained correlations with certain RVA profiles, suggesting that the genes PUL, which affect these indexes, may not undergo mutation. Cloning these mutated genes in the future, especially in z2 and z4, will enhance the genetic network of rice eating quality and hold significant importance for molecular marker-assisted breeding to improve rice quality.Besides increasing grain yield, improving rice ( Rice is one of the most important staple foods for more than half of the world’s population. With economic development, people not only focus on rice yield but also pay more attention to rice quality. Rice quality includes appearance quality, cooking and eating quality (CEQ), milling quality, and nutritional quality. Among them, appearance quality and CEQ have been particularly emphasized by breeders.Chalk5 and WCR1, have been cloned so far [Appearance quality of rice includes grain shape, chalky rate, and chalkiness degree. Among them, the chalkiness degree has the most significant impact on rice quality, as it not only affects the appearance but also reduces milling yield after processing . Chalkind so far ,4. The fd so far , amylopld so far ,8, proted so far ,10, mitod so far ,12, trand so far ,14. Besid so far . High-teWaxy (Wx), plays a very important role in determining AC in endosperm starch [Wx alleles have been identified: aWx, bWx, mqWx, inWx, mpWx, op/hpWx, lvWx, mwWx/laWx and null Wx [The evaluation of CEQ is typically based on three physicochemical characteristics of starch: amylose content (AC), gel consistency (GC), and gelatinization temperature (GT) . AC is tm starch . So far, null Wx .starch synthase IIa (SSIIa), located on the ALK locus of chromosome 6 [Wx locus also affected GT, although GT has not yet been found to be correlated with AC [GT is the temperature range at which the starch granules swell irreversibly in hot water and starch crystalline structures begin to melt, which determines the cooking time . GT can mosome 6 . Wx locu with AC .Wx locus [Wx gene, from C-T, is a significant factor influencing gel consistency [Wx gene controls not only AC and also GC. ALK was also reported as a modifier gene for GC in rice [The GC is a measurement of the hardening of cooked rice when cooled. GC can be classified as soft (>60 mm), adhesive (40–60 mm), or hard (<40 mm). Rice with hard GC is rough and fluffy and becomes hard and dry after cooling, while rice with soft GC is soft and elastic and still keeps soft after cooling. Usually, the larger the GC, the softer, stickier, better taste, and shinier rice . As a quWx locus ; the SNPsistency . Wx gene in rice .Wx gene is a key determinant in the control of RVA profile parameters in rice, while the pullulanase (PUL) gene was shown to play an important role in the control of most of the RVA profile parameters [Starch viscosity profiles tested by a rapid visco analyzer (RVA) have also been utilized to select desirable rice varieties with specific CEQ . RVA is rameters .There are significant correlations among the various sensory qualities of cooked rice. GC and ASV have negative associations with AC . Lower AWx and SSIIa through QTL mapping. However, the phenotypes of chalkiness and floury endosperm are easily identifiable, and these traits usually affect CEQs with potentially low eating qualities [Wx and SSIIa genes for improving rice eating quality in molecular breeding. Eight rice mutants with varying degrees of floury endosperm were used to analyze changes in CEQs under one gene mutated in each mutant. This research provides valuable genetic resources for understanding the fine structure of starch with respect to the textural properties of cooked rice grains.Since it is challenging to directly measure the CEQ traits, currently, the only cloned major genes are ualities . Therefo3 populations created by EMS mutagenesis from varieties ZXYZ  and QJ101  were screened. Moreover, 55 independent mutants were isolated with different floury degrees (FD) endosperm. Among them, eight mutants with single gene mutations  are formed in amyloplasts, which are organelles involved in starch synthesis and storage. A single amyloplast is assembled from dozens of polyhedral SGs that have sharp edges and are easily separable. SGs can be visualized easily under light microscope by staining with iodine solution.z1–z4, q1, and q2  [gif1, bzip58) [ssg4, gpa8, fgr1, flo16) [flo7) [z2 and q2 grains of the same panicle. It was found that this polymorphism in seed morphology remained stable across generations, suggesting it could be influenced by the environment, particularly with temperature in the grain-filling stage. This conclusion is supported by the fact that the majority of rice plants in the experiment exhibited flowering in early September, coinciding with a gradual decrease in temperatures throughout the month.In recent years, some new floury endosperm mutants were obtained through chemical or physical mutagenesis, with phenotypes similar to chalky rice, which can be regarded as extreme chalky phenotypes . These m, flo15) ,39,40, w bzip58) ,42, flou, flo16) ,11,43,44) [flo7) , which cz2 and z4 mutants examined in this study exhibit two distinct phenotypes. In the z2 mutant, nearly all starch granules have adopted a spherical shape  and SRGM (sharp starch granule mutation), respectively. Future research involving the cloning of RSGM and SRGM will deepen our understanding of this process.Rice endosperm amyloplasts produce characteristic compound-type starch granules, which consist of dozens of polyhedral, sharp-edged granules . The molal shape c, a phen mutants . SS4b isgranules e. Accordz2 began to significantly increase in a 5 M urea solution while it significantly decreased in z4, as did the changes in PV, TV, and FV, indicating that changes in starch granule morphology can reflect changes in CEQs. It is hypothesized that the spherical shape of starch granules in z2 may be related to the synthesis of branched chains in amylopectin. The following three reasons can explain it: (1) The urea-induced swelling of starch granules depends on the structure of branched starch rather than the extent or structure of linear starch [ss3a ss4b [z4 mutant’s extremely pointed starch granule morphology and the opposite trends of SU, PV, TV, and FV compared to the z2 mutant can further enhance the understanding of the relationship between starch granule morphology and the length of amylopectin branches. It should be noted that some floury mutants have changed starch granule morphology, but compared with the wild type, there is no significant difference in starch properties [Moreover, compared with the wild-type ZXYZ, SU in r starch . (2) Ther starch . (3) Sphs3a ss4b . Combinioperties ,46. TherWx and ALK, they had varied AC, GC, and ASV, and these parameters showed no correlation among the mutants  varieties QJ101 and ZXYZ through EMS mutagenesis. These mutants were homozygous and self-pollinated for three generations. The wild-type rice and mutants were planted simultaneously in an experimental field at the Zhejiang Academy of Agricultural Sciences in Hangzhou, China. The mature grains were dehulled, and the brown and milled kernels were used as plant materials for this study.The floury endosperm rice mutants were derived from the indica rice . Mature rice seeds were transversely cut using a knife, and samples were examined with a HITACHI TM3000 scanning electron microscope.v/v) glutaraldehyde and 2% (w/v) paraformaldehyde. After dehydration in an ethanol series, samples were embedded in LR White resin , sectioned using an ultramicrotome , and observed under a microscope .Transverse sections of 7 DAF endosperms (approximately 1 mm thickness) of the wild type and eight mutants were fixed overnight in 0.1 M phosphate buffer (pH 7.2) with 2% .Semi-thin sections of 7 DAF endosperm from the wild type and eight mutants were prepared as described by . SectionThe pasting properties of rice flour samples were measured using an RVA analyzer  within a short period of 12.5 min. Approximately 3 g of flour (12% moisture basis) from each rice sample was weighed directly into the aluminum canister and mixed with 25 g of distilled water. The RVA dispersed the samples by rotating the paddle at 960 rpm for the first 10 s of the test, after which the viscosity was sensed using a constant paddle rotation speed of 160 rpm. The idle temperature was set at 50 °C, and the following 12 min test profiles were run in this order: (1) hold at 50 °C for 1.0 min, (2) linearly raise temperature to 93 °C until 5.5 min, (3) hold at 93 °C until 7 min, (4) linearly reduce temperature to 50 °C at 11 min, and (5) hold at 50 °C until the full 12.5 min. All analyses were conducted in triplicate. Viscosity values were recorded in centipose (cp).http://www.megazyme.com/, accessed on 6 February 2022) according to the manufacturer’s protocol. The amylose content was assessed following the method described by [The starch content of rice flour was measured using a starch assay kit .A total of 20 mg of WT and eight mutants flour was mixed with 1 mL of a solution of 0 to 9 M urea, the pH of which was adjusted to 6.0 with acetic acid in an Eppendorf tube. The mixture was incubated at 25 °C for 24 h. The suspension was centrifuged for 20 min at 8000×


Acute-on-chronic liver failure (ACLF) is an acute decompensated syndrome based on chronic liver disease, while neutrophil recruitment is the most critical early step. C-X-C motif chemokine ligand 1 (CXCL1), a cytokine that recruits neutrophils, was significantly upregulated in both ACLF mice and patients with ACLF. This present study aims to explore the role of CXCL1 in the pathogenesis of ACLF.Cxcl1. We employed mass cytometry, flow cytometry, multiplex cytokine and chemokine analysis, Western blot, and reactive oxygen species (ROS) detection in mice blood and liver. ACLF patients (n = 10) and healthy controls (n = 5) were included, and their liver samples were stained using multiplex immunohistochemistry techniques.We established an ACLF mouse model induced by carbon tetrachloride, lipopolysaccharide, and D-galactosamine, and used adeno-associated virus to achieve overexpression and knockdown of CXCL1 was significantly elevated in both ACLF mice and patients. CXCL1 recruits neutrophils by binding to the C-X-C motif chemokine receptor 2 on the surface of neutrophils, affects ACLF prognosis by generating ROS and mitochondrial depolarization and modulating caspase3-related apoptotic pathways. We found that the knockdown of CXCL1 attenuated the infiltration of neutrophils in the mouse liver, reduced the expression of inflammatory cytokines, and also significantly downregulated ROS production and caspase3-related hepatocyte apoptosis, thereby ameliorating the liver injury of ACLF.CXCL1 is a core player in the mobilization of neutrophils in ACLF, and the knockdown of Cxcl1 improves neutrophil infiltration, reduces ROS levels, and reduces hepatocyte apoptosis, thereby attenuating inflammation and liver injury in ACLF. Our results revealed a previously unknown link between CXCL1-induced neutrophil recruitment and ACLF, providing evidencing for potential therapies targeting ACLF. Liver transplantation is the only cure.3 However, the number of liver donors is far less than the number of patients. Thus, ACLF urgently needs new early warning and treatment methods.Acute-on-chronic liver failure (ACLF) is a syndrome of acute decompensation based on chronic liver disease, accompanied by high short-term mortality and multiple organ failure.8 A study by Casulleras et al showed that patients with ACLF had significantly higher neutrophil counts compared with those of healthy individuals and patients with cirrhosis.9 Following bacterial infection and/or tissue injury, neutrophils are rapidly activated, move out of blood vessels, and migrate along a concentration gradient of chemoattractants to target sites, where they recognize and bind to pathogens through pathogen-associated molecular patterns, resulting in phagocytosis of the pathogens.10 During phagocytosis, the NADPH oxidase complex is activated and releases antimicrobial peptides, proteases, and numerous reactive oxygen species (ROS) to kill and destroy bacteria.11 ROS are normal by-products of aerobic metabolism and are produced in mitochondria; however, excess ROS can cause oxidative damage to cells, resulting in intracellular oxidative stress and cell damage. The liberation of cellular contents further exacerbates inflammatory damage. In addition, ROS also promotes the secretion of cytokines, leading to an increase in ROS production, forming a vicious circle and promoting the deterioration of liver disease.As the first barrier of the innate immune system, neutrophils play a vital role in the development of liver injury, including alcohol-associated hepatitis, acute liver failure, and ACLF.12 CXCL1 is highly expressed during the inflammatory response, thereby promoting the inflammatory process. It plays an important role in neutrophil-dependent immune-induced lung inflammation, sepsis, hepatitis, and HSV1-related encephalitis.15 In addition to inducing inflammation, recent studies have demonstrated that CXCL1 is involved in the pathogenesis of liver diseases, such as NASH, HCV-related hepatitis, DILI, and HCC.18 Chang et al demonstrated that blocking CXCL1 reduced hepatic neutrophil infiltration and injury in a mouse model of NASH, whereas Cxcl1 overexpression exacerbated steatohepatitis in mice.19 A study by Hilscher et al showed that the upregulation of CXCL1 promotes neutrophil aggregation and induces hepatic sinusoidal thrombosis and fibrosis through the formation of neuroendocrine tumors.20Several signaling pathways are involved in the chemotaxis and recruitment of neutrophils, and one of the important pathways is the C-X-C motif chemokine ligand 1 (CXCL1)-C-X-C motif chemokine receptor 2 (CXCR2) pathway. CXCL1, a member of the CXC chemokine family, mediates neutrophil recruitment by binding to CXCR2 and is important for neutrophil activation, recruitment, and infiltration.21 However, the unresolved challenge is that we do not know the pathway through which CXCL1 affects the progression of ACLF. In the present study, we established an ACLF mouse model, achieved the overexpression and knockdown of the Cxcl1 gene by adeno-associated virus infection, and confirmed that CXCL1 can recruit neutrophils in the liver. We explored the potential mechanism of CXCL1 affecting the process of ACLF and found that it may trigger some signals that lead to massive death of liver cells, such as excessive production of inflammatory factors and ROS, mitochondrial dysfunction, etc., inducing a large number of liver cell apoptosis, and cause damage to the liver. In addition, we performed further validation in human liver tissue using multiplex immunofluorescence techniques and found that patients with ACLF with low CXCL1 expression had lower levels of neutrophils and ROS in their liver tissue.Previously, in a prospective study, we reported that serum CXCL1 levels and neutrophil counts were significantly higher in patients with HBV-ACLF than in healthy controls and chronic HBV groups and that serum CXCL1 was an independent risk factor for HBV-ACLF prognosis.Liver samples for this study were obtained from 5 healthy donors and 10 patients with ACLF who underwent liver transplantation. The protocol was approved by the Human Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine, and written informed consent was obtained from the subjects (license number: IIT20220273B).Wild-type male C57BL/6 mice, 6–8 weeks old, were obtained from the Animal Experiment Center of Zhejiang Academy of Medical Sciences. They were raised in a room with standard temperature and light, with free access to adequate food and water. All animals were cared for in accordance with the guidelines of the Zhejiang Provincial Institute of Health. Our animal experiment protocol was approved by the Animal Care Committee of the First Affiliated Hospital of Zhejiang University School of Medicine for Animal Experiment Ethics Examination (license number: 2022-1617).4 injection with a single dose of LPS/D-GaIN. Mice were randomly divided into 4 groups: NC group, CCl4 group, D-Gal + LPS group, and ACLF group. The NC group (n = 10) was continuously injected with olive oil for 8 weeks, sacrificed after the last injection, and the serum and liver tissue were collected. Group CCl4 (n = 10) was i.p. injected with a mixture of CCl4  and olive oil twice a week for 8 weeks and then sacrificed. Group D-Gal + LPS (n = 10) received a single i.p. injection of D-GalN  and LPS  and then sacrificed. The mice in group ACLF were i.p. injected with a mixture of CCl4  and olive oil for 8 weeks. At 24 hours after the last injection, they were i.p. injected with D-GalN  and LPS , and their survival was observed every hour. Mice were sacrificed at 0, 3, 6, 9, and 12 h after injection, 10 mice in each time point, and serum and liver tissue were collected.To mimic the clinical progression of ACLF, we combined long-term CClCxcl1 overexpression group (CXCL1-OE); and Group 4: Cxcl1 knockdown group (CXCL1-KD). Mice in group 1 were injected with olive oil twice a week for 8 weeks. Mice in groups 2, 3, and 4 were injected with CCl4/olive oil mixture twice a week for 4 weeks. To achieve Cxcl1 overexpression and knockdown, 20 μg of Cxcl1 overexpression adeno-associated virus  and 20 μg Cxcl1-interfering adeno-associated virus  were injected into the tail vein of mice in groups 3 and 4 in the 5th week. As controls, we set up a CXCL1-OE control group (n = 5) and a CXCL1-KD control group (n = 5). We infected ACLF mice with overexpression control AAV virus  and interference control AAV virus . The mice were then injected with the CCl4/olive oil mixture twice a week for another 4 weeks. At 24 hours after the last injection, mice were i.p. injected with a mixture of D-galactosamine  and LPS , sacrificed 6 h later, and serum and liver tissue were collected.The mice were randomly divided into 4 groups, 10 mice in each group, and the grouping conditions were as follows: Group 1: normal control group; Group 2: ACLF group; Group 3: t-tests were used to compare differences between 2 groups, and 1-way ANOVA was used to compare differences among more than 2 groups. Statistical significance was set as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001.GraphPad Prism 8  was used for graphing and data analysis. Two-tailed unpaired 4 + LPS + D-GalN-induced liver injury mouse model to mimic the disease progression of ACLF , indicating that apoptosis may have occurred. The above data indicated that our ACLF mice had liver fibrosis, acute liver injury, cytokine storm, and poor prognosis, similar to the clinical manifestations of human ACLF.In this study, we used a CClF Figure A. Eight % Figure B. The se% Figure C. The li% Figure D. H&E st% Figure E. To inv% Figure A, while 4+ Dgal/LPS-induced ACLF model reported here, we also detected a significant increase in CD45 + neutrophils in the APAP-induced ACLF mouse model . And it has been reported that neutrophils also play a key role in ACLF caused by liver resection. To explore the underlying mechanism of neutrophil recruitment in ACLF, we analyzed MPO immunohistochemical staining at different time points after acute injury. MPO is a lysosomal protein present in the azurophilic granules of neutrophils and is an activation marker of neutrophils. We found that the amount of MPO + neutrophil infiltration gradually increased, reached a peak at 6 hours, and then decreased . Then, we analyzed the level of CXCL1 in the serum of mice in each group by ELISA and evaluated the relative mRNA expression level of CXCL1 in liver tissue by qRT-PCR. The results showed that the changes in the level of CXCL1 in the mouse liver and serum were in line with our expectations .To explore the role of CXCL1 in neutrophil recruitment and host defense, ACLF mice were infected by tail vein injection of adeno associated virus (AAV) to achieve knockdown or overexpression of 1 Figure A. To enss Figure B. Interes Figure C. To excThen, mass cytometry and flow cytometry analysis revealed local and systemic immune changes in mouse liver after AAV inoculation. The 32 major cell clusters were represented by a t-distributed stochastic neighbor embedding plot on the basis of the CyTOF results . The results showed that after the combined treatment with CCL4+D-GalN+LPS, the levels of pro-inflammatory cytokines and anti-inflammatory cytokines in serum were significantly increased, and there was a correlation with the level of CXCL1. In addition, the levels of granulocyte colony-stimulating Factor (G-CSF) and monocyte chemotactic protein 1 were also significantly increased in the CXCL1-OE group and decreased in the CXCL1-KD group  and mixed lineage kinase domain. It was found that the levels of RIPK3 and mixed lineage kinase domain were indeed significantly upregulated in the ACLF group compared with the NC group but not related to the knockdown and overexpression of CXCL1. Moreover, phosphorylated RIPK3 and phosphorylated mixed lineage kinase domain were barely detectable, suggesting that this pathway was not activated . The autophagy-related marker protein LC3BII/I was also increased in the ACLF group, but there was no significant difference in the CXCL1-OE group and CXCL1-KD . These results demonstrate that knockdown of CXCL1 may alleviate liver injury in ACLF mainly by attenuating hepatocyte apoptosis, rather than through pathways such as cell necrosis and autophagy.The main cause of ACLF is a persistent inflammation of the liver and massive hepatocyte death. To explore the mechanism of ACLF liver injury caused by the massive accumulation of ROS in hepatocytes, we analyzed several main signaling pathways that may be involved, including apoptosis, necrosis, autophagy, etc. Terminal deoxynulceotidyl transferase nick-end-labeling staining indicated that the degree of apoptosis in the liver of ACLF mice was significantly increased, and knockdown of s Figure A. Neutros Figure D, which s Figure B. Westers Figure C–F. KnocIn our previously published study, we showed that serum CXCL1 levels and neutrophil counts were significantly higher in patients with HBV-ACLF than in healthy individuals and patients with chronic HBV or HBV-ACLF–compensated cirrhosis. However, it has not been validated in liver tissue from patients with ACLF. We collected liver tissues from 5 healthy controls and 10 patients with ACLF for further validation. Immunohistochemical staining for CXCL1 was performed. We found that CXCL1 was hardly expressed in the livers of the healthy controls, whereas it was highly upregulated in the livers of patients with ACLF Figure B. Howeve23 Our findings confirmed that the level of CXCL1 was significantly upregulated in both patients with ACLF and ACLF mice, recruiting more neutrophils to produce ROS, which led to the death of hepatocytes. In ACLF mice, overexpression of Cxcl1 markedly increased the number of hepatic neutrophils and aggravated oxidative stress, which in turn activated the downstream caspase3 pathway, leading to hepatocyte apoptosis, liver injury, and inflammation. Knockdown of Cxcl1 reduced the degree of neutrophil infiltration, ROS production, and hepatocyte apoptosis in liver tissue. Therefore, CXCL1 might be a key factor affecting the prognosis of ACLF. Here, we constructed a schematic diagram of the proposed CXCL1-mediated hepatic inflammation and injury in the hepatic microenvironment of ACLF , LPS stimulates the release of inflammatory factors by binding to toll-like receptor 4 on the surface of macrophages.25 Compared with certain existing models, the ACLF mouse model in this study includes features such as chronic liver injury, acute liver injury, bacterial infection, massive neutrophil infiltration, cytokine storm, hepatocyte damage, necrosis and apoptosis, and poor prognosis, which mimics the complex prognostic situation of clinical ACLF.Our animal model combines a double-hit regimen based on long-term CCl4-induced liver injury model, the chemokine CXCL1 did not induce hepatic neutrophil infiltration.18 However, in this study, we provided evidence that CXCL1 plays a key role in neutrophil recruitment and, by promoting hepatic neutrophil infiltration, contributes to liver injury. First, in the CCl4+D-GalN+LPS-induced ACLF mouse model, the level of CXCL1 was observably up-regulated in serum and liver tissue, and the results of immunohistochemical staining and flow cytometry confirmed that neutrophils were also significantly elevated. Then, in the ACLF mouse model, we overexpressed and knocked down the Cxcl1 gene in the mouse liver using AAV. In the CXCL1-OE group, the degree of hepatic neutrophil infiltration was markedly increased, whereas, in the CXCL1-KD group, it was decreased. Furthermore, we found that the level of G-CSF was also significantly increased in the CXCL1-OE group. G-CSF mainly acts on the proliferation, differentiation, and activation of the neutrophil lineage and is one of the most important growth factors for the production of granulocytes and their immediate precursors.26 Our study confirmed that the level of G-CSF in the serum of mice was significantly increased in the case of overexpression of Cxcl1, while the level of G-CSF was markedly decreased in the Cxcl1 knockdown group. Correspondingly, flow cytometry showed that the number of neutrophils in the peripheral blood of the CXCL1-OE group was significantly higher than that of the CXCL1-KD group. At the same time, we performed multiplex immunohistochemistry in the liver samples of patients with ACLF to verify this finding, which showed that both CXCL1 levels and neutrophil levels were significantly increased in patients with ACLF. Our findings also provided visual confirmation that CXCL1 binds to CXCR2 on the surface of CD15+ neutrophils to stimulate neutrophil recruitment. A study demonstrated that blocking CXCR2 inhibited hepatic neutrophil infiltration and injury in a chronic ethanol feeding model precisely because CXCR2 is a receptor for CXCL1.27 In addition, when patients with ACLF were grouped by their CXCL1 levels, their liver ROS levels also correlated positively with CXCL1 levels.Neutrophils act as the first line of defense at sites of infection and tissue damage, and extensive neutrophil infiltration is a feature of ACLF. Interestingly, Stefanovic’s study revealed that in a CCl28 ROS are a normal by-product of aerobic metabolism and play a vital decision in some pathological processes, and excessive ROS can cause oxidative damage to cells, inhibit cellular function, induce mitochondrial phagocytosis, and induce apoptosis.29 The liberation of cellular contents further exacerbates inflammatory injury.30 In addition, ROS promotes the secretion of cytokines, which in turn leads to an increase in the production of ROS, forming a vicious circle and further promoting liver damage. The massive accumulation of neutrophils and excessive production of ROS have been reported to be one of the important causes of various inflammation-related liver diseases, such as alcohol-associated liver disease, hepatic ischemia/reperfusion injury, HCC, and ACLF.32 In addition, damage-associated molecular patterns released after ROS lead to necrosis and apoptosis of hepatocytes. These damage-associated molecular patterns activate innate immune cells in the liver through pattern recognition receptors to produce inflammatory cytokines, including IL-6, TNF-α, and IL-1β, and amplify the inflammatory response, thereby aggravating ACLF. To investigate whether knockdown of Cxcl1 is associated with the inhibition of pro-inflammatory cytokines in ACLF, we detected IL-6, TNF-α and IL-1β in the serum of each group. As expected, in the Cxcl1 knockdown group, they were markedly reduced, which alleviated the hepatic inflammatory response.Our results demonstrate that in ACLF mice, the mitochondrial membrane potential of hepatocytes is depolarized, and there is a large amount of ROS diffusely present in the liver. Further, we examined and analyzed liver tissue from patients with ACLF and found a significant increase in ROS. In ACLF mice, the level of ROS in the CXCL1-OE group was significantly upregulated; and the level of ROS in the CXCL1-KD group was significantly downregulated. After being recruited, neutrophils recognize, bind, and phagocytose pathogens through pathogenic pathogen-associated molecular patterns. During phagocytosis, the NADPH oxidase complex is activated and releases antimicrobial peptides, proteases, and a large amount of ROS to kill and destroy bacteria.33 After detecting and analyzing certain marker molecules of each pathway, we found that under the influence of different levels of CXCL1, the level of hepatocyte apoptosis in ACLF mice changed significantly. The results of terminal deoxynulceotidyl transferase nick-end-labeling staining showed that there was abundant hepatocyte apoptosis in the liver tissue of ACLF mice, and when Cxcl1 was overexpressed, the degree of apoptosis was further aggravated, and when Cxcl1 was knocked down, the level of liver apoptosis was weakened. This result indicated that antagonizing chemokine CXCL1 would be beneficial to ameliorate the degree of apoptosis of hepatocytes caused by excessive ROS production. Further Western blotting analysis showed that the protein levels of NF-κB, cleaved caspase3, cleaved PARP, and BAX decreased in the CXCL1-KD group, indicating that antagonizing CXCL1 has a marked anti-apoptotic effect. PARP is closely related to DNA repair, and its cleavage is considered to be an important indicator of apoptosis. Activated caspase-3 cleaves PARP, thereby promoting apoptosis. BAX is a pro-apoptotic protein whose overexpression can antagonize the protective effect of BCL-2, increasing the tendency of cells to die. It has been demonstrated that lipid peroxidation products can induce apoptosis through different signaling pathways, including the NF-κB pathway, MAPK pathway, and protein kinase C pathway. The NF-κB protein family is widely involved in inflammation, stress response, survival, and cell death and is also responsible for the transcriptional regulation of anti-apoptotic gene expression.34 We also analyzed other cell death pathways. The key regulators of cell necrosis RIPK3 and RIPK3 were indeed up-regulated in the ACLF group, but they were not correlated with the level of CXCL1, and their phosphorylated active proteins were not detected. Autophagy-related marker protein LC3BII/I also did not change significantly. Consequently, we propose that intervention for CXCL1 could affect the prognosis of ACLF by decreasing neutrophil recruitment and infiltration to reduce ROS production, thereby regulating NF-κB and alleviating the downstream caspase3 apoptotic pathway, rather than through pathways such as cell necrosis and autophagy.The next question is, “By which pathway does ROS affect the severity and prognosis of ACLF?” Studies have indicated that ROS-induced lipid peroxidation plays a key role in cell death and participates in the regulation of apoptosis, autophagy, and ferroptosis.Cxcl1 ameliorated CCL4+D-GalN+LPS-induced hepatic neutrophil infiltration and injury, improved ROS levels, and reduced hepatocyte apoptosis.In summary, we found that CXCL1 plays an important role in ACLF. First, the expression level of CXCL1 was significantly upregulated in both ACLF mice and patients with ACLF. Second, multiplex immunohistochemistry confirmed that CXCL1 recruits neutrophils by binding to CXCR2 on their surface. Third, the knockdown of In summary, we successfully constructed a model of ACLF and demonstrated that the expression level of CXCL1 was significantly upregulated in both ACLF mice and ACLF patients. The chemokine CXCL1 regulates neutrophil mobilization by binding to CXCR2 on the neutrophil surface, generating massive oxidative stress, mitophagy, and induction of caspase-associated hepatocyte apoptosis. Whereas Cxcl1 knockdown improved neutrophil infiltration, alleviated ROS levels, and reduced hepatocyte apoptosis, thereby attenuating inflammation and liver injury in ACLF. These results provide a theoretical basis for ACLF research and may provide new ideas for the clinical treatment of ACLF.


Fluids and Barriers of the CNS . We are happy to announce that Dr. Mark Hamilton  will become Co-Editor-in-Chief (joining Drs. Richard Keep and Lester Drewes). Dr. Hamilton, a neurosurgeon, is an internationally recognized expert on hydrocephalus and Past President of the Hydrocephalus Society (ISHCSF). He will have a particular role in overseeing submissions related to hydrocephalus and CNS fluids, as well as guiding the future of the journal. Dr. Hamilton is warmly welcomed as he assumes his new role.Dr. Hazel Jones is retiring as Co-Editor-in-Chief of CSF Research in 2004 [Fluids and Barriers of the CNS. At this stage, Richard Keep and Lester Drewes with their expertise in brain barriers research joined Hazel as Co-Editors-in-Chief. In 2015, the journal became officially affiliated to both the Hydrocephalus Society and the International Brain Barriers Society (IBBS).Hazel was founding editor of what was then  in 2004 . When shThe growth and success of the journal is fundamentally due to Hazel’s indomitable will and the endless hours she was willing to put in to foster its success. Starting a new journal is not easy! However, under Hazel’s guidance, the journal has grown from strength to strength. For example, in 2012, the journal was cited 243 times, in 2017 that was 1024 times, and in 2022 it was cited 3002 times, a 12-fold increase over a decade. There were also about 520,000 downloads in 2021.The journal was sorely needed. Many were finding it difficult to find a ‘home’ for their CSF/hydrocephalus/brain barrier research in ‘mainstream’ journals. Under Hazel’s guidance, the journal not only provided a home for such research, it also helped foster talent entering the field by providing awards for junior investigators attending hydrocephalus and brain barrier conferences. The current editors are ever grateful for Hazel Jones’ drive, determination and commitment to the highest standards of research publication. The whole field owes a great debt of gratitude to Hazel for her founding the journal and her leadership over nearly 20 years. She will be keeping an eye on the journal’s progress!